Analytical Method
of Apremilast: A Review
Kanchan R. Pagar, Poonam M.
Kasar, Sarika V. Khandbahale
R.G. Sapkal Institute of
Pharmacy, Anjaneri, Nashik
*Corresponding Author E-mail: kanchanpagar96@gmail.com
ABSTRACT:
A selective method for
separation and determination of potential related impurities (starting
materials and by-products of synthesis, and degradants) of apremilast drug
substance has been developed and validated. The separation was accomplished on
a Cosmosil C-18 (250 mm × 4.6 mm, 5 μm) column connected to a photodiode
array (PDA) detector using optimized mixture of 0.05% trifluoroacetic acid,
methanol and acetonitrile under gradient elution. Two major degradant
impurities found in force degradation study of apremilast drug substance. Both
degradants were characterized preliminarily by HPLC-MS studies and synthesized
in laboratory. Structure was evidenced by NMR spectroscopy, mass spectrometry
and HPLC method was developed for quantification of the synthesized impurities
along with starting materials. This method can be used for the quality control
testing of drug substance. The performance of the method was validated
according to the ICH guide lines for specificity, limit of detection, limit of
quantification, linearity, accuracy, precision, ruggedness and robustness.
KEYWORDS: Apremilast;
analytical methods, adverse effects.
INTRODUCTION:
Apremilast is
chemically known as N-[2-[(1S)-1-(3-ethoxy-4-methoxyphenyl) -2
(methylsulfonyl)ethyl]-1, 3-dioxo-2, 3- dihydro-1H-isoindol-4-yl]
acetamide. It has an empirical formula of C22H24N2O7S, and a molecular weight
of 460.5 g mole−1.
Apremilast is a
Food and Drug Administration approved drug used for treatment of psoriasis and
psoriatic arthritis. It may also be useful for other immune system related
inflammatory diseases. The drug acts as a selective inhibitor of the enzyme
phosphodiesterase 4 (PDE4) and inhibits spontaneous production of TNF-alpha
from human rheumatoid synovial cells1,2,3. The US-FDA approved
Apremilast for the treatment of moderate to severe plaque psoriasis.
It is also being
tested for its efficacy in treating other chronic inflammatory diseases such
asankylosing spondylitis, Behcet's disease, and rheumatoid arthritis4,5.
Several research papers have been reported in the literature for the
determination of apremilast. These papers were limited to the assay of
apremilast alone performed by UV spectrophotometry where impurity
identification and quantification is not done6. New related
impurities are synthesized and quantification method with HPLC is reported but
the obvious degradants which studied here in present research are not reported7.
Pharmacokinetic study of apremilast in rat plasma has been studied by using
UPLC MS/MS where apremilast is quantified in the blood plasma8,9.
The reported related substance methods are suitable for quantification of some
of related impurities but another degradants formed under the stress conditions
employed were neither discussed nor characterized. Further, no monograph of
apremilast is published in any of the pharmacopoeia for compendia applications.
In present research during force degradation study two major degradant
impurities were observed when the drug substance is exposed to acid and base
degradation. Both degradants were synthesized in laboratory and structure is
elucidated using LCMS, 1H NMR and IR spectroscopy techniques. The RP-HPLC
method is developed for the separation and determination of apremilast and
potential related impurities i.e. raw materials, by-products and degradants.
The proposed analytical method is validated as per International conference on
harmonization guidelines (ICH Q2-R1)10, 11,12. The manuscript describes
a comprehensive investigation on isolation and characterization of a major
process related impurities of Apremilast 3-(acetylamino -2-{[1-(3-ethoxy-4-
methoxyphenyl)-2 (methylsulfonyl)ethyl]carbamoyl}benzoic acid i.e. Impurity-B
and 3-(acetylamino-6-{[1-(3-ethoxy-4-Apremilast, brand name Otezla
among others, is a medication for the treatment of certain types of psoriasis
and psoriatic arthritis. It may also be useful for other immune system related
inflammatory diseases. The drug acts as a selective inhibitor of the enzyme
phosphodiesterase 4 (PDE4) and inhibits spontaneous production of TNF-alpha
from human rheumatoid synovial cells.
Mechanism of action:
Apremilast is a small molecule
inhibitor of PDE413, an enzyme that breaks down cyclic adenosine
monophosphate (cAMP). In inflammatory cells, PDE4 is the dominant enzyme
responsible for this reaction. The resulting increase in cAMP levels
down-regulates expression of a number of pro-inflammatory factors like tumor
necrosis factor alpha (TNFα), interleukin 17, interleukin 23, and many
others, and up-regulates the anti-inflammatory interleukin 10. In ex vivo
models of arthritis, IL-12/IL-23p40 was specifically identified as a downstream
target of apremilast14 The importance of these individual factors for
the clinical effect of apremilast is not clear.
Pharmacokinetics:
Apremilast is absorbed from
the gut well (73%) and independently of food intake, and reaches peak blood
plasma concentrations after 2.5 hours. Plasma protein binding is 68%. It is
metabolised in the liver, mainly via the enzyme CYP3A4, but to a minor extent
via CYP1A2 and CYP2A6. The main metabolite is O-desmethylapremilast
glucuronide15.
The half-life is 6–9 hours.
The substance is eliminated through the kidney (58%) and feces (39%), mainly in
form of its metabolites. Only 3% of the original substance are found in the
urine, and 7% in the feces.
Particle size, polydispersity index (PDI), and zeta
potential (ZP):
The developed APM-loaded PLGA
NPs were freeze dried followed by dispersing them in the Milli-Q water
(20μg/mL). The suspension was then characterized for particle size,
polydispersity index (PDI), and zeta potential (ZP) using the dynamic
light-scattering technique. The Malvern Particle Size Analyzer (Malvern
Instruments Ltd, Holtsville, NY, USA) was used to measure the mean particle
size and the PDI of different developed NPs (F1–F3). The NP samples were
diluted to 200 times with deionized water and sonicated for 10 minutes in order
to obtain clear aqueous dispersion. Each sample (3mL) was The supernatant of
the sample was withdrawn at different time intervals (1, 2, 3, 4, 5, 6, 12, 24,
and 48 hours). The collected sample was centrifuged at 12,000rpm for 5 minutes
and analyzed for the drug content using UV spectroscopy at 229 nm16. The
data obtained from the release study were plotted and fitted in various
kinetic models to obtain the release pattern of the drug from the polymeric
matrix. in transparent disposable plastic cuvettes and the mean particle size
and PDI were measured. The same analyzer was utilized to measure the ZP of the
NPs (F1–F3) but the measurements were done using glass electrode.
ANALYTICAL METHOD
VALIDATION:
Method validation
is closely related to method development. When a new method is being developed,
some parameters are already being evaluated during the “development stage,”
while in fact, this forms part of the “validation stage.” Related substances
method is validated as per ICH guideline9.
Specificity and
force degradation:
The ability of the
method to determine accurately and specifically the analyte of interest in the
presence of other components in a sample matrix that may be expected to be
present in the sample matrix under the stated conditions. Specificity of the
method was evidenced by comparing blank, apremilast and all specified
impurities separate injections as well as spiking all impurities into
apremilast test solution. Force degradation study is performed by exposing the
sample to heat at 105⁰C for 24 hours, sample treated with base 1 N sodium
hydroxide and with acid 1N hydrochloric acid. Sample was exposed to
ultra-violet light for 24 hours and 3% hydrogen peroxide solution. After
exposure samples were tested using the proposed related substances method with
photo diode array detector. The degraded samples were further analyzed to find
out assay of apremilast. Mass balance is calculated by comparison of total
impurities from related substances test and the assay of apremilast.
Solution stability:
Drug stability in
Active Pharmaceutical Ingredient is a function of storage conditions and
chemical properties of the drug and its impurities. Conditions used in
stability experiments should reflect situations likely to be encountered during
actual sample handling and analysis. Stability data are required to show that
the concentration and purity of analyte in the sample at the time of analysis
corresponds to the concentration and purity of analyte at the time of sampling.
The solution stability till twelve hours of apremilast API had been checked by
injecting test solution and standard solution. Test solution was prepared fresh
before injection and immediately injected and same solution was injected after
twelve hours
Linearity:
The ability of the
method to obtain test results proportional to the concentration of the analyte
within a given range. It was evaluated by linear regression analysis, which was
calculated by the least square regression method.
Limit of detection:
The limit of
detection (LOD) is the lowest concentration of analyte in a sample that can be
detected but not necessary quantified. The obtained LOD values of specified
impurities and API is discussed.
LOD = 3.3 × σ
/ S
Where, σ = the
standard deviation of the response and
S= slope of the
calibration curve
Limit of
quantitation:
The limit of
quantitation is the lowest concentration or amount of analyte that can be
determined quantitatively within an acceptable level of repeatability precision
and trueness.
Limit of
quantitation (LOQ) = 10.0 × σ / S
Where, σ = the
standard deviation of the response and
S= slope of the
calibration curve
Precision at LOQ is
confirmed by six replicate analyses of impurities at LOQ level.
Accuracy:
Accuracy can be
defined as the closeness of agreement between a test result and the accepted
reference value. Accuracy of the method was determined by recovery study.
Analytical method may be considered validated in terms of accuracy if the mean
value is within ± 20% of the actual value. During recovery study apremilast API
batch was analyzed and then all specified impurities of known concentration is
spiked in the API at LOQ level, 50%, 100% and 150% with respect to the limit of
specified impurity.
Ruggedness:
The
(intra-laboratory tested) behavior of an analytical process when small changes
in environment and/or operating condition are made. The ruggedness of the
method was evaluated by estimating % RSD of standard solution tested by two
different analysts using different HPLC instrument and columns on different
days. Three validation batches were prepared by each analyst separately. % RSD
of each impurity of preparations of both analysts should not be more than 10%.
Robustness:
Robustness is a
measure of the capacity of the analytical procedure to remain unaffected by
small but deliberate variations in method–performance parameters, which
provides an indication of its reliability during normal usage. Robustness of
the method was determined by analyzing the system suitability solution and
batch analysis with deliberate change in the parameters like (a) flow rate of
mobile phase ± 0.1ml/min and (b) column temperature ± 5°C.
Bioanalytical
methods:
An ultraperformance liquid
chromatography coupled with tandem mass spectrometry (MS/MS) was used for the
quantification of APM in rat plasma samples. Our previously reported assay was
modified for this purpose17. To increase the sensitivity of the
assay, the electrospray ionization was operated in positive mode and the
calibration range was between 1 and 1,000 ng/mL with lower limit of quantification
of 1ng/mL in plasma samples. Due to change in the ionization mode, losartan
was used as the internal standard (IS). The precursor to product ion transition
of 461.16 and178.08 and 423.13 and 207.12 was used for detection and quantification
of analyte (APM) and the IS (losartan), respectively, in the multiple reaction
monitoring (MRM) mode. The optimized MS/MS parameters of capillary voltage
4.00kV, source temperature 150°C, desolvation temperature 350°C, and collision
gas flow rate 0.17 mL/minute were used for sample ionization. A cone voltage of
26 V (both for analyte and IS) and collision energy of 28 and 20 eV were used
for the analyte and the IS, respectively, as compound-specific parameters. Due
to change in ionization mode and IS, the assay was partially validated in terms
of precision and accuracy following the US Food and Drug Administration 2013
guideline for bioanalytical method validation. Both intra- and interday
variation in precision and accuracy was found to be within the acceptable
limits of ±15%.
Diarrhea occurs in about 25%
of patients taking apremilast. Severe gastrointestinal symptoms, when they
occur, typically start within the first few weeks of treatment18, 19.
Worsening depression,
suicidal thoughts, and other mood changes may occur with apremilast20.
Weight loss: Weight loss has
been associated with apremilast. Reports from clinical studies indicated a 5 to
10% decrease in body weight in 10% of patients taking apremilast (compared to
3.3% of patients taking placebo)20.
Common, usually mild to
moderate adverse effects associated with apremilast include headache, back
pain, nausea, diarrhea, fatigue, nasopharyngitis and upper respiratory tract
infections21.
CONCLUSION:
Process related
impurities of apremilast are identified, synthesized and characterized.
Structural elucidations of all synthesized compounds were done by using NMR, IR
and mass spectral data. Impurity of RRT 0.77 is 3-(acetylamino-2-
{[1-(3-ethoxy- 4-methoxyphenyl) -2-(methylsulfonyl) ethyl]carbamoyl}
benzoicacid i.e. Impurity-B. Impurity at RRT 0.79 is 3- (acetylamino-6-{[1-(3-ethoxy-4-
methoxyphenyl)-2-(methylsulfonyl) ethyl] carbamoyl} benzoicacid i.e.
Impurity-C. All process related impurities and degradant impurities are
quantified in the proposed method of analysis. Thus, the regulatory requirement
was fulfilled by characterizing this impurity and the prepared impurity
standard was used during analytical method validation studies. The above
RP-HPLC analytical method satisfies all validation parameters like system
suitability, precision, specificity, accuracy, linearity of detector response,
ruggedness and robustness. At the same time the method satisfies the forced
degradation study. It indicates that the method is stable and suitable for the
apremilast and its related substances determination. Hence, the validated
method can be used for routine analysis of related substances in apremilast in
quality control laboratories in the pharmaceutical industry.
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Received on 13.12.2019 Modified on 31.12.2019
Accepted on 18.01.2020
©Asian Pharma Press All Right Reserved
Asian J. Pharm. Ana. 2020; 10(1):44-47.
DOI: 10.5958/2231-5675.2020.00010.1