Analysis of Gleevac Drug - Essay Example

Gleevec of the Introduction Gleevec is a drug indicated in the therapeutic treatment of chronic myelogenous leukemia (CML). Gleevec contains imatinib mesylate that is a tyrosine kinase inhibitor. Imatinib mesylate is a synthetic drug belonging to the 2-phenyl amino-pyrimidine (PAP) group (Zimmerman et al., 1996). Its molecular formula is presented as C29H31N7O • CH4SO3 with molecular weight 589.7. The structural formula is as figure 1[11] Chemical designation is 4-[(4-methyl-1-poperazinyl) methyl]-N-[4-methyl-3-[[4-(3-pyridinyl)-2-pyrimidinyl] amino]-phenyl] benzamide methanosulphate. It has four pKa values; 1.52, 2.56, 3.73, 8.07, very soluble in buffers of PH 5.5 and slightly soluble in neutral/alkaline aqueous buffers. Gleevec recognizes the auto inhibitory conformation of the activation loop of the protein that regulates the kinase activity. The structure of inactive state is distinct between different kinases. It has a half-life of 15 hours and allows daily administration. Gleevec is used to treat the chronic phase of CML though applications have been found in Advanced and blast crisis phases. Its efficacy has been found to reduce with phase progression.Most patients that don’t respond to gleevec are in advanced stages of CML at the initiation of treatment. Failure rate of imatinib in patient with chronic phase CML remains low(at less than 15%at 18 months) in patients who did not respond to interferon. General synthesis of PAPS Figure 2 syntheses of phenylaminopyrimidines Phenyl aminopyrimidines are a large group of selective anticancer agents that exert action through the selective inhibition of specific kinases. Imatinib Mesylate In the 1980’s and 90’s much skepticism prevailed first on the development of compounds with specificity among protein kinases. Secondly, the targeting of singular molecular defects seemed insufficient in the treatment of highly heterogeneous cancer [11]. Gazit in 1989 reported low molecular weight tyrosine kinase inhibitors with selective activity towards EGFR. These inhibitors were called tyrophostins. Tyrophostins were useful in selective antiproliferation agents for proliferative diseases caused by hyperactivity of protein tyrosine kinases. Though tyrophostins were not clinically developed, they provided the proof of the principle of selective inhibition of tyrosine kinases by using small molecules [16]. Subsequently 2 phenylaminopyrimidines were reported as selective inhibitors for protein kinases C (PKC), Abelson (ABL) and platelet derived growth factor receptor (PDGFR) kinases [3]. Tyrosine kinases mediate many of the signaling pathways by which cancer cells promote their proliferation and survival. They control cellular function and are thought to be too similar to be inhibited in-vivo [15]. Overall imatinib shows activity against PDGF-F, C-KIT LCK, C-Raf, and the three VEGF receptors. Its strong C-KIT inhibition made it to be considered in treatment of inoperable C-KIT positive and metastatic gastrointestinal stromal tumors (GIST). Through the synthesis of chemically related compounds and analyzing their relationship in terms of structure and activity, it was possible to optimize the inhibition action of 2-phenylaminopyrimidines in ABL and PDGFRs. Figure 3 Shows a schematic diagram on optimization. Methyl substitution of amino phenyl ring at the sixth position fig 1(2) caused potent inhibition of ABL and PDGFR kinases but abolishes activity on PKC family. N methyl piperizine group optimizes absorption, distribution, metabolism and excretion (ADME) properties (fig 1(3)). The 3’ pyridyl group at the 3’-position of the pyrimidine enhances cellular activity of the derivatives. Introduction of benzamide at the phenyl ring enhances activity against tyrosine kinases. N methyl piperizine improved solubility and oral bio-availability. See figure 4 for clear indication of groups for optimization. Figure 3 2-phenylaminopyrimidine class Optimization [10] The most potent of compounds were found to be inhibitors of ABL and PDGFR kinases. Based on selectivity against CML cells in vitro and pharmacokinetic and formulation properties Imatinib mesylate was isolated as the lead compound in pre-clinical development [10]. Protein tyrosine kinases Control cellular proliferation, differentiation, and motility. They are highly regulated. Their Dis-regulation is linked to a variety of human cancer. In the pre-clinical trials it emerged that Imatinib is not specific to ABL but also inhibits PDGFR, KIT, ARG and possibly other enzymes. Figure 4 Optimization of the PAP [8] Kinases are functional enzymes that facilitate interaction of a substrate with ATP to effect phosphorylation of the substrate. Protein kinases contain within their catalytic domain a nucleotide binding pocket and an activation loop that controls the activity. Imatinib binds to the ABL nucleotide binding pocket, recognizes and stabilizes a distinct inactive conformation of the activation loop ABL and precludes ATP binding. This prevents Bcr-Abl from phosphorylating tyrosine residues of substrate proteins and therefore interrupt Bcr-ABL signal transduction pathways. Figure 5 Phosphorylation of proteins [8] There are two sites that are targeted by kinase inhibitors. Competitive inhibitors act directly at appropriate binding sites. Allosteric inhibitors interact with residues proximal to the binding thereby causing steric distortions. Figure 6 Protein tyrosine Kinases [8] Based on the works of Hanks, Quin and Hunter 1998, initial expectations were hat it would inhibit kinases closely related to ABL in the tyrosine protein kinases phylogenetic tree, it was also found out that it not only inhibit activated forms of the enzyme but also inactive conformation of ABL [10]. Figure 7 Tyrosine protein kinases phylogenetic tree: kinases known to be inhibited by imatinib are shown in yellow [8] The crystal structures of inactive kinases have remarkable plasticity that allows the adoption of distinct inactive conformations. Imatinib binds and stabilizes the inactive conformations of KIT and PDGFR kinases since they are structurally similar. Due to steric constraints the inactive conformation of ABL is not compatible with ATP binding. Figure 8 why gliveec works [8] Gleevec in treatment of other cancers Gleevec has also found applications in the treatment of other cancers. Due to its specificity towards C-KIT and PDGFR it has found numerous applications: as cited in Luke d, (2006) Disorders related receptor Kinase c-KIT;  Gastrointestinal stromal tumors (GISTs)  Received FDA approval for GIST treatment in 2002  Small cell lung cancer (SCLC) PDGFR (Platelet-Derived Growth Factor Receptor) related diseases;  Chronic myelomonocytic leukemia (CMML)  Glioblastoma Synthesis of Imatinib Mesylate It was first synthesized by Zimmermann in 1993 using the scheme shown below [11] Figure 9 Zimmermann’s scheme for preparation Imatinib base [11] Zimmermann’s process, dealt with virulent cyanamide that was used in the synthesis of phenyl guanidine. The synthesis of enaminone was complex since it involved three steps that required sodium metal and low temperatures. Zimmermann’s paper and his patent failed to elaborate on the yields from each step of his scheme. Loiseleur et al, also proposed a process of preparing the imatinib base. Figure 10 loiseleurs scheme for preparation of imatinib base [11] Compound 10 was obtained by reductive amination of aldehyde using Pt/C catalyst hydrogenation. It also involved the use of Pd2 (dba) 3CH3Cl3 catalyzed C-N coupling reaction in the use of organophosphorous reagent rac BINAP as a ligand. Based on Loiseleurs method Kompella and Szakacs improvise new methods. The two methods above had drawbacks; 1. The use of toxic, hazardous reagents Cyanamide. 2. The high cost palladium as catalyst for C-N coupling reaction makes this process costly in large scale industrial applications Yi-Feng Liu et al., (2008) [11] proposed a process of synthesis that could be applied in large scale industrial applications, and that does not use toxic or hazardous reagents. They developed an inexpensive process of producing imatinib and its analogues with sufficiently good yields. The reaction of enaminone with guanidine gave the pyrimidine amine without using toxic cyanamide. A copper-catalyzed C-N bond-forming reaction of heteroarylamine adopted in the preparation of N-(2-methyl-5-nitrophenyl)-4-(pyridin-3-yl) pyrimidin-2-amine, a key intermediate for the synthesis of imatinib. The N2H4 •H2O/FeCl3/C system was used in the reduction of the intermediate nitro compound. Figure 11. Scheme for the preparation of imatinib and its analogues. [11] Use of Imatinib Mesylate Imatinib is recommended as a first line therapeutic in newly diagnosed chronic phase CML patients. It causes remission of cancer, clears the blood and bone marrow of Philadelphia expressing cells [13]. Its mild adverse effects allow for prolonged use in treatment, sometimes even up to six years. Prolonged treatment is found to be necessary since imatinib does eradicate all Philadelphia expressing cells. They remain detectable in a sensitive PCR protocol. Of importance is the fact that imatinib allows a life threatening disease to be considered as a chronic disorder that does not affect the quality of life. Initially, it was developed as a 2-phenylaminopyrimidine specific for PDGFR. However it demonstrated potency for ABL kinases including the BCR-ABL fusion protein produced as a result of the t(9:22) chromosomal translocation(Philadelphia chromosome)found in CML CML is a clonal disorder of hematopoietic stem cell. The clinical presentation often includes granulocytosis, hypercellular bone marrow and splenomegaly. Imatinib also inhibited C-KIT kinases. BCR-ABL kinases are characteristically expressed by leukemic cells. They are important for progression of CML [1][7]. Bcr-ABL presence in more than ninety percent of CML patients and the identification of its role in pathogenesis of disease provides rationale for targeting in the treatment of CML. Phase I of the study was done in June 1998. It was dose escalation study to establish the maximum tolerated dose. 83 patients with chronic phase CML who had failed standard therapy with interferon α or were tolerant to it were selected. CML patients showed good hematologic response (normalization of blood count) in 95% of the patients and cytogenic response (significant reduction in Philadelphia positive cells) in 45% of the patients [10]. Adverse effects observed were nausea, myalgia, edema and diarrhea Phase II of the study took place in June 1999 where a comparison with the existing standard treatments; interferon and cytarabine in patients with CML was done. Imatinib expressed superior response to the other treatments; Hematologic response of 95% and complete cytogenic response in 75% of the patients. Phase III of the study was the IRIS trial. From the phase I clinical trials it was determined that the threshold dose for there to be significant therapeutic efficacy is 300mg. patients in this regiment present with favorable cytogenic and hematologic response which are used to measure the efficacy of the drug. The increase in cell proliferation causes an increase in white blood cells as the disease progresses through the phases. Gleevec effectiveness decreases with the advancement in disease phase. It is reported that there is a 53% response rate in accelerated phase and 30% in blast crisis phase. There was also 9% relapse in the early stage and a 90% relapse in the late stage patients. The majority of the relapses are attributed to the reactivation of kinase activity due to mutation or Bcr-ABL amplification. The natural course of the disease is the chronic phase, followed by the accelerated phase and lastly the terminal blast crisis phase. It has been determined that the chronic phase lasts from between 3-5 years, accelerated phase lasts between 3-9 months and the terminal blast crisis phase 3-6 months. Treatment of accelerated or blast phase CML with imatinib is less effective but can be associated with some response. In developed countries, more than ninety percent of patients are diagnosed with chronic phase CML and of these eighty percent are able to achieve complete cytogenic response(CCR) when subjected to standard dose treatment with imatinib[13]. It has been observed that the discontinuation of imatinib causes recurrence with the exception of patients that had previously received allogeneic stem cell transplants as cited by Cortes et al, 2004. Also those treated by interferon α prior to imatinib treatment. This cases are key indicators that Bcr- ABL positive cells are leukemogenic. There are cases reported of people who remain RT/PCR negative up to fifteen months from imatinib discontinuation. The diagram below shows the relative response of patients in different phases to Imatinib treatment [2]. Figure 12 Resistance in Advanced Phases as cited in Capdeville R et al, (2002)[8] Imatinib shows significant activity in the treatment of GIST but is ineffective in the treatment of systematic mastocytosis due to ineffective targeting of c- kit kinases with d816v mutation [10]. Response of newly diagnosed chronic phase CML patients is found to be durable though high relapse rates are observed in advanced phase of the disease that is likely to be due to point mutations in critical residues that interfere with the drug binding and therefore reactivate Bcr-ABL. Resistance Cases of resistance to treatment following initial response are common in advanced phases of the disease. They can be attributed to mutations that concern amino acids directly involved in drug binding or that act at a distance from the catalytic site and have allosteric influence. This destabilizes auto inhibition conformation of ABL kinase to which drugs bind[14]. Mutations in the kinase domain (KD) of BCR-ABL impair the binding of Imatinib. There is very low risk of disease progression in Patients with complete cytogenetic remission (CCR) who achieves a reduction in BCR-ABL. However, there remains residual disease that is detectable with reverse transcription polymerase chain reaction (RT-PCR) indicating eradication will be a challenge. Current trends in the prevention of resistance are to shift focus from single highly selective inhibitors toward broad spectrum compounds or cocktails of inhibitors that act in different ways. Resistance Mechanisms have been related to [8] [10] 1. Amplification of the BCR-ABL gene  Increase in Bcr-Abl levels 2. Mutations of Bcr-Abl preventing correct Gleevec binding  Mutation of a direct contact residue  Mutation that prevents formation of inactive conformation 3. Efflux of Gleevec (Multi-Drug Resistance Pumps) 4. Serum protein binding of Gleevec Several strategies have been proposed for remedying resistance, the reactivation of BCR-ABL signaling is a clear indicator that cells depend on specific signaling output of the genetic event that initiated malignant transformation. Therefore, BCR-ABL is still the best therapeutic solution. Strategies to combat resistance are: 1. Dose escalation. From retrospective data, it is evident that the escalation of dose overcomes hematologic and cytogenetic resistance in some patients although the state may not be permanently maintained. Also, dose escalation depends on the mutation. It is more effective in mutations that have a low resistance to imatinib e.g. H386P but not the highly resistant strain T3151 OR E255K 2. Combinational therapy Cytarabine, hemoharringtonine, and IFN are synergistic in vitro. A hypomethylating agent called Decitabine, with activity in CML blast crisis exhibits potential in therapy.. 3. Agents that down-regulate BCR-ABL proteins Geldamycin and 17 AAG lead to degradation of BCR-ABL by inhibiting shock protein 90 4. Alternative ABL inhibitors Figure 13 structures of alternate ABL/SRC kinase inhibitors [10] 5. Combination with other signal transduction inhibitors Strong correlations seem to exist between the disease phase persistence and refractory incidences of hematologic, cytogenetic and molecular levels. Only a small fraction of newly diagnosed patients present molecular remission (negative BCR-ABL by RT-PCR). The future is directed in the use of imatinib and allogeneic transplantation, prediction of the response of imatinib and researching on the unexpected side effects. Figure 14. As cited in Michael D. et al., (2005) shows Rates of complete hematologic remission, complete cytogenetic remission, and molecular remission (defined as RT-PCR negativity) in the phase 2 and 3 trials with the imatinib monotherapy. Blue bars indicate complete hematologic remission (CHR); green bars, complete cytogenetic remission (CCR); red bars, molecular remission (MOR). M-BC indicates myeloid blast crisis, and AP accelerated phase [12]. Figure 1 Refractories and disease persistence incidences are dependent on the phase of the disease and the level of response. Hematologic refractories are rare in chronic phase CML but very common in myeloid blast crisis patients. Blast crisis cells have numerous additional genetic abnormalities that affect oncogenes and tumor suppressor genes. This acts a default to Bcr-ABL reactivation as a primary mechanism to bypass imatinib mesylate. Studies are currently underway to assess predictive nature of treatment with imatinib. Individual response to 400mg imatinib daily dose is being predicted. Patients with leukemia cells tested in-vitro have low IC50 for imatinib are shown to respond better than those with high IC50. Also it is found that patients with normal or significantly high levels of human organic cation transports(HOTC)-1 that is responsible for cellular influx of imatinib fare better than those with lower levels. If such studies can be verified then it could be possible to determine which patients are likely to undergo successful imatinib therapy. Studies are not conclusive as of now[8]. Conclusion In view of the development, in clinical trials Imatinib has proven to be an effective treatment in the targeting of kinases in CML. It presents with high selectivity, improved absorption, pharmacokinetic and formulation properties; its efficacy is supported by the hematologic and cytogenetic response as exhibited by the Phase I and Phase II trials. It also shows efficacy in the treatment of GIST, chronic myelomonocytic leukemia in the rearrangements, in PDGFR and hyperosinophilic syndrome. All these are diseases where imatinib sensitive kinases are key to the pathogenesis. The development and use of imatinib are evidence that molecularly targeted approach in the treatment of cancer is an effective form of therapy. From preclinical studies imatinib shows significant success in human treatment. It has favorable pharmacokinetic profile that allows a once a day oral dosing with minimum side effects. It inhibits BCR/ABL activity in vivo by suppressing down stream signaling in circulating leukemic cells of treatment patients. References [1] Buchdunger E, Cioffi CL, Law N. Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther 2000;295:139–45. [2] David E. Golan (edited) Principles of Pharmacology: The Pathophysiologic Basis of Drug Therapy [3] Deininger MW, O’Brien SG, Ford JM, Druker BJ. Practical management of patients with chronic myeloid leukemia receiving imatinib. J Clin Oncol 2003;21:1637–47. [4] Foulkes JG, Chow M, Gorka C, Frackelton AJ, Baltimore D. Purification and characterization of a protein-tyrosine kinase encoded by the Abelson murine leukemia virus. J Biol Chem 1985;260:8070–7. [5] Heinrich MC, Griffith DJ, Druker BJ, Wait CL, Ott KA, Zigler AJ. Inhibition of c-kit receptor tyrosine kinase activity by STI 571, a selective tyrosine kinase inhibitor. Blood 2000;96:925–32. [6] Joan Boren, Marta Cascante, Silvia Marin, Begon˜ a Comı´n-Anduix, Josep J. Centelles, Shu Lim, Sara Bassilian, Syed Ahmed, Wai-Nang Paul Lee, and La´ szlo´ G. Boros(2001)Gleevec (STI571) Influences Metabolic Enzyme Activities and Glucose Carbon Flow toward Nucleic Acid and Fatty Acid Synthesis in Myeloid Tumor Cells. THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 276, No. 41, Issue of October 12, pp. 37747–37753, 2001 © 2001 by The American Society for Biochemistry and Molecular Biology, Inc. [7] Joshua H. Atkins and Leland J. Gershell 2002 Selective anticancer drugs. page492 | JULY 2002 | VOLUME 1 Retrieved from www.nature.com/reviews/drugdisc [8] Luke D. Lavis Raines (2006) A Proof of Principle for Targeted Molecular Therapy [9] Lydon NB, Adams B, Poschet JF, Gutzwiller A, Matter A. An E.coli expression system for the rapid purification and characterization of a v-abl tyrosine protein kinase. Oncogene Res 1990;5:161–73. [10] Nicholas B. Lydon , Brian J (2004). DrukerLessons learned from the development of imatinib. Leukemia Research 28S1 (2004) S29–S38 [11] Michael Deininger, Elisabeth Buchdunger, and Brian J. Druker (2005) The development of imatinib as a therapeutic agent for chronic myeloid leukemia© 2005 by The American Society of Hematology [12] Yi-Feng Liu,Cui-Ling Wang, Ya-Jun Bai, Ning Han, Jun-Ping Jiao,and Xiao-Li Qi A Facile Total Synthesis of Imatinib Base and Its Analogues. Applied Chemical Institute, Northwest University, Xi’an 710069, P.R. China, School of Life Science, Northwest UniVersity, Xi’an 710069, P.R. China, and Department of Clinical Laboratory, Mental Health Center of Weinan, Weinan 714000, P.R. China Organic Process Research & Development 2008, 12, 490–495. [13] John C. Foreman, Torben Johansen, Alasdair J. Gibb editors(2011). Textbook of Receptor Pharmacology, Third Edition. Published by CRS. Taylor and Francis group. [14] Michael W.N. Deininger (2007). “Signal transduction inhibitors in chronic myeloid leukemia”. Myeloproliferative Disorders. Springer Berlin Heidelberg [15] Stella Pelaganis and Michael (2013). The molecular biology of cancer a bridge from bench to bedside second edition. Published by Wiley-Blackwell. [16]Cornelius F Walker (2010). Imatinib Mesylate. In Uwe M. Martens (2010). Small Molecules of Oncology. p3-7. Springer-verlog Berkun Heidelberg 2010     Read More

 

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