Azhar Int J Pharm Med Sci 2023; Vol 3 (1):15-31

(Research Article)
Print ISSN: 2735-4598
Online ISSN: 2735-4601

Formulation and evaluation of colon targeted pulsincap delivery of

Carvedilol for treatment of hypertension
Amira F. Elghamry 1, *, Zeinab Alkasaby Zalat 2, Amal S. Abu El-Enin 3, and Hosny A. Elewa 4
1 Chest Disease Hospital, Zagazig, Sharkia City, Egypt.
2 Department of Clinical Pharmacy, Faculty of Pharmacy (Girls), Al-Azhar University, Cairo, Egypt.
3 Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy (Girls), Al-Azhar University,
Cairo, Egypt.
4 Department of Pharmacy Practice, Faculty of pharmacy, Horus university, New Damietta city, Egypt.

* Correspondence: [email protected]

Article history: Received 2021-12-13 Revised 2022-03-13 Accepted 2022-03-22

Abstract: The objective of this work is to ensure the controlled release of carvedilol at a given time and
location for treating hypertension as a pulsincap delivery system. The capsule body was made of insoluble
hard gelatin and filled with carvedilol microspheres, then a hydrogel plug was used to close it. The
microspheres were made by oil/water emulsification followed by a solvent evaporation method in different
drug: polymer ratios using Eudragit L100 and S100 as polymers. Direct compression was used to make the
hydrogel plugs. Various assessment parameters, FTIR analysis, and in-vitro release experiments were
performed on the produced microspheres. The results showed that formulated microspheres had satisfactory
results for various micrometric properties. The microsphere formulations showed the highest entrapment
efficiency were F9, F14 and F15 (89.18±2.25, 83.91±2.13 and 92.2±2.28% respectively). FTIR results
revealed that carvedilol was molecularly dispersed into the polymeric matrix. In-vitro release studies of
carvedilol microspheres showed sustained action over 8 hours. The pulsincap system was evaluated for the
in-vitro release studies. PF9 had the highest cumulative release after 12 hours (94.161±0.83 %) and reduced
drug release during lag time so, PF9 was chosen for the in-vivo study. The in-vivo study was carried out on 16
hypertensive patients using HPLC method to measure carvedilol conc. in the blood and compare PF9 with
commercially available tablets of the same strength. The in-vivo study revealed that the prepared pulsincap
has shown better pharmacokinetics parameters compared with the marketed formulation with a delayed
release pattern for the effective treatment of hypertension.

Keywords: Carvedilol; Colon-targeted; Hypertension; Microspheres; Pulsincap.

This is an open access article distributed under the CC BY-NC-ND license

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take their medicine before bedtime will experience

1. INTRODUCTION less morning discomfort and may have a lower
Hypertension is a long-term condition whose cardiovascular risk3. Therefore, we developed a
symptoms are most obvious early in the morning and unique pulsincap formulation that releases the
there is a need to control the morning surge1. It has medicine according to a predefined schedule. It
been recommended to treat hypertension by using consists of a water-insoluble capsule body filled with
the concept of chronopharmacotherapy to ensure that medication content and sealed with a hydrogel plug
the maximum concentration of the drug should be at the opening end, which is covered by a
present in the bloodstream during the activation of water-soluble cap. The whole unit was enteric coated
sympathetic tone which results in a rapid increase in to avoid the problem of variable gastric emptying 4.
blood pressure, known as morning blood pressure When the capsule enters the small intestine, the plug
surge (MBPS)2. Antihypertensive individuals who is ejected by swelling or erosion, and the medication
is released.

Cite this article: Elghamry A., Zalat Z., Abu El-Enin A., Elewa H. Formulation and evaluation of colon targeted pulsincap delivery
of Carvedilol for treatment of hypertension. Azhar International Journal of Pharmaceutical and Medical Sciences, 2023; 3(1):15-31.
doi: 10.21608/AIJPMS.2022.110955.1100 15
DOI : 10.21608/AIJPMS.2022.110955.1100 https://aijpms.journals.ekb.eg/
Colon targeted pulsincap carvedilol delivery system

Carvedilol is a nonselective β-adrenergic 2. METHODS

blocking agent with α1-blocking activity and is
2.1. Materials
indicated for the treatment of hypertension and mild
or moderate heart failure of ischemic or Carvedilol was kindly donated from Global
cardiomyopathic origin. Because of the carbazole NAPI pharmaceuticals, Cairo (Egypt). Eudragit L
moiety in its structure, carvedilol can also be 100, Eudragit S100, Hydroxy Propyl
considered a powerful antioxidant5. The drug is methylcellulose (HPMC K4M), dibutyl phthalate,
rapidly absorbed and undergoes extensive first-pass cellulose acetate phthalate and lactose were kindly
metabolism in the liver. It reaches a peak supplied by Egyptian International Pharmaceutical
concentration 1 to 2 hours post dose and has an Industries Company (EIPICO), Cairo (Egypt).
elimination half-life of about 4-7 hours6. It is also Polyvinyl Alcohol (PVA), Potassium dihydrogen
poorly soluble in water resulting in a very low orthophosphate, Di-sodium hydrogen
bioavailability (it has a bioavailability of 25-35 %), orthophosphate anhydrous, Dichloromethane,
dose frequency, patient incompliance, and decreased Ethanol, hydrochloric acid, formaldehyde solution
stability. Microspheres provide prolonged and (37 % formaldehyde solution stabilized by 15%
constant therapeutic effect, as well as particle size methanol, Merck), acetyl acetone, ammonium
reduction for improving drug solubility. acetate, glacial acetic acid, acetone, and potassium
Microspheres minimise dose frequency, improving permanganate were purchased from El Gomhoureya
patient compliance, provide a better therapeutic for Drugs Trade & Medical Equipment, Cairo
effect for medications with a short half-life while (Egypt). Acetonitrile, Ethanol, and methanol, HPLC
also protecting the medication from enzymatic and grade; Merck. Darmstadt, Germany. Diethyl ether,
photolytic degradation. Drug absorption in the ethyl acetate, dichloromethane, butanol, sodium
stomach is reduced, resulting in less local side hydroxide, trichloroacetic acid, and chloroform were
effects. In general, microspheres prevent the first purchased from El Nasr Chemical Co.
pass metabolism, improve the biological half-life Triethanolamine and Ortho-phosphoric acid,
and also improve the bioavailability7. Microspheres analytical grade; Riedel-dehaene, Germany.
are matrix systems in which the medication is 2.2. Formulation and in-vitro evaluation of the
uniformly dispersed, either dissolved or suspended.
pulsincap system
Its structure is made up of solid or liquid particles
that are dispersed or dissolved in a matrix8. Emulsion 2.2.1. Preparation of Carvedilol microspheres
solvent evaporation techniques have proven to be An accurately weighed amount of carvedilol
more useful when compared to other methods of and polymers (Eudragit L100 and Eudragit S100) in
preparing microspheres. In this technique, the drug is various proportions were dissolved in 25 mL of the
dissolved in the polymer/solvent system. Then it is organic solvent (Ethanol+ Dichloromethane 1:1) at
added dropwise to the aqueous phase by continuous room temperature as shown in table (2). The mixture
agitation until the solvent is evaporated. This process was stirred with a magnetic stirrer (Wise stir
results in hardened microsphere which contains the ®MSH-20 D DAIHAN scientific, Co., Ltd., Korea)
drug9. to make a homogeneous polymer dispersion of the
The aim of the present study is to formulate and medication. The previous organic phase was added
evaluate pulsincap drug delivery system containing drop-by-drop to 100 mL of distilled water containing
carvedilol microspheres. This system can release the 0.5% polyvinyl alcohol as an emulsifying agent. A
drug at a predetermined time after a lag period, so mechanical lab stirrer (HS-120A, DAIHAN
that the drug will be released from the formulation Scientific Co., Ltd., Korea) was used to maintain
according to the physiological needs of the body, constant stirring at 1000 rpm until the organic phase
which offers benefits like controlled administration was evaporated. The microspheres were filtered,
of therapeutic dose at the desired rate, reduction of washed with distilled water, and left to dry
side effects, minimization of dosing frequency, and overnight10.
decreasing cardiovascular risk, especially in the
early morning. 2.2.2. Evaluation of Carvedilol Microspheres
2.2.2.1. Determination of percentage yield and
entrapment efficiency: The yield of the microspheres
was calculated using the following equation11,

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Elghamry et al., Azhar Int J Pharm Med Sci 2023; Vol 3 (1):15-31

Weight of the produced microspheres reflector in the range 4000-400 cm-1 with resolution
Percantage yield = X 100
Total weight of drug and polymer used 4 cm-1, refractive index 2.4 (National research
For the evaluation of entrapment efficiency, 25 Centre).
mg microspheres were weighed and dissolved in 10 2.2.2.5. Particle size analysis and determination of
mL of the organic solvent (Ethanol + flow properties of microspheres
Dichloromethane 1:1). Add 20 mL of 0.1 N HCl (pH
2.2.2.5.1. Particle size analysis: Optical microscopy
1.2) to the beaker containing the organic solvent to
equipped with a calibrated ocular micrometer was
precipitate the polymer. The organic solvent was
used to determine the average particle size of the
evaporated by heating the beaker on a magnetic
microspheres. A small number of microspheres were
stirrer then the solution was filtered through a
put on a glass slide and were determined in each
double-layered filter paper (Whatman 42), in a 100
formula by making use of Edmondson’s equation:
mL volumetric flask, and the volume was adjusted to
D mean =Ʃnd/Ʃn
100 mL using (0.1 N HCl). The solution was filtered,
and from the filtrate, the absorbance was measured at
Where “n” means the number of counted
240 nm 12.
microspheres, and “d” stands for the mean size
range12.
Actual drug content (mg)
% Entrapment Efficiency = X 100
Theoretical drug content (mg)
2.2.2.5.2. Angle of repose: The angle of repose was
determined by a fixed funnel method. In this
2.2.2.2. Saturation solubility study: The Solubility
method, microspheres were poured through the walls
was investigated using shake-flask method13 for a
of a funnel which was fixed at a position such that its
blank formula of pure carvedilol and carvedilol
lower tip was at a height of exactly 2.0 cm above
microspheres (F9, F14, and F15) as they had the
hard surface. The sample was poured till the time
highest entrapment efficiency. In 250 mL conical
when upper tip of the pile surface touched the lower
flasks containing 25 mL of distilled water, an excess
tip of the funnel. Draw a circle around the conical
quantity of drug and microspheres was introduced.
heap and measure its diameter. The height of the pile
At 37 ± 0.5ºC, the sealed flasks were shaken for 24
(h) and the radius were determined. Then, the angle
hours. Following that, aliquots were filtered using
of repose was calculated using the following
Whatman filter paper. Carvedilol concentration was
equation15:
evaluated using a UV spectrophotometer set at 240
nm. A saturation solubility study was also performed Tan θ=h/r
in ethanol, 0.1 N HCl (pH 1.2), and phosphate buffer
solutions (pH 6.8 and 7.4). 2.2.2.5.3. Bulk Density and Tapped density: Bulk
density was measured by gently introducing a known
2.2.2.3. Study of the external morphology (Scanning
sample mass through a glass funnel into a 10 mL
electron microscopy and microscopic images):
graduated cylinder and leaving the powder without
Scanning electron microscopy (SEM) (JEOL
compacting it. The apparent untapped volume is then
JSM-6480LV, Japan) was used to study the surface
read to the nearest graduated unit.
shape and structure of microspheres. Microspheres
were placed on a sample holder before being Bulk density = (Weight of the powder)/ (Bulk volume)
sputter-coated with a conducting metal (platinum). Tapped density was determined by pouring gently a
After that, the sample was examined for particle size specified quantity of sample through a glass funnel
and surface morphology with a focused fine electron into a 10 mL graduated cylinder. The initial volume
beam14. The microscopic images were captured was observed, and then the cylinder was allowed to
using optical microscope (Labomed 9131040 40x stroke. The tapping was continued until no further
Semi-Plan Achromatic Objective, USA) change in volume was noted. Volume occupied by
the sample after tapping was recorded and the tapped
2.2.2.4. Fourier transformed infrared specrtroscopy
density was calculated 16.
(FTIR): FTIR spectra of pure carvedilol, Eudragit
L100, Eudragit S100, and the prepared microspheres Tapped density = (Weight of the powder)/(Tapped volume)
were determined using Bruker VERTEX 80 2.2.2.5.4. Carr’s index (CI)17:
(Germany) combined platinum diamond ATR,
comprises a diamond disk as that of an internal Carr’s index = (Tapped density-Bulk density)/(Tapped density)

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Colon targeted pulsincap carvedilol delivery system

2.2.2.5.5. Hausner’s ratio (HR)18: time in decreasing the solubility of capsule shell.
Then formaldehyde exposed capsule bodies were
Hausner’s ratio = (Tapped density)/(Bulk density)
dried in hot air oven. The time at which the capsule
2.2.2.6. In-vitro release study of microspheres: became soluble was noted 21.
Carvedilol microspheres were tested for in-vitro
2.2.4.3. Qualitative estimation of free formaldehyde:
release using dialysis membrane method. A dialysis
Residual content in treated gelatin capsule bodies
tube (2.5 cm in diameter and 6 cm in length) acted as
(Colorimetric Estimation of Formaldehyde):
a donor compartment. A dialysis membrane
Standard formaldehyde solution: 3 mL of
(semi-permeable cellophane membrane) which had
formaldehyde solution (37% formaldehyde solution
been pre-soaked for 30 minutes in warm water (to
stabilized by 15% methanol, Merk) were put in 50
open the pores of the membrane) was inserted over
mL volumetric flask and the volume was made up to
the glass tube's lower end and made watertight by a
50 mL by distilled water to give a standard reference
rubber band. The receiver compartment held 100 mL
solution containing 20 µg/mL concentration of
of phosphate buffer solution pH 6.8 (release media).
formaldehyde (The limit for residual formaldehyde
Microspheres equivalent to 12.5 mg of carvedilol
according to FDA is 0.002%).
were dispersed into 5 mL of pH 6.8 buffer and
positioned in the donor compartment. The system Sample solution: formaldehyde treated bodies
was maintained at 37 ± 0.5ºC for 8 hours in a (about 25 in number) were cut into small pieces and
thermostatic shaker water bath at 50 rpm. Samples of taken into a beaker containing 25 mL distilled water.
5 mL were taken at intervals of 0.5, 1, 2, 3, 4, 5, 6, 7, This was stirred for 1 hour with a magnetic stirrer to
and 8 hours. To maintain constant volume, the solubilize the free formaldehyde. The solution was
volume of each sample was replaced with the same then filtered into a 50 mL volumetric flask and
amount of new buffer. The samples were analyzed volume was made up to 50 mL with distilled water.
spectrophotometrically at 240 nm19.
Acetyl acetone Reagent: Prepared by dissolving
2.2.3. Preparation of cross-linked gelatin capsules 15.4 g of ammonium acetate in 50 mL of reagent
with formaldehyde treatment water in a 100-mL volumetric flask. To this solution,
Hard gelatin capsules of size 1 were taken. The add 0.20 mL of acetyl acetone and 0.30 mL of glacial
caps were detached from their bodies. To create acetic acid. Mix the solution thoroughly, then dilute
formalin vapors, 25 mL of formaldehyde solution to 100 mL with reagent water.
(37% formaldehyde solution stabilized by 15% Method
methanol) was placed in desiccators and potassium To 1 mL of sample solution, 9 mL of water was
permanganate was added. Unfilled capsule bodies added. 1 mL of resulting solution was taken into a
were subjected to formalin fumes. Because the caps test tube and mixed with 4 mL of water and 5 mL of
were not exposed, they remained water-soluble. The acetyl acetone reagent. The test tube was warmed in
bodies were removed after 6 hours of reaction and a water bath at 40 oC and allowed to stand for 40
dried at 50°C for 30 minutes in a hot air oven. After minutes and the color intensity of the sample solution
that, the bodies were dried at an ambient temperature was compared with the standard formaldehyde
to remove any remaining formaldehyde20. solution which was prepared and processed in the
same procedure as the test solution using 1 mL of
2.2.4. Tests for unfilled capsules treated with
standard solution in place of the sample solution 22.
formaldehyde
2.2.4.1. The capsules' diameter, Length and weight
2.2.5. Preparation of the hydrogel plug
were measured before and after formaldehyde
Each hydrogel plug was made by compressing
treatment using a caliper.
an equal amount of HPMC K4M and lactose in a
2.2.4.2. Solubility studies of treated capsules: Both single punch tablet machine (Korsch pressen,
treated and untreated capsules were stirred in a EKO-DMS, USA) to make tablets with a flat surface
beaker containing 100 mL dissolution medium by weighing 70-100 mg and compressed with a 7 mm
using a lab stirrer. The dissolution medium taken punch. A 2 mg talc powder was added to act as
were 0.1 N HCL (pH 1.2), phosphate buffer solution lubricant23. Table (1) and figure (1) show the
(pH 7.4 and 6.8). The capsule bodies were subjected composition of hydrogel plug tablets.
to formaldehyde solution for different hours ranging
from 2 -12 hours and checking the effect of exposure
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 Elghamry et al., Azhar Int J Pharm Med Sci 2023; Vol 3 (1):15-31

Table 1. Composition of the hydrogel plug tablets. capsules were thoroughly coated with 5% Cellulose
Acetate Phthalate (CAP) 26.
Ingredients Hydrogel Hydrogel Hydrogel
(mg) plug 1 plug 2 plug 3
2.2.8. Coating of Pulsincap
HPMC Cellulose acetate phthalate solution (5% w/v)
34 44 49 was prepared by adding 5 gm CAP to 0.75 gm
K4M
dibutyl phthalate as a plasticizer using acetone:
Lactose 34 44 49 ethanol (8:2) as a solvent. Dip coating method was
used. The capsules were alternatively dipped in 5 %
Talc 2 2 2
CAP solution and dried. Coating was repeated until
Total weight an expected weight gain of 8-12 % was obtained 27.
70±0.34 90±0.25 100±0.33
(mg)

2.2.6. Evaluation of the prepared hydrogel plugs

2.2.6.1. Thickness and hardness test: Thickness and
Hardness were measured using electronic digital
tablet hardness tester (pharma test, PTB 311,
Germany).
2.2.6.2. Swelling index: Hydrogel plug tablets were
sequentially immersed in media of three different pH
(pH 1.2, pH 6.8, and pH 7.4). For each formulation,
one tablet was weighed (dry weight) and placed in a
beaker containing 200 mL of buffer solution. After
each hour the hydrogel plug tablet was removed from
beaker and weighed again up to 6 hours. The tablets
were wiped off to remove excess of surface water by A
using filter paper. The percentage weight gain by the
hydrogel plug tablet was calculated 24.

% swelling = ((Wet weight-Dry weight))/(wet

weight) X 100
B C D
2.2.6.3. Lag time: The lag time test was calculated
indirectly by measuring the time necessary for
ejection of hydrogel plug from the impermeable
capsule body mouth completely. For the study, an
insoluble capsule body containing carvedilol
microspheres in the bottom and HPMC K4M
hydrogel plug tablet tightly put in the opening of the
impermeable capsule body was connected to the
paddle of the USP apparatus II by a thread and
suspended in phosphate buffer solution (pH 6.8) for
6 hours 25.

2.2.7. Formulation of a pulsatile drug delivery

system (modified pulsincap)
Carvedilol microspheres equivalent to 12.5 mg
were weighed and manually hand-filled into the
previously formaldehyde-treated bodies. The
microsphere-containing bodies were subsequently Figure 1. A) Hydrogel plugs with three different
filled using hydrogel plugs. Then, using a tiny thicknesses., B) empty capsule, C) formulated pulsincap
amount of the 5 % ethyl cellulose ethanolic solution, before coating, D) pulsincap after coating with 5%
seal the capsule body and cap together. The sealed cellulose acetate phthalate solution.

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Colon targeted pulsincap carvedilol delivery system

was calculated from the highest values of the

2.2.9. Evaluation of the modified Pulsincap obtained correlation coefficients.
2.2.9.1. Weight variation: 10 capsules were chosen
2.3. In-vivo therapeutic effect and bioavailability
at random from each batch and weighed individually.
study
2.2.9.2. Coating thickness of cellulose acetate
phthalate: Was measured using a screw gauge. The aim of this study is to study the effect of
2.2.9.3. Drug content: This test was done to ensure carvedilol on blood pressure especially in the early
that Equivalent weight of microspheres introduced morning when the cardiovascular risk increases so,
into the capsule was within the pharmacopoeial limit we formulate pulsincap to enable the patient to take
(95-105%). Microspheres (equivalent to 12.5 mg of the medicine at bedtime and the drug will be released
carvedilol) were immersed in 50 mL of 0.1 N HCl for from the pulsincap after a lag time in the early
30 minutes before sonication using a probe sonicator morning. The study included 16 hypertensive male
(Model 275 T, Crest Ultrasonics Crop, Trenton, adults ranging in age from 33 to 47 years and weight
USA) for 10 minutes to break the microspheres and from 72 to 84 kg (non-obese). Each patient signed a
facilitate extraction of the drug. The solution was written consent form. Each participant underwent a
centrifuged using a centrifuge (Phoenix CD-0412-50 thorough physical and clinical examination. All the
GMbH, Germany) and was filtered. The clear patients were supervised by a physician who was
supernatant solution was analyzed responsible for their safety and the collection of
spectrophotometrically at 240 nm28. samples during the study. Each patient's liver
function was assessed by measuring ALT and AST
2.2.10. In-vitro release studies of carvedilol because carvedilol is primarily eliminated through
pulsincap the liver. The patients were instructed not to take any
Release studies of carvedilol pulsincap were over-the-counter medications for 72 hours before the
done using the USP dissolution type II apparatus study. The study was approved by the Research
(Copley, NG 42JY, Nottingham, U.K.) paddle type. Ethics Committee of Al Azhar University- Faculty of
A cotton thread was used to attach a capsule to the Pharmacy (Girls) in Cairo, Egypt no. 38 on
paddle, ensuring that it was thoroughly submerged in 18-11-2015 and was carried out in the outpatient
dissolution media making them all in one level. clinic_internal medicine department_Al Hussein
Three dissolving media with pH 1.2, 7.4, and 6.8 Hospital. The patients were randomly numbered and
were employed consecutively to imitate pH divided into two dosing groups of equal size, each
variations along the GI tract, known as the with eight patients. They were then treated as
"sequential pH change approach." The pH 1.2 follows: Group I: They received the best-prepared
medium was utilised for 2 hours (since the usual formulation of Pulsincap (PF9) with a cup of water,
gastric emptying duration is 2 hours) before being and after a 7-day washout period, they received the
removed and replaced with a fresh pH 7.4 phosphate conventional marketed tablet (Carvipress® 12.5 mg)
buffer solution. The medium was withdrawn after 3 in the second period. Group II: Orally administered
hours (typical small intestine transit time) and the standard marketed tablet in the first period and
replaced with a new pH 6.8 dissolving medium for the pulsincap (PF9) in the second period.
the subsequent hours. 900 mL of dissolving medium
were used, at each time. The temperature was 2.3.1. Sample collection
maintained at 37±0.5ºC with a spinning speed of For Group I, blood samples were collected in
rpm. At specific time intervals, 5 mL of dissolution heparinized tubes using a vein puncture cannula
medium was removed and replaced with new before carvedilol administration (blank), as well as at
dissolution media. At 240 nm, the withdrawn 4,5, 6, 7, 8, 10, 12, and 14 hours after the dose.
samples were spectrophotometrically examined, and Sample withdrawal at 4 hours post dose was to
the total percentage release of the drug was confirm that there was no release of the drug.
computed29.
For Group II, blood samples were collected
using a vein puncture cannula before carvedilol
2.2.11. Kinetic modelling of drug release
administration (blank), as well as at 0.5, 1, 1.5, 2, 4,
The mathematical modeling and the in-vitro
6, and 8 hours after the dose. The sampling time was
drug release kinetics of carvedilol pulsincap were
different because pulsincap is a controlled release
calculated according to zero order, first order and
dosage form so, the appearance of carvedilol in
Higuchi's diffusion model. The best kinetic order
plasma was after 5 hours and that was confirmed by
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Elghamry et al., Azhar Int J Pharm Med Sci 2023; Vol 3 (1):15-31

withdrawal of blood sample before 5 hours. It was AUMC0→∞ is the area under the first moment curve
found that no drug release in plasma. However, in from time zero to infinity.
conventional tablet the drug appears rapidly after 1-2 MRT is the mean residence time.
hours. R.B is the relative bioavailability.

2.3.2. Instrumentation and chromatographic 3. RESULTS

conditions
3.1. In-vitro evaluation of the pulsincap system
HPLC (Agilent 1100, Germany) instrument
that was equipped with G1313A Autosampler, 3.1.1. Evaluation of carvedilol microspheres
G1315B DAD Detector, G1316A Column 3.1.1.1. Determination of percentage yield and
Compartment, G1322A Vaccum Degasser, G1311A entrapment efficiency: The prepared microspheres'
quaternary pump, and solvent tubing. The percentage yield ranged from 75±1.13 % to
chromatographic analysis was performed on a 94.66±1.46 %. The best value was found to be for F9
reversed phase column, Kromacil® 250x4.6mm (94.66±1.46). The results are revealed in table (2)
(i.d), 5µm with a guard column (4×3 mm i.d., and showed that the entrapment efficiency increased
Phenomenex) packed with the same material at a when the polymer concentration was increased until
flow rate of 1.0 mL/min and the injection volume it reached a particular limit, after which it declined.
was 20μL. The mobile phase consisted of water – The formulations had the highest entrapment
acetonitrile – methanol – ethanol-1M- triethylamine efficiency were F9, F14 and F15 (89.18±2.25,
(83:58:55: 3:1), the pH was adjusted to 2.5 with 83.91±2.13 and 92.2±2.28 % respectively). By
ortho-phosphoric acid. The mobile phase was increasing carvedilol: Eudragit L100 ratio from 1:1
filtered using 0.45-micron membrane filter paper and (F1) to 1:3 (F3), the entrapment efficiency increased
degassed for 15 minutes30. Detection was performed from 39.17±1.011 to 68.93±2.22. However,
at 240 nm. carvedilol: Eudragit L100 ratio of 1:4 (F4) and 1:5
(F5) gave lower values (37.975±1.14 and 28.69±1.17
2.3.3. Pharmacokinetic parameters and statistical respectively). By increasing carvedilol: Eudragit
analysis S100 ratio from 1:1 (F6) to 1:4 (F9), the entrapment
The pharmacokinetic parameters were analysed efficiency increased from 50.114±1.13 to
by the statistical package for the Social Sciences 89.18±2.25. However, carvedilol: Eudragit S100
(SPSS) version 26 (IBM Corp., Armonk, NY, USA). ratio of 1:5 (F10) gave lower value (34.89±0.02). By
The mean and standard deviation were used to increasing carvedilol: Eudragit L100: Eudragit S100
summarise the data. Unpaired t-test was used to ratio from 1:0.5:0.5 (F12) to 1:2:2 (F15), the
compare the two groups. P-values less than 0.05 entrapment efficiency increased from 21.59±1.22 to
were considered statistically significant. The 92.2±2.28. However, carvedilol: Eudragit L100:
calculated pharmacokinetic parameters obtained for Eudragit S100 ratio of 1:2.5:2.5 (F16) and 1:3:4
pulsincap and conventional tablet were: (F11) gave lower values (78±2.25 and 25.15±1.04
respectively).
Cmax is the maximum observed plasma
concentration.
3.1.1.2. Saturation solubility study: The results of
Tmax is the time to reach the maximum observed
saturated solubility study are shown in table (3).
plasma concentration.
3.1.1.3. Study of external morphology (Scanning
Kel is the apparent terminal elimination rate constant.
Electron Microscopy and microscopic images):
(t1/2) el is the apparent terminal half-life.
Examination of the formulae (F9) by SEM showed
AUC0-t is the area under the plasma
that the microspheres were found to be round and
concentration-time curve from time zero to the time
resembled aggregates or distinct particles.
of the last measurable concentration.
Microscopic images and SEM photograph of the
AUC0→∞ is the area under the plasma
carvedilol microspheres are revealed in figure (2).
concentration-time curve from time zero to infinity.

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 Colon targeted pulsincap carvedilol delivery system

Table 2. Composition, percentage yield, and entrapment efficiency of different formulations for Carvedilol microspheres.

Carvedilol Eudragit Eudragit Ratio (Drug: Total Percentage Entrapment

Formulation
(mg) L100 (mg) S100 (mg) polymer) weight (mg) yield (%) efficiency (%)

F1 300 300 ------ 1:1 600 83.33±2.74 39.17±0.01

F2 300 600 ------ 1:2 900 94.44±2.33 54.87±0.08

F3 300 900 ------ 1:3 1200 91.66±1.19 68.93±0.22

F4 300 1200 ------ 1:4 1500 80.00±1.23 37.97±0.14

F5 300 1500 ------ 1:5 1800 91.66±2.45 28.69±0.17

F6 300 ------ 300 1:1 600 80.00±1.67 50.11±0.13
F7 300 ------ 600 1:2 900 91.11±1.25 63.93±0.12
F8 300 ------ 900 1:3 1200 83.33±1.43 73.00±0.05
F9 300 ------ 1200 1:4 1500 94.66±1.46 89.18±0.25
F10 300 ------ 1500 1:5 1800 80.55±1.14 34.89±0.02
F11 300 900 1200 1:3:4 2400 87.50±1.20 25.15±0.04
F12 300 150 150 1:0.5:0.5 600 75.00±1.13 21.59±0.22
F13 300 300 300 1:1:1 900 86.66±1.06 66.29±0.19
F14 300 450 450 1:1.5:1.5 1200 83.33±2.33 83.91±0.13
F15 300 600 600 1:2:2 1500 93.33±2.97 92.20±0.28
F16 300 750 750 1:2.5:2.5 1800 83.33±1.30 78.00±0.25

Figure 2. A) SEM photograph of Carvedilol microsphere, B) Microscopic images of carvedilol microspheres (F9, F14, and
F15).
3.1.1.4. Fourier transformed infrared spectroscopy present in the pure carvedilol disappeared and a
(FTIR): The spectrum of pure carvedilol, Eudragit broad peak at the same wavenumber range (around
S100, Eudragit L100, and microspheres are shown in 3366.21 cm−1) was observed.
figure (3). In the spectra of the produced
microspheres, the secondary amine sharp peak

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Elghamry et al., Azhar Int J Pharm Med Sci 2023; Vol 3 (1):15-31

Table 3. Saturated solubility of blank formula for pure carvedilol and carvedilol microspheres.

Saturated solubility (mg/mL)

Solvent
Pure carvedilol F9 F14 F15

Distilled water 0.0045±0.0 0.0067±0.0003 0.0052±0.0001 0.0031±0.0001

01
Ethanol 10.9± 1.2 12±0.25 12.7±0.14 11.3±0.15

pH (1.2) 0.271±0.00 0.009±0.006 0.005±0.002 0.002±0.004

2
pH (6.8) 0.153±0.00 0.8±0.005 0.75±0.001 0.71±0.005
2
pH (7.4) 0.064±0.00 0.7±0.004 0.68±0.008 0.66±0.003
1

A
C

B
D

Figure 3. FTIR spectrum of A) pure carvedilol, B) Eudragit L100, C) Eudragit S 100, D) microspheres.
.
3.1.1.5. Particle size analysis and determination to 249.9±1.91 μm. The evaluation results of
of flow properties of microspheres: The mean prepared microspheres are shown in table (4).
particle size of the microspheres increased The calculated properties were all satisfactory
dramatically as the polymer concentration in all formulations and showed good flow
increased, ranging from 79±1.50 properties 31.

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 Colon targeted pulsincap carvedilol delivery system

Table 4. Flow properties of carvedilol microspheres.

Formulations Particle Angle of Bulk density Tapped Hausner’s Carr’s

size(μm) repose (g/cm3) density(g/cm3) ratio index(%)

F1 79.00±1.50 27.64±0.04 0.398±0.034 0.477 ±0.066 1.195±0.035 16.56±0.201

F2 85.00±3.20 26.93±0.03 0.432±0.032 0.526 ±0.051 1.375±0.030 17.87±0.125

F3 89.80±1.28 22.10±0.08 0.474±0.059 0.586 ±0.031 1.370±0.015 19.11±0.059

F4 97.33±1.64 26.75±0.09 0.456±0.055 0.541 ±0.087 1.307±0.020 15.71±0.236

F5 110.00±0.70 20.52±0.02 0.399±0.051 0.449 ± 0.026 1.120±0.010 11.13±0.221

F6 83.54±0.30 26.14±0.03 0.767±0.058 0.898±0.033 1.170±0.013 14.58±0.142

F7 95.36±1.28 25.71±0.07 0.523±0.032 0.604±0.025 1.150±0.045 13.41±0.246

F8 102.50±0.64 26.71±0.06 1.320±0.025 1.530±0.02 1.159±0.017 13.72±0.075

F9 108.66±1.20 25.42±0.03 1.110±0.025 1.250±0.01 1.126±0.054 11.20±0.207

F10 114.25±0.63 26.81±0.01 1.160±0.065 1.600±0.021 1.379±0.024 12.29±0.033

F11 249.90±1.91 26.52±0.01 0.519±0.041 0.606±0.085 1.167±0.012 13.64±0.122

F12 132.55±1.80 26.64±0.02 0.749±0.031 0.884±0.053 1.180±0.029 15.28±0.155

F13 148.90±0.71 23.43±0.02 0.492±0.057 0.578±0.075 1.174±0.018 14.87±0.143

F14 167.80±1.30 26.32±0.02 0.781±0.007 0.903±0.036 1.156±0.048 13.49±0.186

F15 178.46±1.20 23.95± 0.05 0.248±0.048 0.279±0.016 1.125±0.036 11.11±0.136

F16 189.00±0.66 21.52±0.02 0.508±0.064 0.591±0.024 1.163±0.160 13.98±0.131

capsules were found to be slightly thicker. It was

3.1.1.6. In-vitro release study of carvedilol
found that the length of capsules increased from
microspheres: The in-vitro release is shown in figure
20±0.3 mm (before formaldehyde treatment) to
(4) and it was biphasic, with a quick phase followed
20.3±0.1mm (after formaldehyde treatment), also the
by a delayed phase. The initial rapid effect may be
capsule external diameter was found to increase after
desired to guarantee therapeutic medication plasma
formaldehyde treatment from 7±0.1 mm to 7.3±0.2
concentrations at the start of treatment. The drug
mm. The weight of empty capsule bodies increased
released from F9 (91.7±0.69%) after 8 hours was
from 74.51 ±0.23 mg to 75.36±0.25 mg after
higher than F14 and F15 (75.256±0.48 and
formaldehyde treatment.
73.716±0.03% respectively).
3.1.2.2. Solubility studies for the treated capsule
3.1.2. Evaluation of unfilled capsules treated with bodies
formaldehyde The results are revealed in table (5) and showed that
both untreated capsule bodies and caps dissolved
3.1.2.1. The diameter, length, and weight of empty within less than 20 minutes while formalin-treated
capsule bodies were measured before and after capsules bodies remained intact for longer time.
formaldehyde treatment. The formaldehyde treated

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 Elghamry et al., Azhar Int J Pharm Med Sci 2023; Vol 3 (1):15-31

Table 5. Solubility studies of formaldehyde-treated capsule bodies.

Time needed for Time needed for

Time of exposure to Time needed for Time needed for
solubility in solubility in
formaldehyde solution solubility in solubility in 0.1 N
phosphate buffer phosphate buffer
in hours distilled water HCL (pH 1.2)
solution ( pH 7.4) solution ( pH 6.8)
2 4:15 h 50 minutes 1h 1:10 h
4 7:30 h 1:25 h 1:30 h 1:15 h
6 10:20 h 2h 2:10 h 2:15 h
8 15:10 h 3:20 h 3:25 h 3:30 h
10 17 h 5:15 h 5:30 h 5:20 h
12 22 h 6h 6h 6:10 h

3.1.2.3. Qualitative estimation of free formaldehyde than the standard solution indicating that less than
residual content in treated gelatin capsule bodies): 20μg free formaldehyde is present in 25 capsules.
The test showed that a yellow-colored solution was
3.1.3. Physical evaluation of the hydrogel plugs:
produced for both sample and standard solutions.
Hydrogel plug 3 showed the highest %swelling
The sample solution was not more intensely colored
index and the longest lag time of 5.35±0.12 as shown
in table (6).

Table 6. The thickness, hardness, lag time and percentage swelling index of the prepared hydrogel plug.

Hydrogel plug Thickness Hardness % Swelling index after 6 h

No. (mm) (Kg/cm2) Lag time (h)
In pH 1.2 In pH 6.8 In pH 7.4
Hydrogel plug 1 2.80±0.042 2.4±0.014 73.28±3.27 73.14±0.16 72.19±2.33 4.30±0.13
Hydrogel plug 2 3.05±0.035 2.7±0.075 78.13±2.41 79.96±0.19 79.08±2.77 5.05±0.18
Hydrogel plug 3 3.60±0.021 2.9±0.023 84.09±1.17 84.13±0.17 83.98±0.22 5.35±0.12

3.1.4. Evaluation of the prepared pulsincap improves the water resistance of this coating
Dibutyl phthalate was used as a plasticizer. The material, and formulations using such plasticizers are
percentage of the plasticizer is 15% of cellulose more effective than when cellulose acetate phthalate
acetate phthalate. The addition of plasticizers is used alone.
Table 7. Evaluation of the prepared pulsincap.

Weight of Weight of Weight of

Thickness Average
empty microspheres hydrogel Total weight
Formulation of CAP weight after % Drug
capsule equivalent to plug of capsule
No. Coating coating with 5 content
(mg) 12.5 mg tablet (mg)
(mm) % CAP (mg)
carvedilol (mg)

PF9 75.36±0.25 70.07±0.15 100±0.11 245.43±0.51 0.63±0.061 269.973±3.58 99.31±1.36

PF14 75.22±0.18 59.591±0.11 100±0.21 234.811±0.5 0.62±0.032 258.292±2.17 98.54±3.74

PF15 75.95±0.31 67.78±0.42 100±0.13 243.73± 0.86 0.65±0.015 268.103±3.11 98.45±2.13

3.1.5. In-vitro release studies of pulsincap cumulative release in pH 6.8after 12 h (94.161±0.83

The in-vitro release of pulsincap for selected %) compared to PF14 and PF15 (84.262±1.23 and
formulations (PF9, PF14, and PF15) was performed 80.068±0.67 %) respectively and reduced drug
and it was found that PF9 had the highest % release during lag time. The release in pH 1.2 after 2
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Colon targeted pulsincap carvedilol delivery system

hours was zero for the three formulations. While released 4.88±0.05% of the drug after 5 hours in pH
PF14 released 4.964±0.22% of the drug and PF15 7.4, as shown in figure (4).

A B

Figure 4. A) Percentage of carvedilol released from loaded microspheres (F9, F14, and F15), B) B In vitro release profile
of pulsincap (PF9, PF14, and PF15).

3.1.6. Kinetic modeling of drug release 3.2.1. Plasma Concentration-Time data

The kinetic analysis of all release profiles Figure (5) show the mean plasma concentration
follows zero order models. of carvedilol following oral administration of 12.5
mg conventional tablet and pulsincap by sixteen
3.2. In-vivo therapeutic effect and bioavailability patients.
study

Figure 5. A) The mean plasma concentration of carvedilol (ng/ml) versus time (hours) following administration of 12.5 mg
conventional tablet by sixteen patients, B) The mean plasma concentration of carvedilol (ng/ml) versus time (hours)
following administration of 12.5 mg pulsincap (PF9) by sixteen patients.

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 Elghamry et al., Azhar Int J Pharm Med Sci 2023; Vol 3 (1):15-31

Results of pharmacokinetic parameters are

3.2.2. Pharmacokinetic parameters and statistical
presented in table (8).
analysis

Table 8. The pharmacokinetic parameters of 12.5 mg carvedilol oral administration for both conventional tablet and
pulsincap (F9) were measured and calculated in sixteen patients.

Pharmacokinetic Conventional Pulsincap (PF9)

p-Value
Parameters tablet (mean ± S.D) (mean ± S.D)

Cmax (ng/mL) 60.00±4.1 60.73±2.84 0.558

Tmax (hr.) 1 10 <0.001*

Kel (hr-1) 0.296±0.015 0.090±0.097 <0.001*

(t1/2) el (hr.) 2.343±0.096 7.683±0.042 <0.001*

AUC0→∞(ng.hr/mL) 165.445±9.391 559.22±9.01 <0.001*

AUMC0→∞(ng.hr2/mL) 559.22±9.01 6207.819±98.265 <0.001*

MRT (hr) 3.38±0.084 11.01±0.043 <0.001*

Cmax/AUC0→t (hr-1) 0.408±0.014 0.178±0.006 <0.001*

Relative bioavailability (R.B) 338%

4. DISCUSSION
In the preparation of microspheres, surfactant For the entrapment efficiency, as the ratio of
concentration used was 0.5% PVA. The goal in drug-to-polymer increased, encapsulation efficiency
preparing emulsions must be to reduce the increased as seen in the formulations (from F1 to
interfacial tension to promote a more intimate F3), (From F6 to F9) and (from F12 to F15); this is
blending of the two phases. This can be achieved by due to the fact that higher ratio of drug-to-polymer
reducing the viscosity of the internal phase to make would produce large size droplets with decreased
a good emulsion form. Low PVA concentration surface area, such that diffusion of drug from such
(0.2% w/v) might be insufficient to stabilize the microsphere will be slow, resulting in higher
droplets, which led to low encapsulation efficiency. encapsulation efficiency33. While further increase in
PVA concentration of 0.5% w/v was found to be polymer content leads to a decrease in the
sufficient to stabilize the droplets leading to high encapsulation efficiency as seen in the formulations
encapsulation. Moreover, high PVA concentration (F4, F5, F10, F11 and F16). This can be due to the
(1% w/v) can affect the encapsulation efficiency of fact that an additional increase in polymer content
lipophilic drug due to increased viscosity and led to an enhancement of the concentration gradient
formation of molecular aggregates or micelles in between the emulsion droplets and the continuous
aqueous phase. The micelles have tendency to phase; as a result, increasing the amount of drug
solubilize lipophillic drug molecules resulting in partitioning into the continuous phase34.
increased solubility of lipophillic drugs thereby
leading to poor encapsulation of drug 32.

The results of saturated solubility study of the stomach). However, in basic pH, carvedilol
revealed that carvedilol exhibits pH-dependent may precipitate in a crystalline form under digestion
solubility. It is a weak base (pKa = 7.8), and hence condition. Lower solubility of carvedilol in buffers
it is ionisable only at very low PH values (acidic pH at high salt concentrations might be related to the
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 Colon targeted pulsincap carvedilol delivery system

higher ionic strength values of the dissolution During estimation of residual formaldehyde
media, where it has been shown that solubility content, formaldehyde reacts with acetylacetone in
decreases with an increase in ionic strength. presence of ammonium acetate leading to the
Solubility of microspheres in distilled water and pH formation of 3,5-diacetyl-1,4-dihydrolutidine
1.2 exhibit very low values. This could be due to the (DDL) which is characterized by yellow color
presence of the drug in a relatively more localized (Hantzsch reaction) 40.
way in the core of the microspheres. And neither
Eudragit S100 nor Eudragit L100 polymers Swelling behavior of hydrogel plugs was
dissolved in distilled water or the acidic medium assessed by the weight method. A gradual increase
(1.2). When the pH of the dissolution medium was in the swelling indices was achieved with increasing
increased to the slightly basic buffer medium (pH the amount of HPMC K4M and lactose attributable
6.8 and pH 7.4), the solubility increased. This may to the ability of HPMC to absorb water due to the
be due to the increase of the porosity of the presence of hydrophilic groups in its structure
microspheres due to the enhanced solubility of leading to increased viscosity, more hydration and
Eudragit S 100 and L100 in this medium and its gel formation around the surface of the tablet,
subsequent gradual removal from the microsphere attributing to high swelling index. Due to high
matrix structure35. viscosity, matrix integrity is maintained for a longer
duration leading to least erosion (stronger
The results of FTIR analysis showed that in diffusional layer that is resistant to diffusion or
the spectra of the produced microspheres, the erosion) 41. The swelling equilibrium (maximum
secondary amine sharp peak present in the pure swelling index) is reached when the osmotic forces
carvedilol disappeared and a broad peak at the same of the functional groups are balanced by the
wavenumber range (around 3366.21 cm−1) was restrictive forces of the higher ordering of the
observed, which is indicative of formation of polymer chains42.
hydrogen bond (which is a physical bond) between
the O- and NH groups of the drug and polymers36. The enhanced bioavailability of carvedilol
This might be evidence indicating a breakdown in may be due to the preparation of microspheres using
the crystalline structure of carvedilol, leading to the Eudragit, which improves the drug's solubility and
formation of the amorphous state within the provides a high level of protection against early
microspheres. These results could explain that the drug release in the stomach and small intestine.
reduction in crystallinity of drug led to a decrease of Carvedilol microspheres formed with Eudragit
the energy required in the dissolving process and convey the majority of the drug load to the colon,
also to a highly dispersed state of the drug. This allowing for medication release at the appropriate
bonding is most probably related to the controlled spot after adequate transit time20. Also, the
release of the drug37.The carbonyl stretching peak of hydrogel plug creates a lag phase (no release of
Eudragit L100 and S100 appears as a strong band at drug) that delays the drug release. Cross-linking of
1722.23 cm-1in microspheres with minor shifting the capsule body by formaldehyde vapour and
which confirm that carboxylic moieties are linked to coating the entire capsule with cellulose acetate
the carvedilol backbone chain in microspheres. phthalate (5%CAP) modified the solubility of the
Therefore, the microspheres could envelop capsule and was effective in delaying the drug
carvedilol, and strong chemical interactions release.
between carvedilol and polymers were absent38.
These movements that occur during the process may 5. CONCLUSIONS
indicate that Eudragit S100 and L100 polymers
It can be concluded that pulsatile drug
have been changed from a linear chain to a
delivery systems provide a solution for the
polymeric matrix form 39.
distribution of medications with
The main aim of formaldehyde treatment was chronopharmacological behavior, significant first-
to modify the solubility of hard gelatin capsules. pass metabolism, night-time dosing requirements, or
Cross-linking of gelatin molecules was achieved by GIT absorption window. The carvedilol pulsincap
exposing to formalin vapors. Cross-linking involves was successfully administered to the colon region,
the reaction of amino groups in gelatin molecular meeting the chronotherapeutic strategy for better
chain with aldehyde groups of formaldehyde by a hypertension therapy.
“Schiff’s base condensation” forming an
Funding: No significant financial support was
irreversible complex so that the gelatin becomes
received for this work.
water insoluble giving enough time for the polymer
to swell and extend the drug release29.
28

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Elghamry et al., Azhar Int J Pharm Med Sci 2023; Vol 3 (1):15-31

Acknowledgments: I would like to express my Author Contribution: The authors confirm

gratitude to my supervisors, who guided me contribution to the paper as follows: Study
throughout this project. I would also like to thank conception and design: Amira F. Elghamry, Zeinab
my family who supported me and offered deep Alkasaby Zalat , Amal S. Abu El-Enin and Hosny
insight into the study. A. Elewa; Data collection: Amira F. Elghamry,
Zeinab Alkasaby Zalat , Amal S. Abu El-Enin and
Conflicts of Interest: All authors declare that they Hosny A. Elewa; Analysis and interpretation of
have no conflicts of interest. results: Amira F. Elghamry carried out the
experimental analysis and performed the numerical
Ethical Statement: This study was approved by the calculations for the suggested experiment.;
Research Ethics Committee of Al Azhar University- Manuscript preparation: Amira F. Elghamry wrote
Faculty of Pharmacy (Girls) in Cairo, Egypt no. 38 the manuscript with input from all authors. All
on 18-11-2015 and was carried out in the outpatient authors reviewed the results and approved the final
clinic_internal medicine department_Al Hussein version of the manuscript. All authors provided
Hospital. This material is the authors' own original critical feedback and helped shape the research,
work, which has not been previously published analysis and manuscript.
elsewhere. The paper is not currently being
considered for publication elsewhere. The paper
reflects the authors' own research and analysis in a
truthful and complete manner.

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