A synergistic formulation for leukemia with reduced side effects and preparation method thereof

Abstract

ABSTRACT A SYNERGISTIC FORMULATION FOR LEUKEMIA WITH REDUCED SIDE EFFECT AND PREPARATION METHOD THEREOF Present invention discloses an approach of combining Imatinib with two natural anti-depressants i.e., quercetin and Imatinib to combat Imatinib induced depression. The composition of present invention is formulated in the form of tablet comprising 80mg Imatinib, 100 mg thymoquinone and 300mg quercetin as active pharmaceutical ingredients and 0.5% crospovidone and 2.5% polyvinylpyrrolidone as excipients. The preparation process involves weighing and mixing of pharmaceutical ingredients with excipients, adding water to form paste, drying and sieving the paste to obtain powder and subjecting the powder to table punching / forming machine. The disclosed formulation is capable of maintaining levels of dopamine, norepinephrine and epinephrine levels, while maintaining levels of blood cells, thus the same exhibit promising anti-depressive effects. Neurotransmitter estimation, behavioral studies, hematological analysis and histopathology studies affirmed the Tri combination is multifaceted impact, demonstrating osteogenic, hepatoprotective, immunomodulatory, and anti-leukemic effects.

Description

FIELD OF INVENTIONThe present invention relates to the field of synergistic formulations. Moreparticularly, it pertains to a synergistic formulation for leukemia with reduced sideeffects and preparation method thereof. The same consists of a combination ofImatinib, quercetin and thymoquinone effective in combating Imatinib induceddepression during chemotherapies.BACKGROUND OF THE INVENTIONLeukemiaLeukemia is a broad term for cancer of blood cells and usually affect the bloodforming tissues including bone marrow. Leukemia is of various types such as acuteacute lymphoblastic leukemia, acute myeloid leukemia and chronic lymphocyticleukemia based on the rapidity of the proliferation and cell of origin. Leukemia is10th most common type of cancer worldwide and is responsible for a number ofdeaths globally. Thus treatment and effective management of leukemia is verycrucial. (https: / / www.ncbi.nlm.nih.gov / books / NBK560490 / )Treatment method for LeukemiaTreatment of leukemia depends on the type of leukemia but generally involveschemotherapy. Several other treatment method involves immunotherapy, stem celltransplant combined with chemotherapy, donor lymphocyte infusion, surgicalintervention and treatment with tyrosine kinase inhibitors. The primary andcommonly employed and very efficacious treatment for chronic phase chronicmyeloid leukemia (CML) is the utilization of tyrosine kinase inhibitor (TKI).Imatinib, sold under the brand name Gleevac and Glivic is a Tyrosine kinaseinhibitor (TKI), which is effective in blocking the growth of cancers. Imatinib bindsto BCR-ABL kinase domain and prevent BCR-ABL protein to exert their role inoncogenic pathway in leukemia. Imatinib bind the BCR-ABL domain bypreventing transfer of phosphate group to tyrosine on the protein substrate and alsoprevent subsequent activation of phosphorylated protein, which results in theblockage of transmission of proliferative signals to the nucleus and thus leukemiacell apoptosis is induced.(https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3894842 / #:~:text=Imatinib%20binds%20to%20BCR%2DABL,leukemic%20cell%20apoptosis%20is%20induced.)Drawbacks associated with use of ImatinibDespite of potency of Imatinib, it is associated with some non-selective toxicities,notably depression. Consumption of imatinib result in psychological side effects,such as anxiety, being easily emotional or depression.(https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC10788803 / #:~:text=Furthermore%2C%20imatinib%20itself%20might%20also,2023)%2C%20yet%20this%20has%20neve) Depression is a recognized as a serious psychological disorder associatedwith various characteristic features such as persistent sad mood, decreased pleasurein day to day activities, reduced concentration, inability to take decisions,helplessness, loss of interest and persistent thoughts of committing suicide etc.Various studies have documented that tyrosine kinase activation modulates theanxious and depressed behavior and imatinib may affects the behavior of cancerpatients. Further, evidences has shown that discontinuation of tyrosine kinaseinhibitors including imatinib leads to the anxiety or depression in chronic myeloidleukemia patients.There are several alternative drugs to Imatinib such as SSRIs (selective serotoninreuptake inhibitor) however there is a concern that selective serotonin reuptakeinhibitor (SSRI) treatment itself may increase the risk of suicide attempts or deaths,hence the use of same is not suitable.As depression is associated with various behavioral changes, which affect the lifeof the humans, thus in view of depression induced because of Imatinib, there was aneed to disclose such an innovative formulation, which is effective in combatingthe side effects associated with use of Imatinib, particularly depression and anxiety.Innovative Approach by Inventors:In order to combat Imatinib induced depression, inventors of present inventioncame up with an innovative approach of combining two natural anti-depressantagents i.e., quercetin and thymoquinone along with adequate doses of Imatinib.Thus inventors of present invention discloses a novel pharmaceutical blendcomprising synergistic combination of Imatinib, Thymoquinone, and Quercetinalleviating Imatinib-induced depression. Quercetin is capable to potentiallyalleviating depression through its antioxidant properties and modulation ofneurotransmitters, while thymoquinone, with antioxidant and anti-inflammatoryeffects, shows promise in protecting against oxidative stress associated withdepressive symptoms.OBJECT OF THE PRESENT INVENTIONThe primary object of the present invention is to disclose a synergisticpharmaceutical composition comprising combination of Imatinib, quercetin andthymoquinone.Another object of present invention is to disclose combination of Imatinib withantidepressants thymoquinone and quercetin, which effectively manage imatinibinduced depression.SUMMARY OF THE INVENTIONThe treatment of leukemia often involves use of Imatinib, a Tyrosine KinaseInhibitor (TKI) renowned for its efficacy against cancer. Despite of potency,Imatinib is associated with non-selective toxicities, notably depression. Thus tocombat / overcome Imatinib induced depression, present invention discloses a novelapproach of combining Imatinib with two natural anti-depressants i.e., quercetinand Imatinib to combat Imatinib induced depression. The composition of presentinvention is formulated in the form of tablet comprising 80mg Imatinib, 100 mgthymoquinone and 300mg quercetin as active pharmaceutical ingredients and 0.5%crospovidone and 2.5% polyvinylpyrrolidone as excipients. The preparationprocess involves weighing and mixing of pharmaceutical ingredients withexcipients, adding water to form paste, drying and sieving the paste to obtainpowder and subjecting the powder to table punching / forming machine. Thedisclosed formulation is capable of maintaining levels of dopamine, norepinephrineand epinephrine levels, while maintaining levels of blood cells, thus the sameexhibit promising anti-depressive effects. Neurotransmitter estimation, behavioralstudies, hematological analysis and histopathology studies affirmed the Tricombination is multifaceted impact, demonstrating osteogenic, hepatoprotective,immunomodulatory, and anti-leukemic effects.BRIEF DESCRIPTION OF DRAWINGSFigure 1: Graph depicting the One-way anova results of Weights of rats at differentdose and day intervalsFigure 2: Graph depicting one way anova results of FST at different dose and dayintervalFigure 3: Graph depicting the One-way anova results of TSTFigure 4: Graph depicting one way anova results of EPMT at diff dose and daysintervalFigure 5: Graph depicting the One-way anova results of NSFTFigure 6: Graphical representations of blood parameters(A) Haemoglobin (B) RBC count (C) Haematocrit (HCT) (D) MCV (E) MCH)(F) MCHC (G) RDW-CV (H) PLATELET COUNT (I) WBC (J) Neutrophils(K) Lymphocytes (L) Monocytes (M) Eosinophils (N) BasophilsFigure 7 (A to D): Images for histopathology of kidneyFigure 8 (A to D): Images for histopathology of liverFigure 9 (A to D): Images for histopathology of spleenFigure 10 (A to B): Images for histopathology of heartFigure 11 (A to B): Images for histopathology of lungsFigure 12 (A to G): LCMS graphs for group 1, 2, 4, 5, 6, 7 & 8 respectivelyDETAILED DESCRIPTION OF THE INVENTIONThe inventors of present invention innovatively by thorough research came withan approach of combining two natural anti-depressant agents i.e., quercetin andthymoquinone along with adequate doses of Imatinib in order to combat Imatinibinduced depression. The specific use of quercetin and thymoquinone is as follows:Specific use of Quercetin and ThymoquinoneThymoquinone- Thymoquinone is a phytochemical substance derived from theNigella sativa plant is the main active ingredient of volatile oil of black seed. It hasbeen recognized for its pain-relieving, cancer-fighting, free-radical-fighting,inflammation-reducing, and fever-reducing properties. Further, thymoquinonesignificantly contributes to anti-depressant properties, it prevents elimination ofbrain neurotransmitters affecting depression such as serotonin and dopamine.(https: / / journals.lww.com / aptb / fulltext / 2022 / 12120 / antidepressant_and_anti_nociceptive_effects_of.1.aspx#:~:text=It%20has%20been%20reported%20that,thymoquinone%20has%20also%20been%20documented.)Quercetin- Quercetin is an inherent pigment found in numerous fruits, vegetables,and grains. One of the most plentiful antioxidants in the diet, it has a crucial functionin assisting the body in fighting against the harm caused by free radicals, which areassociated with chronic illnesses. Quercetin is utilized in the management of cancer,allergic responses, inflammation, arthritis, and cardiovascular problems. Theflavonoid also has a significant impact on the process of platelet aggregation, aswell as the peroxidation of lipids, and it also stimulates the biogenesis ofmitochondria. Quercetin acts as a natural antioxidant with anti-depressant effect asit is used to protect BDNF (Brain derived neurotrophic factor) from in vivooxidation and increases the antidepressant activities of BDNF. Quercetin results inantidepressant-like actions because of its antioxidant, anti-inflammatory, andneuroprotective effects. It can have antidepressant effects by inhibiting the HPAaxis. (https: / / www.healthline.com / nutrition / quercetin)METHOD FOR PREPARATION25The method for preparation of synergistic combination of Imatinib with antidepressantagents i.e., Quercetin and Thymoquinone as disclosed in presentinvention comprises the following steps:Procurement of raw materials:Imatinib (KVB Asta Life Sciences), quercetin, Thymoquinone, Crospovidone andpolyvinylpyrrolidone (PVP) were purchased from Hychem Laboratories.Composition of present invention:The synergistic composition of present invention is formulated in the form of tabletand the same comprises of following active pharmaceutical ingredients andexcipients in following ratios:Sr. No. Constituent (APIs) Amount (mg)1. Imatinib 80 mg2. Thymoquinone 100 mg3. Quercetin 300 mgSr. No. Constituents (Excipients) Amount (%)4. Crospovidone (Excipient) 0.5%5. polyvinylpyrrolidone (Excipient) 2.5%Table 1- Composition of per 500mg Imatinib-quercetin-thymoquinone tableFurther the tablets preparation method comprises following steps:- weighing and mixing the active pharmaceutical ingredients with excipientsin china dish;- adding adequate amount to water to said mixture in order to prepare a thickpaste;- drying the paste and sieving through sieve no. 12 to obtain granulatedpowder;- Feeding the dried powder to the tablet punching / forming machine andcollecting the finally prepared tablets.BEHAVIORAL ANALYSIS:An extensive in vivo study was conducted on six animal groups giving varioustreatments, with Imatinib (80mg / kg) as the standard, Quercetin (300mg / kg),Thymoquinone (100 mg / kg).Before behavioral analysis tests, the animals are categorized into various groupsdepending upon treatment to be provided. The grouping of animals is as follows:Table 2: Grouping of animals in different groupsWeight of test subjects:Initially, the weight of the selected animals i.e., wistar rats was determined andgraphical representation showing the weight of rats of different groups is depictedin figure 1, which represent, that rats treated with pharmaceutical composition ofpresent invention has higher weight in the span of 30 days with 29 doses.Average body weight of different groups at dose 1, 15 and 29 is as below in table3:Table 3: Group Average body weights of ratsValues are expressed in Mean ± SEM for six animals in each group. aAll groupsvs Normal control; bAll groups vs Disease control (Only FST); cAll groups vsanimals with Imatinib, *P<0.05, *P<0.01, *P<0.001, NS, not significant.Following behavioral tests on male wistar rats were conducted to determine theeffectiveness of the synergistic composition as disclosed in present invention incombating Imatinib induced depression:Forced Swimming test (FST)Male wistar rats were placed in water-filled cylinders for 3 minutes to adapt. Theywere observed for climbing, swimming, and immobility during the ForcedSwimming Test. The test was repeated four times over 30 days. After each test, ratswere dried and cared for. Figure 2 discloses the results for forced swimming test(FST), and it was observed that the rats treated with composition of presentinvention shows highest climbing and swimming ability, but lower immobility.Table 4: FST: Overall Mean group wiseValues are expressed in Mean ± SEM for six animals in each group. aAll groups vs Normalcontrol; bAll groups vs Disease control (Only FST); cAll groups vs animals with Imatinib,*P<0.05, *P<0.01, *P<0.001, NS, not significant.Tail Suspension test (TST)Rats were adapted by securing them upside down on a rod stand with plaster. TheTail Suspension Test (TST) was conducted, observing immobility time, movement,and active mobility. Weights were measured to ensure rat welfare and avoid harm,with care taken throughout the experiment, including their tails. The TST resultsare depicted in figure 3, which indicates that rats treated with composition ofpresent invention show highest immobility time, movement, and active mobility.Group Tail Suspension Test (TST)Day 15, Dose 141 250 ± 0.96612 261 ± 1.528 a****3 273 ± 0.9661 a****, b****4 230 ± 1.932 a****, b****, c****5 208 ± 0.9661 a****, b****, c****6 195 ± 0.9661 a****, b****, c****7 242 ± 1.713 a**, b****, c****8 239 ± 1.713 a****, b****, c****Table 5: Tail Suspension Test (TST): Mean group wiseValues are expressed in Mean ± SEM for six animals in each group. aAll groups vs Normalcontrol; bAll groups vs Disease control (Only FST); cAll groups vs animals with Imatinib,*P<0.05, *P<0.01, *P<0.001, NS, not significant.Elevated plus-maze test (EPMT)The rats were placed on an Elevated plus maze apparatus in a controlledenvironment without external interference. The experiment was recorded via phonefor behavioral analysis. Observations included time spent in open / closed arms,entries into arms, and instances of standing on hind legs. Prior to the experiment,rats were familiarized with the setup. The test was conducted on multiple days, anddata were collected for comprehensive analysis. Results are depicted in figure 4,which depicts, when the rats were treated with composition of present invention,they spent highest time in open arms and comparatively lesser time in closed arms.Groups Average Group wiseOpen time On hind leg No of entries1 86 ± 2.082 28.4 14.22 77.66 ± 8.413 aNS 24.83 13.93 74.60 ± 11.33 aNS, bNS 17.665 11.4154 92.383 ± 7.672 aNS, bNS, cNS 34.165 16.3335 99.22 ± 9.081 aNS, bNS, cNS 39.245 276 108.331 ± 4.806 aNS, bNS, cNS 44.995 29.087 89.72 ± 7.448 aNS, bNS, cNS 29 15.338 90.72 ± 7.702 aNS, bNS, cNS 31 15.91Table 7: Elevated Plus Maze Test (EPMT): Overall Mean group wiseValues are expressed in Mean ± SEM for six animals in each group. aAll groups vs Normalcontrol; bAll groups vs Disease control (Only FST); cAll groups vs animals with Imatinib,*P<0.05, *P<0.01, *P<0.001, NS, not significant.Novelty Suppressed feeding test (NSFT)The fasting period commenced on Day 14 of the experiment, with no food or waterprovided to the animals. After 18 hours of fasting, rats were observed for their firstbite, indicating novelty-suppressed feeding behavior. The time of the first bite foreach rat was recorded to assess their emotional response to food deprivation andnovel environments. This test was conducted for all six rats in each of the eightgroups. The results are provided in table below and figureTable 6: NSFT: Mean group wiseValues are expressed in Mean ± SEM for six animals in each group. aAll groups vs Normalcontrol; bAll groups vs Disease control (Only FST); cAll groups vs animals with Imatinib,*P<0.05, *P<0.01, *P<0.001, NS, not significant.Sucrose Preference Test (SPT)The Sucrose Preference Test was conducted following the Novelty SuppressedFeeding Test on Day 16. Each rat cage had two bottles: one with water and the otherwith a 10% sucrose solution. Over one hour, rat consumption of both liquids wasobserved and recorded. The 10% sucrose solution was prepared by dissolving 1gram of sucrose in 5 ml of water, then adjusting the volume to 100 ml.HISTOPATHOLOGICAL AND HEMATOLOGICAL STUDIESThe rats were anaesthetized using ketamine xylazine. Further, for histopathologicaland hematological studies, rats were sacrificed, their brain samples are isolated,homogenized, centrifuged and filtered.HEMATOLOGICAL STUDIES:Further, blood samples were also collected, animals were dissected and organsincluding kidney, liver, spleen, heart and lungs were collected and stored in 40%formalin and 60% water solution for storage and histopathological analysis.Figure 6 (A-N) discloses graphical representation of hematological results ofvarious test groups. From figure 6, it is identified, that hemoglobin, RBC count,hematocrit, MCV, MCH, MCHC, RDW-CV, platelet count, neutrophils,monocytes, eosinophils, basophils are higher in Imatinib-quercetin-thymoquinonetreated groups in comparison to disease control group, whereas WBC count andlymphocytes count was lesser in Imatinib-quercetin-thymoquinone treated groupsin comparison to disease control group.HISTOPATHOLOGICAL STUDIES:Histopathological studies of kidney:Normal morphology of glomerulus (red arrow) and tubules of kidney in cortexregion (green arrow) was observed in group 1, 2, 5, 6 & 8 and same is depicted infigure 7 (a) and (b). Tubular dilation and degeneration of cortical tubule epithelialcell characterized by vasculation of cytoplasm and pykonsis of nuclei (arrow) incortex region of kidney was observed in group 3 and same is depicted in figure 7(c)and multifocal tubular / intestinal inflammation (red arrow) with infiltration ofinflammatory cells was observed in group 4 and 7, same is depicted in figure 7(d).Histopathological studies of liver:Normal morphology of hepatocytes in portal, peri-portal and centri lobular regionwith red arrow was observed in group 1, 2, 6 & 8 and same is revealed in figure8(a). Multifocal foci of necrosis of hepatocytes in centri lobular and peri portalregion of liver observed in group 3 & 7 as depicted in figure 8(b). Mild sinusoidaldilatations and haemorrhages (red arrows) in group 4 and 5 and same is depicted infigure 8(c) and group 6 also normal hepatocytes morphology (red arrow) and muitifociperi portal infiltration of inflammatory cells (green arrow) as depicted in figure8(d).Histopathological studies of spleen:Figure 9(a) reveals normal morphology of lymphatic follicles in cortex region ofspleen (red arrow) as observed in group 1, 2, 5 & 6. Figure 9(b) and (c) depicts mildhyperthrophy of lymphatic follicles in cortex region of spleen (red arrow) asobserved in group 3,4 & 8 and figure 9(d) reveals mild hyperthrophy of lymphaticfollicles with hyperplasia of lymphocytes in cortex region of spleen (red arrow) asobserved in group 7Histopathological studies of Heart:Figure 10(a) reveals normal morphology of myocardium (green arrow) and cornoryartery (red arrow) of heart as observed in group 1, 2, 4, 5, 6, 7 & 8 and figure 10(b)shows multifocal mild myocardial hemorrhages of heart (red arrow) as observed ingroup 3.Histopathological studies of Lungs:Figure 11 (a) reveals normal morphology of alveoli (red arrow) and bronchioles(green arrow) of lungs as observed in group 1, 2, 5, & 6 and figure 11(b) revealsmoderate alveolar / interstitial pneumonia: alveolar / interstitial (b / w alveoli)inflammation with infiltration of alveolar macrophage and inflammatory cells andfibrosis noticed in lungs in group 3 and mild alveolar / interstitial pneumonia:alveolar / interstitial (b / w alveoli) inflammation with infiltration of alveolarmacrophage and inflammatory cells and fibrosis noticed in lungs was observed ingroup 4, 7 & 8.LCMS and DIP MS:The isolated brain samples further undergoes Liquid chromatograph massspectroscopy (LCMS) and DIP MAS i.e., Direct Insertion Probe MassSpectroscopy. The Liquid chromatograph mass spectroscopy (LCMS) results aredepicted in figure 12 (a) to (g) for groups 1, 2, 4, 5, 6, 7 & 8 respectively. Fromfigure 12 (e), it is being analyzed that the rats treated with composition of presentinvention possesses significant dopamine and norepinephrine levels i.e., 153.1019dopamine level in 0.439 minutes, Epinephrine level of 183.204 at 0.236 minutesand 169.0582 norepinephrine level in 0.182 minutes, which indicates thatcomposition of present invention maintains adequate levels of these enzymes, lowerlevels of which causes depression, thus it is concluded that present invention iseffective in combating depression.Further, DIP MS results are provided in table 7 below:Table 7: Result of DIP Mass (check mark) indicates presence of compound and x indicatesabsence of such compoundFrom aforementioned table, it is interpreted that, group 6 treated models i.e., theone treated with composition of present invention show presence of all threehormones i.e., Dopamine, Epinephrine and norepinephrine, adequate levels ofwhich are important to provide a feeling of satisfaction, pleasure and motivationand decrease the symptoms of depression. The normal group i.e., group 1 and group6 treated with composition of present invention show presence of all threehormones, whereas all three hormones are lacking in group 3 treated with Imatinibonly, which indicates that Imatinib contributes to depression, whereas whensynergistic composition of present invention is used, then the same is capable ofcombating Imatinib induced depression.Notably, a significant reduction in imatinib induced depression was observed, whenthe wistar rats were treated with formulation disclosed in present invention.The disclosed formulation exhibited promising anti-depressive effects.Neurotransmitter estimation, behavioral studies, hematological analysis andhistopathology studies affirmed the Tri combination is multifaceted impact,demonstrating osteogenic, hepatoprotective, immunomodulatory, and antileukemiceffects. By harnessing Thymoquinone's multi-striking attributes andQuercetin's neuroprotective effects, this synergistic composition has shownsuperior efficacy compared to Imatinib alone or in combination with other two dosecombinations of Thymoquinone and Quercetin. These results underscore theinnovative combination potential as a promising strategy for mitigating Imatinibinduceddepression. Leveraging Quercetin's promotion of BDNF and its binding tothe high-affinity receptor tyrosine kinase receptor B (TrkB) for neural progenitorcell survival and Thymoquinone's multi-faceted impact, this combination offers apromising avenue to minimize adverse psychological effects associated withImatinib therapy, potentially enhancing the overall well-being of treated subjects.The technological advancement lies in disclosure of pharmaceutical composition,wherein Imatinib is being used along with antidepressant agents i.e., quercetin andthymoquinone, which is capable of treating leukemia and in turns combat thedepression induced due to the use of Imatinib alone. Quercetin inducesantidepressant-like effects due to its antioxidant, anti-inflammatory, andneuroprotective properties. By suppressing the hypothalamic-pituitary-adrenal(HPA) axis, it can exert antidepressant effects. Thymoquinone potentially relievesdepression by functioning as an antioxidant, safeguarding the brain againstoxidative stress. Additionally, it has the ability to regulate neurotransmitters suchas serotonin and dopamine, as well as have anti-inflammatory capabilities.The industrial use of the present invention is evident, as it offers significantadvantages to the medical sector by providing a combination which is not onlyefficient in managing leukemia by also combat major side effect associated withuse of Imatinib drug.

Claims

1. A synergistic pharmaceutical formulation for leukemia with reduced side effect wherein the same consists of: - 80mg Imatinib with 100 mg thymoquinone; - 300mg Quercetin; - 0.5% crospovidone; - 2.5% polyvinylpyrrolidone.

2. The synergistic composition as claimed in claim 1 wherein the same induces Dopamine level of 153.1019 at 0.439 minutes, Epinephrine level of 183.204 at 0.236 minutes and norepinephrine level of 169.0582 at 0.182 minutes.

3. The synergistic composition as claimed in claim 1 wherein the same is in the form of tablet.

4. The method for preparation of synergistic composition as claimed in claim 1 wherein the same consists of following steps: - weighing and mixing the 80mg Imatinib, 100 mg thymoquinone, 300mg quercetin with 0.5% crospovidone and 2.5% polyvinylpyrrolidone in china dish; - preparation of thick paste by adding adequate amount to water to said mixture; - drying the paste and sieving through sieve no. 12 to obtain granulated powder; - feeding dried powder to the tablet punching / forming machine and collecting the finally prepared tablets.