| Research Article |
Open Access |
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| In-Vitro Release and Pharmacokinetics of Anti-tubercle
Drug Ethionamide in Healthy Male Subjects |
| Mahmood Ahmad*, Asad Ullah Madni, Muhammad Usman |
| Department of Pharmacy, the Islamia University of Bahawalpur, Pakistan-63100 |
| *Corresponding author: |
Dr. Mahmood Ahmad,
Department of Pharmacy,
the Islamia University of Bahawalpur,
Pakistan-63100,
E-mail: ma786_786@yahoo.com |
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| Received December 26, 2009; Accepted December 30, 2009; Published December 30, 2009 |
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| Citation: Ahmad M, Madni AU, Usman M (2009) In-Vitro Release and Pharmacokinetics of Anti-tubercle Drug Ethionamide in Healthy Male Subjects. J Bioanal Biomed 1: 046-049. doi:10.4172/1948-593X.1000010 |
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| Copyright: © 2009 Ahmad M, et al. This is an open-access article distributed
under the terms of the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are credited. |
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| Abstract |
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| The aim of study was to assess the pharmacokinetics of
ethionamide in the local population of healthy human subjects.
Serum samples were taken from each of the selected
subject at different time intervals. These samples were
analyzed by using High Performance Liquid Chromatography
consisting of reverse phase C18 column, UV detector
set at 291nm. The mobile phase was consisted of 0.02
M disodium hydrogen phosphate and acetonitrile (75:25)
and delivered at a rate of 1.5ml/min. The value of Cmax was found to be 1.941 ± 1.487 μg/ml (mean ± SEM) and
Tmax was 1.75 ± 1.487 hours (mean ± SEM). The area under
the curve (AUC) was 8.745 ± 0.536 (mean ± SEM).
The elimination half life (t½) was found out as 1.995 ±
1.157 hours (mean ± SEM). The total body clearance (Cl)
was determined as 32.591 ± 0.298 ml/hr/kg (mean ± SEM).
It was concluded that ethionamide (Ethomid® Schazoo-
Lahore, Pakistan) found in consistent with the values reported
in the available literature. The study will be beneficial
and valuable in designing dosage regimen for the
patients on ethionamide therapy and can be utilized as
guideline in accessing the bioavailability and pharmacokinetics
parameters in clinical situations. |
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| Keywords |
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| Ethionamide (ETA); Pharmacokinetics; Serum;
High performance liquid Chromatography (HPLC) |
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| Introduction |
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| Ethionamide is an isonicotinic acid derivative of thioamide
class. It is the second line orally administered drug used in the
treatment of clinical tuberculosis that has failed to respond to
adequate first line therapy. Ethionamide is often combined with
other anti tuberculous agents for the treatment of multidrug resistant
organisms. Ethionamide is active against tubercle bacilli
that are growing with in human macrophages (Auclair et al., 2001; Conte et al., 2000). It is used to cure tuberculosis, a disease that
infects more than a third of the world’s population (Vannelli et
al., 2002). Infections caused by Mycobacterium avium intracellular
complex (MAI) and drug resistant mycobacterium are increasingly
common in different parts of the world and are fueled
with spread of Acquired Immunodeficiency Syndrome (AIDS),
as a result of this second line antimicrobial agents such as ethionamide
are being used much more frequently. In-vitro, ethionamide
is active against most of the Mycobacteria including, Mycobacterium
tuberculi and MAI (Mycobacterium Avium
Intracellulare complex) in the concentration range of 0.6-1.2 mg/
L while Bovine strains and BCG are more resistant and are inhibited
only by a concentration of 5.0 mg/L, thus displaying broad
range of potential clinical applications (Conte et al., 2000; Rebecca et al., 1999; Harnansingh et al., 1970). Extremely, limited data available in literature on the pharmacokinetics of
ethionamide in healthy subjects and in patients with tuberculosis
thus made such studies important to be conducted (Auclair et
al., 2001). |
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| Subjects, Materials and Methods |
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| Subjects |
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| Twelve healthy adult male human volunteers participated in
this study. The ages of volunteers ranged from 21 to 30 years
and their body weights in the range of 56-70kg. On the basis of
medical history, clinical examination and laboratory investigations
(hematology, blood biochemistry and urine analysis), none
of the participants had any revealed medical abnormality. In addition,
the included subjects had no history of hospitalization
and involvement in any medical trial within twelve weeks prior
to the initiation of the study, and none of them had received any
regular course of drug therapy within the preceding four weeks. |
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| Subjects were excluded from the study if they had a history of
serious illness, were taking any medications (including OTC)
within one week prior to the start of the study, or having a history
of any kind of allergy, history of hypersensitivity to ETA,
Clofazimine, Cylcoserine, Paraminosalicylic acid or pyridoxine,
had donated blood within 30 days prior to the study. Written
consent was obtained from the volunteers. The Pharmacy ethical
committee of Faculty of Pharmacy and Alternative Medicine,
the Islamia University of Bahawalpur approved the protocols
of this study. |
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| In-vitro studies |
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| The drug formulation was tested and passed the Compendial
requirements with respect to its content uniformity and weight
variation. Dissolutions studies were performed in 0.1 N HCl
(Dissolution media) by placing six tablets in USP apparatus-I at
37°C. The apparatus was adjusted at 100 rpm. Samples were
then drawn at designated time intervals at 5, 15, 30 and 45 minutes
and analyzed for their Ethionamide content spectrophotometrically
at 290 nm against the dissolution medium as blank
(USP-NF, 2004). Concentrations were determined with reference
to a standard curve. The values are given in Table 1 and
shown in Figure 1. |
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Figure 1: Percentage release of Ethionamide Tablets determined by the dissolution
studies. |
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| Table 1: Dissolution profile of Ethionamide tablets (ETT-011). |
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| In vivo studies |
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| The study was an open, single dose, complete one period of
treatment dosing. Subjects received single dose of Ethionamide
hydrochloride (Ethomid® Schazoo Laboratories (Pvt.) Ltd.,
Lahore–Pakistan) Tablets of 250 mg (Batch No.ETT-011). Drug
administration began approximately at 7.30 a.m. on the study
day after an overnight fast of 10 h. Written informs consent was
obtained from each subject before the study. An indwelling cannula
was inserted into a forearm vein before the ingestion of
Ethionamide tablet with 250 ml of water. The subjects were asked
to remain fasting for 4 hours into the study. No water was permitted
2 hours after dosing. A light breakfast was allowed at 2
hours after dosing. A light breakfast was allowed at 4 hours followed
by standardized lunch and evening tea with refreshment.
Cigarettes and food or beverage containing caffeine were not
allowed over 24 hour. |
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| Sample collection |
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| A 20-gauge venous cannula was inserted into a forearm for
the collection of blood samples and 3 ml blood samples were
collected through the syringe before dosing (zero time) and at
0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 4.0, 6.0, 8.0, 12.0 and 24.0 hours after
dosing of Ethionamide tablet. Following clot retraction, the
samples were centrifuged at 4000 rpm for 15 min. The obtained
serum was frozen at -20°C pending analysis. Samples were kept
refrigerated during the period of collection. |
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| High performance liquid chromatography |
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| Analyses were performed by using Shimadzu Liquid Chromatography,
with a pump LC 10 A.S, SPD 10 A. Shimadzu U.V.
Visible Detector set at 291 nm. A reverse phase system was used
consisting of a 5-μm Hypersil ODS (C18) column (250mm x
4.6 mm I.D.) precede by 5-μm Hypersil ODS (C18) cartridge
Guard (10mm x 4.6 mm I.D.) column. The mobile phase was
0.02 M disodium hydrogen phosphate buffer–Acetonitrile (75:25), which was filtered through 0.45μm membrane filter,
degassed by sonicator, and delivered at a rate of 1.5 ml/min.
injection of 20-μm were injected, with a run time of 6 min.(Auclair et al., 2001; Charles et al., 1991). |
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| HPLC analysis |
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| Serum concentrations of Ethionamide were quantitated by a
rapid and sensitive high performance liquid chromatographic
(HPLC) method (Charles et al., 1991). The mobile phase consisted
of disodium hydrogen phosphate 0.02 M and Acetonitrile
in the ratio of 75:25. 0.02 M disodium Hydrogen phosphate was
prepared by dissolving 3.588 g of di sodium hydrogen phosphate
in about 200ml of distilled water and mix thoroughly until
complete solubility in distilled water, then make the volume to
1000 ml with distilled water. Now take 750 ml of this buffer
solution and 250 ml of Acetonitrile to make 1000 ml with Acetonitrile.
Filter the mobile phase by passing through filtration
assembly under vacuum pressure of 150-200 torr using 0.45μm
membrane filter (Sartorius). Now degas the mobile phase by placing
it in the sonicator for 2-3 min. until complete degassing of
the mobile phase is ensured. |
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| Stock solution (100μg/ml) were prepared fresh daily by dissolving
the drug in the acetonitrile. Extraction procedure was
simple based on precipitation method. In the extraction procedure
1 ml of the drug solution was spiked with 1 ml of the blank
serum in the 8 ml glass centrifuge tube and mixes well, then
centrifuge for 10 min. Separate the upper layer (organic layer)
by micropipette, filter by using of the filtration syringe and take
the filtrate in 1.5 ml eppendorf tubes. Evaporate the sample by
using vacuum evaporator to dryness under the steam of nitrogen.
Reconstitute the sample with 70μL of the mobile phase;
vortex mix and Inject in to the HPLC injection port 20μl of the
volume by injection syringe. |
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| Preparation of standard curve |
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| Standard curve was constructed to encompass the anticipated range of serum ethionamide concentration found in healthy Subjects.
Blank serum was spiked with ethionamide drug solution
to give the concentrations of 0.1, 0.5, 1.0, 2.0, 3.0, and 4.0μg/
ml. The extraction procedure was same as described earlier. The
standard curve is constructed for each of the subjects to avoid
variation and to ensure precision and accuracy in the results,
also average of the twelve volunteer is also calculated by taking
mean of the twelve subjects. |
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| Injections of 20μl were injected and spectra were taken of
each concentration. The peak areas are noted for each concentration.
The absolute recovery of ETA was 86%. The within day
precision {coefficient of variation (CV)} of validation samples
was 0.42-5.26%, and the overall validation precision was 0.65-
3.89 pattern was determined form samples assayed over the
course of study. The specificity of the HPLC assay for ethionamide
was determined by testing spiked samples (Auclair et al.,
2001; Charles et al., 1991). |
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| Safety Analysis |
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| Health assessment, including vital signs, physical examination
and clinical laboratory testing was performed before and
seven days after the study. Subjects were interviewed at the beginning
and end of each study period and were monitored throughout of the confinement period to determine any adverse
events potentially related to study medication of procedures
(Auclair et al., 2001). |
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| Pharmacokinetic Analysis |
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| Pharmacokinetics analyses were performed by non-compartmental
method of analysis. Maximum concentration of Ethionamide
in serum (Cmax) and times to these concentrations (Tmax),
area under the concentration time curve (AUC0-∞) and other pharmacokinetic
parameters were calculated by using Kinetica Software. |
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| Results |
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| The in vitro results indicate that the Ethionamide formulation
met the specifications for content uniformity and weight variation
stipulated by compendial standards. Ethionamide concentration
and peak height ratios of Ethionamide were linear throughout
the concentration range of 0.1 to 4μg/ml. Standard curve of
Ethionamide in serum was constructed to determine the slope.
The limit of detection of the assay was calculated to be 10 ng of
Ethionamide per ml serum. Mean serum concentrations following
the oral administration of Ethionamide to 12 volunteers are
presented in Table 2 and plotted on rectangular co-ordinate graph & on a semi logarithmic scale in Figure 2 and Figure 3, respectively.
The ration of AUC0-12h /AUC0-∞ for all individuals was >
98% indicating an adequate sampling time, since the extrapolated
portion of the total AUC is less than 2%. Maximum concentration
in all the twelve subjects found in the range of 1.229
to 2.7527μg /ml with a mean value of 1.941 ± 1.3857μg/ml at
time ranging from 1 to 2.5 hours with a mean value of 1.75 ±
1.487 hours. |
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| Table 2: Mean ± SEM (n=12) Serum concentration of Ethionamide HCl administered
in the oral dose of 250 mg in Healthy Human Volunteers. |
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Figure 2: Serum Concentration vs time profile of Ethionamide HCl plotted
on rectangular co-ordinate graph, administered in the oral dose of 250 mg in
healthy human volunteers (n=12). |
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Figure 3: Concentration vs time of Ethionamide HCl plotted on semi-log
graph, administered in the oral dose of 250 mg in healthy human volunteers
(n=12). |
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| The bioavailability and pharmacokinetic parameters of
Ethionamide are calculated by using non-compartmental model
of pharmacokinetic analysis for accessing Cmax, Tmax and Area
under the curve (AUC). The absorption of Ethionamide was excellent
with mean serum concentration (Cmax) of 1.941μg/ml
obtained at average maximum time (Tmax) of 1.75 hours. The
geometric mean (± SEM) of bioavailability and pharmacokinetic
parameters (AUC0-∞, AUMC0-∞, Ka, t½(abs), MAT, t½(elim), MRT, Vd
and Cl) following the administration of Ethionamide are presented
in Table 3. |
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| Table 3: Pharmacokinetic and Bioavailability Parameters of Ethionamide HCl
administered in a dose of 250 mg in healthy human volunteers (n=12). |
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| Discussion |
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| Although less information is available in literature on the
bioavailability and pharmacokinetics of ethionamide in healthy
human subjects as well as in patients with tuberculosis. However,
the bioavailability and pharmacokinetic parameters observed
in this study are in accordance with the earlier reported
values. |
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| The absorption of Ethionamide is rapid and complete after
oral administration and more than 90% of the given dose absorbed
from gastro-intestinal tract. The absorption rate constant
(Ka) in this study found in the range of 0.272 to 0.598 hr-1 with a
mean value of 0.384 ± 3.185 hr-1 which shows that absorption is
rapid and measurable quantities of drug reaching blood with in
15 to 20 minutes. Absorption rate constant (Ka) is an important
parameter having an influence on the Cmax, Tmax and AUC. Shorter
the value of Tmax, faster will be the absorption rate. Cmax also increases
with higher values of Ka. The Mean absorption time
(MAT) 1.671 to 3.554 hours (2.758 ± 1.23) shows that absorption
of the drug completed within 2 to 3 hours. The maximum plasma concentration Cmax is calculated to be in the range of 1.229
to 2.753μg/ml (1.941 ± 1.857) and the time to reach maximum
concentration (Tmax) ranging from 1 to 2.5 hours. These values
are in consistent with the previously reported values (Auclair et
al., 2001; Zhu et al., 2002). Area under the curve (AUC) reflects
the total amount of drug that reaches the systemic circulation.
AUC is directly proportional to the dose of the drug. As dose of
the drug increases, AUC also increases. Ethionamide is rapidly
and widely distributed through out the body. The values for AUCo-∞ are calculated in this study ranging from 5.231-15.615μg.h/ml
with mean values of 8.745 ± 0.566μg.h/ml. This value is in accordance
with the previously reported value ranging from 5.4 to
17μg.h/ml with a mean value of 10 ± 0.360μg.h/ml (Auclair et
al., 2001). The elimination rate constant (Ke) is an important
parameter having effect on Cmax, Tmax and AUC. With an increase
in the elimination rate, the values of Cmax, Tmax and AUC will
decreased and vice versa. The mean ± SEM values of elimination
rate constant and elimination half life were calculated to be
0.39 ± 2.864 hr-1 and 1.8 ± 1.362 hours, respectively. The mean
± SEM values reported by Zhu et al., (2001) for elimination rate
constant (Ke) and elimination half life (t½) is 0.43 ± 2.105 hr-1 and 1.94 ± 1.362 hours which are in line with the present findings.
The values of clearance reported in literature lies in the
range of 27.5 to 66.5 L/hr/Kg (32 ± 0.698 L/hr/Kg, mean ± SEM).
The clearance is a useful index for the measurement of drug
removal from the body. Faster the clearance, lower will be volume
of distribution and elimination half life. If elimination half
life is decreased the accumulation of the drugs in the body will
also decreased and the clearance will be increased. Volume of
distribution is also related with the dose of the drug. As dose of
the drug increases, the volume of distribution (Vd) also increased.
The volume of distribution Vd estimated in this study ranging
from 52.78 to 75.70 L/kg. Volume of distribution relates plasma concentration to the amount of drug in the body. A large value of
volume of distribution shows that the drug is readily soluble and
completely absorbed from the routes of administration. Volume
of steady state shows the transfer of the drug from the tissue to
central compartment. The volume of steady state, Vss worked out
in the range of 4.128-38.388 L/kg with mean value of 18.154 l/
kg ± 0.2958 L/kg. |
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| The bioavailability and pharmacokinetic parameters assessed
in the present study shows a similarity with the previous reported
parameters. The drug shows better absorption profile and more
than 90% of the drug absorbed. The drug also shows excellent
distribution and elimination profiles. These parameters will be beneficial and valuable in designing dosage regimen for the patients
on ethionamide therapy and can be utilized as guideline
for assessing the bioavailability and pharmacokinetic parameters
in clinical situations. These studies must be conducted in the
real clinical conditions in local population to determine its safety
and tolerability. |
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| Acknowledgement |
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| The Authors wish to thank to the Management of Schazoo
Laboratories (Pvt.) Ltd. Lahore-Pakistan for providing all kind
facilities for the completion of this research project. |
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