King Saud University
College of Pharmacy
Department of Pharmaceutical Chemistry
Novel Microwell-Based Assays with High-Throughput for Quality Control of Crizotinib
‫معايرات مستحدثة عالية اإلنتاجية معتمدة على األبيار الدقيقة لضبط جودة الكريزوتينيب‬
M. Sci. Research Proposal Submitted to the Department of
Pharmaceutical Chemistry
By
Jamilah Mohamed Alshehri
B. Pharm. Sci. (………. H)
1434 (H)
2013 (G)
1. Introduction
Cancer is one of the greatest health concerns as it is the principal cause of mortality
among men and women worldwide; it accounted for 7.4 million deaths (around 13% of
all deaths). Deaths from cancer worldwide are projected to continue rising, with an
estimated 12 million deaths in 2030 [1]. Worldwide, lung cancer is the most common
cancer in terms of both incidence and mortality in men and women. In 2008, there were
1.61 million new cases, and 1.38 million deaths due to lung cancer [2]. The most common
causes of lung cancer are long-term exposure to tobacco smoke, genetic factors, radon
gas, asbestos, and air pollution [3,4]. Most cancers that start in lung, known as primary
lung cancers, are carcinomas that derive from epithelial cells. The main types of lung
cancers are small-cell lung cancer and non-small cell lung cancer (NSCLC). NSCLC
accounts for approximately 85% of all lung cancers. These cancer cells grow quickly and
spread early in the course of the disease. Surgery, radiotherapy, and chemotherapy are the
main treatment options for NSCLC. Surgery and radiotherapy are usually considered for
localized stage I and stage II of NSCLC, however, it is not eligible for advanced
NSCLC [5,6]. Chemotherapy is considered for approximately 80% of all patients with
NSCLC [7]. Multiple randomized, controlled trials and large meta-analyses all confirmed
the superiority of chemotherapy regimens for advanced NSCLC [8,9]. In advanced
metastatic or localized NSCLC, chemotherapy is used as first-line treatment and it
improves survival and quality of life, provided the patient is well enough for the
treatment [8]. Typically, the first generation of tyrosine kinase inhibitors (e.g. gefinitinib,
erlotinib) have been used [10,11]. However, a considerable number of patients with
NSCLC have a chromosomal rearrangement that generates a fusion gene between EML4
(echinoderm microtubule-associated protein like 4) and ALK (anaplastic lymphoma
kinase), which results in constitutive kinase activity that contributes to increased cell
carcinogenesis and drives the malignant phenotype [12-14]. Patients with this gene fusion
are typically younger non-smokers who do not have mutations in either the epidermal
growth factor receptor gene (EGFR) or in K-ras gene [3,4]. The kinase activity of the
fusion protein is not inhibited by the first generation of tyrosine kinase inhibitors, thus a
research was directed to discover an alternative novel drug for NSCLC including patients
whose tumors harbor EML4-ALK fusion proteins.
1
Crizotinib (CZT); 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4ylpyrazol-4-yl)pyridine-2-amine (Fig. 1) is a novel small-molecule inhibitor of tyrosine
kinases. It functions by competitive binding with the ATP-binding pocket of target
kinases [15-18]. It caused tumors shrining in 90% of patients carrying ALK fusion
gene [19]. Based on two successful clinical multi-center studies, CZT was granted an
accelerated approval by the Food and Drug Administration (FDA) on August 26, 2011
(under the trade name of Xalkori® capsules made by Pfizer, Inc.) for the treatment of
patients with advanced local or metastatic NSCLC that is ALK-positive as detected by
FDA-approved test (Abbot Molecular, Inc.) [20].
NH
Cl
N
F
N
Cl
O
H2N
N
Figure 1. Chemical Structure of Crizotinib (CZT).
The effective and safe therapy with CZT is basically depending on the quality of its
pharmaceutical preparations (Xalkori® capsules). For pharmaceutical QC of CZT, proper
analytical method with high-throughput is required. Literature review revealed that there
was no any method available for determination of CZT in neither its bulk form nor
capsules. Therefore, this research proposal is devoted to the development of proper
methods for QC of CZT.
Photometry has considerable importance in drug analysis, and photometric methods
are extensively applied for the determination of active substances in bulk drugs and
pharmaceutical preparations [21]. The importance of photometric methods in the field of
pharmaceutical QC has greatly increased, due to the fact that these methods can be very
readily automated. Automated analyzers equipped with photometric detection are widey
used for the serial analysis of pharmaceutical preparations, especially for studying the
contents uniformity and dissolution characteristics of the solid dosage forms [22]. It is
obvious that the chemical structure of CZT contains a weakly isolated chromophoric
2
moities, and it is anticipated to exhibit low molar absorptivity. Therefore, the
development of a selective photometric method for determinetion of CZT based on its
native ultraviolet (UV) light absorption would be difficult. As well, the development of
visible-photometric method for CZT is important to enable the use of simple analyzers
equipped with photometric detection systems. For these reasons, the present research
proposal is directed towards the
development of visible-photometric methods for
determination of CZT.
The
molecular
interactions
between
the
electron-donating
pharmaceutical
compounds and electron-accepting reagents are generally associated with the formation
of intensely colored charge-transfer (CT) complexes, which usually absorb radiations in
the visible region. The rapid formation of these complexes leads to their widespread
utility in the development of visible-photometric methods for QC of many
pharmaceutical compounds [23-26]. Literature survey revealed that the CT reactions of
CZT have not been investigated yet. These facts promoted my interest in employment
CT-reactions as basis for the development of new photometric methods for QC of CZT.
However, all the conventional CT-based photometric methods that have been reported so
far are not automated and consequently their throughput is low, thus their applications in
QC laboratories are limited. Moreover, these methods suffer from the consumption of
large volumes of organic solvents, which leads to high analysis cost, and more
importantly, the incidence of exposure of the analysts to the toxic effects of the organic
solvents [27-31]. For these reasons, the present research proposal is devoted to
investigate the CT reaction of CZT, and its employment in the development of novel nonconventional photometric method with high analysis throughput and can reduce the
consumption of organic solvents in the QC of CZT.
In previous studies, Darwish et al. [32-34] has successfully apopted a microwell plate
reader equipped with photometric detection in the development of high-throughput
photometric assays for measuring the active drug contents in pharmaceutical
preparations. For these reasons, the present research project is designed to employ this
methodology for CZT. The proposed photometric methods will be carried out in 96microwell plates (200 l volume). For high throughput analysis, the solutions will be
dispensed by 8-chanel pipette, and the absorbances will be measured by a plate reader.
The reader is able to read the 96 well simultaneously in ~ 30 seconds.
3
2. Research Objectives
The major tasks of this research work will undertake complete investigations required
for the development of novel microwell-based assays with high-throughput for QC of
CZT based on its CT reactions. The objectives can be pointed out as follows:
– Investigation of the perspective of CT reactions of CZT with electron-acceptors,
optimization of the different factors that can affect the reactions, and proposing the
reactions mechanisms.
– Employment of the optimum reactions conditions in establishing the analytical
procedures in 96-microwell assay plates, and measuring the signals by plate reader.
– Validation of the proposed assays, in terms of the sensitivity, accuracy, specificity,
precision, ruggedness, and robustness according to the guidelines of The International
Conference of Harmonization (ICH) for validation of the analytical procedures [35].
– Application of the developed assays for analysis of CZT in its bulk form and in
pharmaceutical dosage forms (Xalkori® capsules).
3. Material and Methods
3.1. Materials
– Crizotinib (Pfizer Inc., NY, USA).
– 7,7,8,8-Tetracyanoquinodimethane (TCNQ; Aldrich Chem. Co, Milwaukee, USA).
– 1,3,5-Trinitrobenzene (TNB; Prodotti Chimiol Pet Analist Scientifico, Milano, Italy).
– 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ; Merck, Munich, Germany).
– p-Chloranilic acid (pCA, BDH Chemicals, Poole, UK).
– Tetracyanoethylene (TCNE; Nacalai Tesque, Kyoto, Japan).
– Bromanil (Hopkin & Williams Ltd, UK).
– Chloranil (Sigma Chemical Co, St. Louis, USA).
– 2,4,7-Trinitro-9-fluorenone (TNF; Fluka, Switzerland).
–
Xalkori® capsules (Pfizer Inc., NY, USA) labeled to contain 250 mg CZT per
capsule.
– Analytical grade solvents and other chemicals (Fischer Scientific, USA).
– Microwell plates (Corning/Costar, Inc., Cambridge, USA).
– Finnpipette adjustable 8-channel pipette (Sigma Chemical Co. St. Louis., USA).
4
3.2. Apparatus
– Microplate Reader (Spectramax M5, Molecular Devices, California, USA)
empowered by Spectramax M5 software, provided with the instrument.
– Spectrophotometer (V530, JASCO, Tokyo, Japan), double beam.
3.3. Methods
3.3.1. Design of the assays, strategy for their development, and expected advantages
The proposed assays are designed to employ 96-microwell assay plates as the CT
reactions will be carried out in microwells of the assay plates (200-µl reaction volume)
instead of the conventional volumetric flasks (10,000-µl volume). The solutions will be
dispensed by 8-channel pipette, and the color signals will be measured by microwellplate reader that enables the measuring of all the 96 wells in ~ 30 seconds.
In this research proposal, CZT was selected based on its therapeutic importance,
clinical success, and its expected good electron-donating ability because of the presence
of multiple basic amino groups in the chemical structure of CZT (Fig. 1). Previous
studies involving CT reactions with polyhalo-/polycyanoquinone electron n-acceptors
revealed that their reactivity are widely varied according to the basicity of the electrondonor and the involved site of interaction [25]. Therefore, various acceptors (Fig. 2) will
be investigated in the development of the proposed assays for CZT. The 96-microwell
design of the proposed assays for CZT was based on the previous success of Darwish
et al. [32-34] in the utility of this design for the QC of other pharmaceutical compounds.
It is anticipated that the proposed assay will offer the following advantages:
– Providing a high throughput property that can facilitate the processing of large
number of samples in a relatively short time. This property is attributed to the use of
multi-channel pipettes for efficient dispensing the solutions, carrying out the
analytical reactions in 96-well plates (as reaction vessels), and measuring the color
signals in all the 96 wells at ~ 30 seconds by the plate reader.
– Reduction in the consumption of organic solvents, accordingly reduction in the
exposures of the analysts to the toxic effects of organic solvents.
– Reduction in the analysis cost by 50-folds which can be reflected on the price for the
finished dosage forms, thus it can reduce the expenses for the medications.
5
O
O
O
HO
Cl
Cl
OH
Cl
Cl
Cl
Br
Cl
Br
Br
Br
O
O
O
Chloranil
p-Chloranilic acid (pCA)
O
N
N
NO2
Bromanil
N
Cl
Cl
NO 2
O 2N
N
N
N
O
1,3,5-Trinitrobenzene
Tetracyanoethylene
2,3-Dichloro-5,6-dicyano-1,4-
(TNB)
(TCNE)
benzoquinone (DDQ)
N
N
O
O
O
N
N
O
O
N
N
N
O
O
7,7,8,8-Tetracyanoquinodimethane
2,4,7-Trinitro-9H-fluoren-9-one
(TCNQ)
(TNF)
Figure 2. Chemical structures of the polyhaloquinone and polycyanoquinone
electron acceptors that will be investigated in the present study.
3.3.2. Proposed studies
The reactivity of CZT with electron acceptors in 200-µl volume (in a microwell plate)
will be investigated, and the absorption spectra for the formed CT complexes will be
recorded for determination of their maximum absorption peaks. The optimum conditions
of the reactions required for achieving the highest possible sensitivity for each assay will
be established. The measurements of the colors will be performed by plate reader. The
site of interaction between CZT and acceptors will be determined by UV-visible, IR,
1
H-NMR spectrometry, and by energy-minimization computational molecular modeling.
Based on the obtained results, the reactions mechanisms will be proposed.
6
3.3.3. General analytical procedure
One hundred microliters of the standard or sample solution of CZT will be transferred
into each well of the 96-microwell assay plate. One hundred microliters of the acceptor
solution will be added, and the reaction will be allowed to proceed at the optimum
temperature for the optimum time. The absorbances of the resulting solutions will be
measured at the maximum absorption peaks by the microwell-plate reader. Blank wells
will be treated similarly except 100 µl of solvent will used be instead of the sample, and
the absorbances of the blank wells will be subtracted from those of the experiment wells.
3.3.4. Validation of the proposed assays
Linearity, detection and quantitation limits. The calibration curves for the analysis of
CZT by the proposed assays will be constructed. Regression analysis for the results will
be performed for calculating the intercepts, slopes, and correlation coefficients. The
limits of detection and limits of quantitation will be determined using the guidelines
recommended by ICH [35].
Accuracy and precision. The accuracy of the proposed assays will be evaluated by
standard addition method. The intra- and inter-assay precisions will be determined by
replicate analysis. The relative standard deviation (RSD) should not be more than 2%.
Interference studies. Interference liabilities will be carried out to explore the effect of
excipients on the analytical performance of the proposed assays.
Robustness and ruggedness. Robustness will examined by evaluating the influence of
small variation in the experimental parameters on the analytical performance of the
proposed assays. Ruggedness will be tested by applying the proposed methodology to the
assay of CZT using the same operational conditions but at two different elapsed times.
The results obtained from day-to-day will be evaluated by statistical analysis.
Statistical analysis. Statistical analysis for the results of the proposed methodology will
be carried out using t- and F-tests for evaluation of the accuracy and precision,
respectively. As far as there is no a reported reference method for CZT analysis for the
statistical comparison, each particular proposed assay will be independent compared with
another assay that will be developed in this study.
7
4. References
[1]
World Health Organization, Geneva, Report 2005. http://www.who.int/cancer/en.
[2]
J. Ferlay, H.R. Shin, F. Bray, D. Forman, D.M. Parkin. Estimates of worldwide
burden of cancer in 2008: GLOBOCAN 2008. Int. J. Cancer 127; 2893-2917 (2010).
[3]
A.J. Alberg, J.M. Samet. Murray & Nadel's Textbook of Respiratory Medicine, 5th
edition, Saunders Elsevier (2010).
[4]
K.M. O'Reilly, A.M. Mclaughlin, W.S. Beckett, S.J. Sime. Asbestos-related lung
disease. Am. Fam. Phys. 75; 683–688 (2007).
[5]
N.P. Rowell, C.J. Williams. Radical radiotherapy for stage I/II non-small cell lung
cancer in patients not sufficiently fit for or declining surgery (medically inoperable):
a systematic review. Thorax. Aug. 56; 628-638 (2001).
[6]
T.E. Stand, P.F. Brunsvig, D.C. Johannessen, et al. Potentially curative radiotherapy
for non-small cell lung cancer in Norway: a population-based study of survival. Int.
J. Radiat. Oncol. Biol. Phys. 80; 133-141 (2011).
[7]
W.W. Tan, J.E. Harris, S. Huq. Non-small cell lung cancer. Medscape Reference.
http://emedicine.medscape.com/article/279960-overview. Retrieved Jan. 26, 2013.
[8]
NSCLC Meta-Analyses Collaborative Group. Chemotherapy in addition to
supportive care improves survival in advanced non-small-cell lung cancer: a
systematic review and meta-analysis of individual patient data from 16 randomized
controlled trials. J. Clin. Oncol. 26; 4617-4625 (2008).
[9]
Non-Small Cell Lung Cancer Collaborative Group. Chemotherapy and supportive
care versus supportive care alone for advanced non-small cell lung cancer. Cochrane
Database of Systematic Reviews (5): CD007309.
http://onlinelibrary.wiley.com/doi/10.1002/14651858.CD007309.pub2/abstract.
Retrieved Jan. 26, 2013.
[10] M.H. Cohen, G.A. Williams, R. Sridhara, G. Chen, W.D. McGuinn Jr, D. Morse, S.
Abraham, A. Rahman, C. Liang, R. Lostritto, A. Baird, R. Pazdur.. United States
Food and Drug Administration drug approval summary: gefitinib (ZD1839; Iressa)
tablets. Clin. Cancer Res. 10;1212-1218 (2004).
[11] J.R. Johnson, M. Cohen, R. Sridhara, et al. Approval summary for erlotinib for
treatment of patients with locally advanced or metastatic non-small cell lung cancer
after failure of at least one prior chemotherapy regimen. Clin. Cancer Res. 11;
6414-6421 (2005).
[12] Maintenance therapy for non-small cell lung cancer. Medscape CME.
http://cme.medscape.com/viewarticle/720896_transcript. Retrieved Jan. 26, 2013.
8
[13] W. Ron. Advances come in war on cancer. The Wall Street Journal.
http://online.wsj.com/article/SB10001424052748704002104575291103764336126.ht
ml?mod=WSJ_WSJ_US_News_3. Retrieved Jan. 26, 2013.
[14] Pfizer oncology to present new clinical data from ten molecules across multiple
tumor types" (Press release). Pfizer Oncology.
http://media.pfizer.com/files/news/press_releases/2010/asco_curtain_raiser_052010.
pdf. Retrieved Jan. 26, 2013.
[15] I.O. Sai-Hong. Crizotinib: a novel and first-in-class multitargeted tyrosine kinase
inhibitor for the treatment of anaplastic lymphoma kinase rearranged non-small cell
lung cancer and beyond. Drug Design, Dev. Ther. 5; 471-485 (2011).
[16] S.J. Rodig, G.I. Shapiro. Crizotinib, a small-molecule dual inhibitor of the c-Met
and ALK receptor tyrosine kinases. Curr. Opin. Investig. Drugs 11; 1477-1490
(2010).
[17] E.L. Kwak, Y.J. Bang, D.R. Camidge, et al. Anaplastic lymphoma kinase inhibition
in non-small-cell lung cancer. New Eng. J. Med. 363; 1693-1703 (2010).
[18] J.J. Cui, M. Tran-Dube´, H. Shen, et al. Structure based drug design of crizotinib
(PF-02341066), a potent and selective dual inhibitor of mesenchymal-epithelial
transition factor (c-MET) kinase and anaplastic lymphoma kinase (ALK). J. Med.
Chem. 54; 6342-6363 (2011).
[19] ALK inhibitor crizotinib has high response rate in patients with ALK-positive
NSCLC. HemOncToday. http://www.healio.com/hematology-oncology/lungcancer/news/online/%7B7F093ECD-D7CD-420A-86E2F47AC2497224%7D/ALK-inhibitor-crizotinib-has-high-response-rate-in-patientswith-ALK-positive-NSCLC. Retrieved Jan. 26, 2013.
[20] FDA approval for crizotinib. Posted on Aug. 29, 2011.
http://www.cancer.gov/cancertopics/druginfo/fda-crizotinib. Retrieved Jan. 26, 2013.
[21] S. Görög. Ultraviolet-Visible Spectrophotometry in Pharmaceutical Analysis, CRC
Press, New York (1994).
[22] D. Perez-Bendito, M. Silva, A. Gomez-Hens. Automated kinetic-based
determinations for routine analysis: recent developments. Trends Anal. Chem. 8;
302-308 (1989).
[23] I.A. Darwish, I.H. Refaat. Spectrophotometric analysis of selective serotonin
reuptake inhibitors based on formation of charge-transfer complexes with
tetracyanoquinondimethane and chloranilic acid. J. AOAC Int. 89; 362-333 (2006).
[24] I.A. Darwish. Kinetic spectrophotometric methods for determination of trimetazidine
dihydrochloride. Anal. Chim. Acta 551; 222-231 (2005).
9
[25] I.A. Darwish. Analytical study for the charge transfer complexes of losartan
potassium. Anal. Chim. Acta 549; 212-220 (2005).
[26] I.A. Darwish, H.A. Abdel-Wadood, N.A. Abdel-Latif. Validated spectrophotometric
and fluorimetric methods for analysis of clozapine in tablets and urine. Annali di
Chim. 95, 345-356 (2005).
[27] A.T. Fidler, E.L. Baker, R.E. Letz. Neurobehavioural effects of occupational
exposure to organic solvents among construction painters. Br. J. Indust. Med. 44;
292-308 (1987).
[28] H. Wennborg, J.P. Bonde, M. Stenbeck, J. Olsen. Adverse reproduction outcomes
among employee in biomedical research laboratories. Scand. J. Work Environ.
Health 28; 5-11 (2002).
[29] M.L. Lindbohm, H.T. Taskinen, M. Sallman, K. Hemminki, Spontaneous abortions
among women exposed to organic solvents. Am. J. Indust. Med. 17; 449-463 (2007).
[30] H. Wennborg, B. Lennart, V. Harri, A. Gösta. Pregnancy outcome of personnel in
swedish biomedical research laboratories. J. Occup. Environ. Med. 42; 438-446 (2000).
[31] P. Kristensen, B. Hilt, K. Svendsen, T.K. Grimsrud. Incidence of
lymphohaematopoietic cancer at university laboratory: a cluster investigation. Eur. J.
Epidemiol. 23; 11-15 (2008).
[32] I.A. Darwish, A.M. Mahmoud, A.A. Al-Majed. A novel analytical approach for
reducing the consumption of organic solvents in the charge transfer-based
spectrophotometric analysis: Application in the analysis of certain antihypertensive
drugs. Acta Pharm. 60; 493-501 (2010).
[33] I.A. Darwish, T.A. Wani, N.Y. Khalil, A. Al-Shaikh, N. Al-Morshadi.
Development of a novel microwell assay with high throughput for determination of
olmesartan medoxomil in its tablets. Chem. Cent. J. 6:1; 1-7 (2012).
[34] I.A. Darwish, T.A. Wani, N.Y. Khalil, A.H. Backeit. Novel 96-microwell
spectrophotometric assays with high throughput for determination of irbesartan in
tablets. Digest J. Nanomaterials & Biostructures 7; 415-421 (2012).
[35] ICH Guideline, Q2(R1): Validation of Analytical Procedure: Text and Methodology,
London (2005).
10