Patent Application: US-201615548959-A

Abstract:
mebendazole is an antiparasitic drug with over 40 years of safe use . recently mebendazole was repurposed for glioblastoma therapy . three polymorphs of mebendazole exist , but the relative polymorph content for existing drugs varies , and the therapeutic anti - cancer relevance of the different polymorphs was unknown . as an oral drug mebendazole polymorph c is a superior form , and it reaches the brain and brain tumors in effective concentrations . efficacy is further improved by combining mebendazole with a p - glycoprotein inhibitor . mebendazole may also be used for therapy of other cancers , as well as a chemo - preventative agent .

Description:
the inventors have developed efficacious and safe formulations for treating tumors , particularly tumors of the brain , breast , and lung . other tumors may also be treated including but not limited to colorectal , ovarian , sarcomas , gastric , esophageal , prostate , pancreatic , liver , and thyroid tumors . polymorph c of mebendazole has been found to be the most potent of the polymorphs for treating tumors . often , however , it appears that the potency of a preparation decreases over time due to loss of polymorph c or conversion to other polymorphs . preferably a preparation of mebendazole that is used in the invention will be at least 90 % polymorph c . in some cases it may be at least 91 %, 92 %, 93 %, 94 %, 93 %, 96 %, 97 %, 98 %, or 99 % polymorph c . the mebendazole may optionally be granulated . this provides a suitable formulation for adding to comestibles and providing a palatable medicament . it may also increase gastric absorption . optionally the granulated form may be coated . this may increase the palatability of the medicament . typical materials used for enteric coatings include fatty acids , waxes , shellac , plastics , and plant fibers . any such enteric coatings used in the art may be used . p - glycoprotein ( p - gp ), the permeability glycoprotein or plasma glycoprotein is an active , efflux , membrane bound transport protein pump . it is a member of atp binding cassette ( abc ) super family . it goes by many names including abcb1 , mdr1 , pgy1 , and cd243 . it is involved in multidrug resistance in tumors . in that context it may be referred to as a multidrug resistant pump . any inhibitor of p - gp can be used in formulations with mebendazole , including but not limited to elacridar , progesterone , gomasin a , piperine , apocyanin , amprenavir , quinidine , and valspodar . the p - gp inhibitor may be co - coated along with the mebendazole or the p - gp inhibitor may be uncoated or separately coated . the two agents may be administered at the same time , in combination or separately . the two agents may be delivered within days or weeks of each other as part of a combined regimen . in some formulations and for some uses , such as prophylactic uses , polymorph c can be formulated with a non - steroidal anti - inflammatory drug . these include , without limitation , aspirin , choline and magnesium salicylates , choline salicylate , celecoxib , dielofenac potassium , dielofenac sodium , dielofenac sodium with misoprostol , diflunisal , etodolac , fenoprofen calcium , flurbiprofen , ibuprofen , indomethacin , ketoprofen , magnesium salicylate , meclofenamate sodium , mefenamic acid , meloxicam , nabumetone , naproxen , naproxen sodium , oxaprozin , piroxicam rofecoxib , salsalate , sodium salicylate , sulindac , tolmetin sodium , and valdecoxib . the combination is potent in prophylactic effect . individuals who have higher risk of developing colorectal cancer are those with any of a variety of environmental , behavioral , and genetic factors . these include , without limitation , overweight or obese , physical inactivity , a diet that is high in red meats ( such as beef , pork , lamb , or liver ) and processed meats , smoking , heavy alcohol use , personal history of colorectal polyps or colorectal cancer , personal history of inflammatory bowel disease , family history of colorectal cancer or adenomatous polyps , family cancer syndromes , familial adenomatous polyposis ( fap ), lynch syndrome , attenuated fap , turcot syndrome , peutz - jeghers syndrome , mutyh - associated polyposis , and type 2 diabetes . these individuals may benefit from the prophylaxis with mebendazole , in particular with polymorph c , and more particularly with combination therapies of mebendazole and non - steroidal anti - inflammatory drugs . the two agents may be administered at the same time , in combination or separately . the two agents may be delivered within days or weeks of each other as part of a combined regimen of prophylaxis . a racemic mixture of mebendazole or even a composition comprising more of polymorphs a or b than c may be used for prophylactic and / or therapeutic anti - cancer treatments . application of a formulation to food may encompass any means known in the art . sprinkling , shaking , spraying , dowsing , or mixing , for example , can be used to apply the formulation to the food . administration or dispensing of a formulation for oral ingestion may comprise , for example , delivering in a cup , on a plate , or directly into the mouth of the subject . brain tumors which may be treated include astrocytoma ; atypical teratoid rhaboid tumor ( atrt ); chondrosarcoma ; choroid plexus ; craniopharyngioma ; cysts ; ependymoma ; germ cell tumor ; glioblastoma ; glioma ; hemangioma ; lipoma ; lymphoma ; medulloblastoma ; meningioma ; metastatic brain tumor ; neurofibroma ; neuronal & amp ; mixed neuronal - glial tumors ; oligoastrocytoma ; oligodendroglioma ; pineal tumors ; pituitary tumors ; pnet ; and schwannoma . human tumors which may be treated with the formulations include acute lymphoblastic leukemia ( all ); acute myeloid leukemia ( aml ); adolescents , cancer in ; adrenocortical carcinoma ; childhood ; aids - related cancers ; kaposi sarcoma ; lymphoma ; anal cancer ; appendix cancer ; astrocytomas , childhood ; atypical teratoid / rhabdoid tumor , childhood , central nervous system ; basal cell carcinoma — see skin cancer ( nonmelanoma ); childhood ; bile duct cancer , extrahepatic ; bladder cancer ; childhood ; bone cancer ; ewing sarcoma family of tumors ; osteosarcoma and malignant fibrous histiocytoma ; brain stem glioma , childhood ; brain tumor ; astrocytomas , childhood ; brain and spinal cord tumors treatment overview , childhood ; brain stem glioma , childhood ; central nervous system atypical teratoid / rhabdoid tumor , childhood ; central nervous system embryonal tumors , childhood ; central nervous system germ cell tumors , childhood ; craniopharyngioma , childhood ; ependymoma , childhood ; breast cancer ; childhood ; male ; pregnancy , breast cancer and ; bronchial tumors , childhood ; burkitt lymphoma — see non - hodgkin lymphoma ; carcinoid tumor ; childhood ; gastrointestinal ; carcinoma of unknown primary ; childhood ; cardiac ( heart ) tumors , childhood ; central nervous system ; atypical teratoid / rhabdoid tumor , childhood ; embryonal tumors , childhood ; germ cell tumor , childhood ; lymphoma , primary ; cervical cancer ; childhood ; childhood cancers ; chordoma , childhood ; chronic lymphocytic leukemia ( cll ); chronic myelogenous leukemia ( cml ); chronic myeloproliferative neoplasms ; colon cancer ; colorectal cancer ; childhood ; craniopharyngioma , childhood ; cutaneous t - cell lymphoma — see mycosis fungoides and sézary syndrome ; duct , bile , extrahepatic ; ductal carcinoma in situ ( dcis ); embryonal tumors , central nervous system , childhood ; endometrial cancer ; ependymoma , childhood ; esophageal cancer ; childhood ; esthesioneuroblastoma , childhood ; ewing sarcoma ; extracranial germ cell tumor , childhood ; extragonadal germ cell tumor ; extrahepatic bile duct cancer ; eye cancer ; intraocular melanoma ; retinoblastoma ; fallopian tube cancer — see ovarian epithelial , fallopian tube , and primary peritoneal cancer ; fibrous histiocytoma of bone , malignant , and osteosarcoma ; gallbladder cancer ; gastric ( stomach ) cancer ; childhood ; gastrointestinal carcinoid tumor ; gastrointestinal stromal tumors ( gist ); childhood ; germ cell tumor ; central nervous system , childhood ; extracranial , childhood ; extragonadal ; ovarian ; testicular ; gestational trophoblastic disease ; glioma — see brain tumor ; childhood brain stem ; hairy cell leukemia ; head and neck cancer ; childhood ; heart cancer , childhood ; hepatocellular ( liver ) cancer ; histiocytosis , langerhans cell ; hodgkin lymphoma ; hypopharyngeal cancer ; ilntraocular melanoma ; islet cell tumors , pancreatic neuroendocrine tumors ; kaposi sarcoma ; kidney ; renal cell ; wilms tumor and other childhood kidney tumors ; langerhans cell histiocytosis ; laryngeal cancer ; childhood ; leukemia ; acute lymphoblastic ( all ); acute myeloid ( aml ); chronic lymphocytic ( cll ); chronic myelogenous ( cml ); hairy cell ; lip and oral cavity cancer ; liver cancer ( primary ); childhood ; lung cancer ; childhood ; non - small cell ; small cell ; lymphoma ; aids - related ; burkitt — see non - hodgkin lymphoma ; cutaneous t - cell — see mycosis fungoides and sézary syndrome ; hodgkin ; non - hodgkin ; primary central nervous system ( cns ); macroglobulinemia , waldenström ; male breast cancer ; malignant fibrous histiocytoma of bone and osteosarcoma ; melanoma ; childhood ; intraocular ( eye ); merkel cell carcinoma ; mesothelioma , malignant ; childhood ; metastatic squamous neck cancer with occult primary ; midline tract carcinoma involving nut gene ; mouth cancer ; multiple endocrine neoplasia syndromes , childhood ; multiple myeloma / plasma cell neoplasm ; mycosis fungoides ; myelodysplastic syndromes ; myelodysplastic / myeloproliferative neoplasms ; myelogenous leukemia , chronic ( cml ); myeloid leukemia , acute ( aml ); myeloma , multiple ; myeloproliferative neoplasms , chronic ; nasal cavity and paranasal sinus cancer ; nasopharyngeal cancer ; childhood ; neuroblastoma ; non - hodgkin lymphoma ; non - small cell lung cancer ; oral cancer ; childhood ; oral cavity cancer , lip and ; oropharyngeal cancer ; osteosarcoma and malignant fibrous histiocytoma of bone ; ovarian cancer ; childhood ; epithelial ; germ cell tumor ; low malignant potential tumor ; pancreatic cancer ; childhood ; pancreatic neuroendocrine tumors ( islet cell tumors ); papillomatosis , childhood ; paraganglioma ; childhood ; paranasal sinus and nasal cavity cancer ; parathyroid cancer ; penile cancer ; pharyngeal cancer ; pheochromocytoma ; childhood ; pituitary tumor ; plasma cell neoplasm / multiple myeloma ; pleuropulmonary blastoma , childhood ; pregnancy and breast cancer ; primary central nervous system ( cns ) lymphoma ; prostate cancer ; rectal cancer ; renal cell ( kidney ) cancer ; renal pelvis and ureter , transitional cell cancer ; retinoblastoma ; rhabdomyosarcoma , childhood ; salivary gland cancer ; childhood ; sarcoma ; ewing ; kaposi ; osteosarcoma ( bone cancer ); rhabdomyosarcoma ; soft tissue ; uterine ; sézary syndrome ; skin cancer ; childhood ; melanoma ; merkel cell carcinoma ; nonmelanoma ; small cell lung cancer ; small intestine cancer ; soft tissue sarcoma ; squamous cell carcinoma — see skin cancer ( nonmelanoma ); childhood ; squamous neck cancer with occult primary , metastatic ; stomach ( gastric ) cancer ; childhood ; t - cell lymphoma , cutaneous — see mycosis fungoides and sézary syndrome ; testicular cancer ; childhood ; throat cancer ; thymoma and thymic carcinoma ; childhood ; thyroid cancer ; childhood ; transitional cell cancer of the renal pelvis and ureter ; unknown primary , carcinoma of ; childhood ; unusual cancers of childhood ; ureter and renal pelvis , transitional cell cancer ; urethral cancer ; uterine cancer , endometrial ; uterine sarcoma ; vaginal cancer ; childhood ; vulvar cancer ; waldenström macroglobulinemia ; and wilms tumor . kits are means of packaging two or more items in a single container . the kit may comprise multiple internal containers to keep components separate . the kit may comprise instructions or other printed matter to facilitate use , such as standard curves . information may also be provided on an electronic storage medium such as a disk or drive . information may be provided by reference to a website . additional components that are useful for treating cancers may also be provided . tools for administration may be included . vessels for mixing components may be provided in the kit . mebendazole can be prepared by any means known in the art . in one method , p - chlorotoluene is used as a starting material . see fig8 . typically for purification , polymorph c is crystalized using methanol at room temperature . mebendazole is commercially available . in this study , we demonstrate that mbz can reach the brain tissue in significant concentrations and with high brain to plasma ratios . between 1 and 8 hours after the oral administration , mbz - c maintained the brain levels above 0 . 767 μg / g ( equivalent to 2 . 7 μm ), with a c max of 2 , 016 μg / g ( equivalent to 7 . 1 μm ). these exceeded the ic 50 ( 4 . 3 μm ) of mbz on vegfr2 kinase in vitro and the ic 50 ( 0 . 11 - 1 μm ) in a series of glioma and medulloblastoma cell lines in tissue culture ( 3 , 4 ). furthermore , mbz - c emerged as the most efficient polymorph , achieving an acu 0 - 24 h b / p ratio of 0 . 82 . this is encouraging since temozolomide , the standard treatment for high - grade gliomas , was measured of having a b / p ratio of 0 . 408 in mice and a cerebrospinal fluid ( csf )/ plasma ratio of 0 . 2 in human ( 21 , 22 ). in our study , the distributions of mbz in the gl261 brain tumor and in the normal brain tissue did not differ significantly . it is worth mentioning that advanced growth of gl261 glioma results in substantial amount of blood in the tumor , similarly to other glioma models and a thorough perfusion was essential to eliminate the contamination of mbz from the blood . among the three polymorphs , mbz - a showed no efficacy in gl261 glioma model , explained by the very low plasma presence at only 19 % of auc 0 - 24 h measured with mbz - c . mbz - a &# 39 ; s low bioavailability and inferior anti - tumor efficacy are in line with previous reports of its poor performance in anti - parasitic applications ( 10 , 12 ). in comparison , mbz - b was able to reach 165 % of mbz - c &# 39 ; s auc 0 - 24 h in the plasma , while showing a similar brain concentration demonstrated by the measurement at 6 h . this could explain the elevated toxicity of mbz - b in gl261 glioma - bearing mice as the anti - brain tumor efficacy remained essentially the same compared to mbz - c . thus , we suggest that mbz - c is a better choice in brain tumor therapy . as a practical matter , the tablets made by mbz - c should be stored under lower temperature ( 13 ), since the mbz tablets of teva brand may have lost its efficacy under the standard rt condition within 3 years likely due to the conversion to polymorph a , although we do not know the original concentration of polymorph c in these tablets that used to be efficacious in our previous study ( 3 ). mbz &# 39 ; s small size ( 295 daltons ) and lipophilic property favor brain penetration ( 2 ). it is remarkable that other benzimidazoles tested so far , such as albendazole , thiabendazole , flubendazole , oxifendazole and fenbendazole sharing similar physical properties , failed or only marginally improved the survival of gl261 glioma - bearing mice , even at higher doses than mbz ( supplementary fig1 ) ( 3 ). as we previously made the observation that fenbendazole in feed impaired the intake of the implantation of a medulloblastoma cell line in athymic nude mice ( 3 ), it only made a very marginal and statistically insignificant survival improvement in gl261 glioma model by gavaging 5 days after the implantation . there are several factors potentially contributing to the stark discrepancy in the brain tumor therapy with various benzimidazoles . for one as shown with mbz polymorph a , low bioavailability likely due to the poor absorption could be detrimental to the therapeutic performance of this class of drugs . second , the brain penetration of these benzimidazoles has not been well studied and could be insufficient for any significant therapeutic effects . furthermore , mbz has been implicated in inhibiting multiple tyrosine kinases in recent reports , whereas albendazole showed lack of such ability , indicating differences in anti - tumor mechanisms among benzimidazoles ( 4 , 7 , 8 ). p - glycoprotein ( p - gp , abcb1 ) is an atp - binding cassette ( abc ) transporter and plays an important role in limiting drug uptake into the brain . ( 23 ) elacridar is a 3 rd generation inhibitor of p - gp efflux transporters and also inhibits the breast cancer - resistant protein ( bcrp , abcg2 ) that is another key efflux transporter in bbb ( 24 ). previous studies demonstrated that co - administration of elacridar in rodents has markedly increased by multiple folds the brain distribution of a number of cancer drugs , such as sunitinib , pazopanib , erlotinib and crizotinib , which were determined as the substrates of p - gp and abcg2 by in vitro and animal studies ( 25 - 28 ). furthermore , elacridar has been found safe in phase i clinical trials ( 19 ). in this study , we investigated the combination of elacridar with mbz to potentially enhance its therapeutic efficacy . we found that the combination greatly improved the survival in two orthotopic brain tumor models . however , in this limited study , the b / p ratio and brain auc 0 - 8 h of mbz did not show statistically significant differences with co - administration of elacridar , despite its ability to significantly increase survival in brain cancer bearing mice . when analyzing the metabolites , mbz - nh 2 , one of the two major metabolites in rodents and human ( 20 ), was significantly elevated in terms of b / p ratio ( 2 . 5 folds ) and auc 0 - 8 h ( 2 . 4 folds ) as a result of co - administration of elacridar . also noticeable is our finding that mbz - n 2 was preferentially accumulated in the gl261 brain tumor vs the normal brain tissues . although these data could indicate that mbz - nh 2 is a potential substrate of p - gp and / or abcg2 , the significance of this finding is unclear at this point . a possible direct cytotoxic effect of mbz - nh 2 appears unlikely as further testing displayed only a marginal cytotoxicity with cultured gl261 cells . however , increased toxicity through mbz - nh 2 &# 39 ; s preferential accumulation in the acidic tumor environment cannot be excluded and requires further investigations . further investigations include the study of mbz and elacridar interactions , particularly the potential substrate profile of efflux transporters with mbz , in order to better understand and thereby improve the combination with mbz . mbz - c is the most efficacious polymorph in brain tumor therapy . the combination of mbz - c with elacridar , a p - glycoprotein inhibitor , can greatly improve efficacy . this combination may be used to treat , inter alia , high grade glioma and / or medulloblastoma . the combination may be co - administered or separately administered as part of a regimen of treatment . the above disclosure generally describes the present invention . all references disclosed herein are expressly incorporated by reference . a more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only , and are not intended to limit the scope of the invention . mbz tablets ( 500 mg ) from janssen pharmaceuticals ( pantelmin ®) and medley pharmaceuticals were purchase from local pharmacies in brazil in 2013 and stored at − 20 ° c . freezer . mbz tablets ( 100 mg ) from teva pharmaceuticals usa were purchased from the outpatient pharmacy at the johns hopkins hospital in 2011 and stored at room temperature ( rt ). teva has discontinued mbz in the us market since oct . 2011 . aurochem laboratories ltd . ( mumbai , india ) manufactured mbz tablets ( 500 mg ) s2015 containing the current active pharmaceutical ingredient ( api ) that typically has mixed polymorphs , and s2017 ( polymorph c ) with specific api revealed . aurochem also kindly supplied us with mbz polymorph a , b and c . elacridar ( eld ; gf120918 ; n -( 4 -( 2 -( 1 , 2 , 3 , 4 - tetrahydro - 6 , 7 - dimethoxy - 2 - isoquinolinyl ) ethyl ) phenyl )- 9 , 10 - dihydro - 5 - methoxy - 9 - oxo - 4 - acridine carboxamide )) was purchased from sigma ( st . louis , mo ., usa ). thiabendazole ( tbz ), flubendazole ( flz ), oxifendazole ( oxz ) and fenbendazole ( fbz ) were purchased from sigma ( st . louis , mo ., usa ). cell lines for this study were obtained as previously described : mouse glioma cell line gl261 and the human medulloblastoma xenograft d425med ( d425 ). ( 3 , 14 ) gl261 and d425 cells were maintained in dmem media supplemented with 10 % fetal bovine serum and antibiotics at 37 ° c . in humidified air containing 5 % co2 . gl261 - luc cells expressing firefly luciferase were described previously ( 3 ). a direct detect ™ infrared ( ir ) spectrometer was used ( millipore , billerica , mass ., usa ). mbz powder or tablets ground to powder was mixed with water first , applied to the card and air dried following the manufacturer &# 39 ; s instructions . the spectra of — c ═ o and — nh were analyzed and compared as described before . ( 9 ) all animal studies were approved by the animal care and use committee ( acuc ) of the johns hopkins university . the intracranial implantation of gl261 - luc in the frontal lobe and d425 cells in the cerebellum of the mouse brain followed the procedure described before ( 3 , 4 ). five days after tumor implantation , mice were gavaged with mbz or the other benzimidazoles at 50 mg / kg five days a week . mbz and other benzimidazoles were prepared by either mixing the power with pbs and sesame oil ( 1 : 1 , v : v ) ( sigma ) or by grinding the tablets to powder and resuspending in the aforementioned pbs / sesame oil mixture . elacridar was prepared as a 10 mg / ml suspension in 0 . 5 % hydroxypropylmethylcellulose and 0 . 5 % tween 80 in pbs similarly as described before ( 15 ). female c57bl6 mice , 5 - 6 weeks of age , were purchased from ncl . animal experimentation was conducted under an approved iacuc protocol and complied with local and national guidelines . all mbz polymorphs and tablets were administered by oral gavage at a dose of 50 mg / kg . elacridar was administered by oral gavage at 50 mg / kg 2 hours prior to the administration of mbz - c . mice ( 3 animals / time point ) were first anesthetized via intraperitoneal injection of 60 μl of a stock solution containing ketamine hydrochloride ( 75 mg / kg ) ( 100 mg / ml ; ketamine hcl ; abbot laboratories , chicago , ill ., usa ) and xylazine ( 7 . 5 mg / kg ) ( 100 mg / ml ; xyla - ject ®, phoenix pharmaceutical , st . joseph , mo ., usa ) in a sterile 0 . 9 % nacl solution . then the blood samples were taken by puncturing and aspiring from the left heart ventricle . blood samples were mixed with 5 mm edta and centrifuged at 10000 g for 5 min to obtain the plasma for further analysis . for brain distribution studies , mice were perfused under anesthesia with 20 ml ice - cold saline supplemented with 20 μl of 0 . 02 % heparin by injecting slowly into the left heart ventricle using a 20 gauge needle . the right atrium was cut open before to allow the blood outflow . the yellow color of kidney indicated a good perfusion quality that was essential to deplete blood from the brain tissue . in gl261 tumor - bearing mice , gl261 tumor was distinguished from the normal brain by easily recognizable differences in color and shade . gl261 tumor was separated with a scalpel and the normal brain tissue was cut from the contralateral hemisphere . all brain samples were weighed and stored at − 80 ° c . before processing . blood , brain and brain tumor tissues were harvested as a function of time after mbz administration . to compare the pharmacokinetics of mbz polymorphs , three cohorts of mice each were administered a single dose of 50 mg / kg by oral gavage . for the initial comparison studies , plasma samples were obtained at 1 , 2 . 5 , 4 , 6 , 8 , 15 , and 24 hours after mbz administration while brain tissue was only collected at 6 hours . for the comparison studies of polymorph c with or without eld , plasma and brain tissue samples were obtained at 2 . 5 , 4 , and 8 hours after mbz administration . brain tumor tissue samples were also obtained for polymorph c alone . mbz and the two metabolites , 2 - amino - 5 - benzoyl - benzimidazole ( mbz - nh 2 , cas 52329 - 60 - 9 ) and rac dihydro mebendazole ( mbz - oh , cas 60254 - 95 - 7 ), were quantified in plasma , brain and brain tumor tissue . tissue homogenates were prepared at a concentration of 200 mg / ml in plasma prior to extraction . mebendazole and metabolites were extracted from 50 μl of plasma or tissue homogenates with 0 . 1 ml of methanol containing 0 . 5 μg / ml of the internal standard a620223 . 69 . after centrifugation , the supernatant ( 60 μl ) was mixed with water ( 40 μl ) and then transferred into autosampler vials . separation was achieved with an atlantis dc18 ( 2 . 1 × 100 mm , 3 μm ) column at room temperature with methanol / water mobile phase ( 60 : 40 , v : v ) containing 0 . 1 % formic acid using isocratic flow at 0 . 25 ml / min for 5 minutes . the analytes were monitored using an ab sciex triple quadrapole ™ 5500 mass - spectrometric detector ( applied biosystems , foster city , calif ., usa ) using electrospray ionization operating in positive mode . the spectrometer was programmed to allow the [ mh +] ions of mbz , mbz - nh 2 , mbz - oh , and a620223 . 69 at m / z 296 . 0 , 238 . 0 , 298 . 0 , and 287 . 2 , respectively to pass through the first quadrupole ( q1 ) and into the collision cell ( q2 ). the daughter ions for mbz ( m / z 263 . 9 ), mbz - nh 2 ( m / z 105 . 1 ), mbz - oh ( m / z 266 . 0 ), and a620223 . 69 ( m / z 124 . 1 ) were monitored through the third quadrupole ( q3 ). calibration curves for mbz and metabolites were computed using the area ratio peak of the analysis to the internal standard by using a quadratic equation with a 1 / x weighting function over the range of 5 to 500 ng / ml ( mbz ) and 1 to 500 ng / ml ( metabolites ) with dilutions of up to 1 : 100 ( v : v ). if one or more concentrations were below limits of quantification , a value of ½ the limit of quantification was assigned for pharmacokinetic calculations . if two consecutive time points were below limits of quantification , the last one was excluded from the analysis . mean plasma and brain concentrations were calculated at each time point for both mbz and its metabolites . 1 . 045 g / ml was used as the average wet rodent brain tissue density ( 16 ). pharmacokinetic parameters were calculated from mean mbz and its metabolites concentration - time data using noncompartmental methods as analyzed in phoenix ® winnonlin ® version 6 . 3 ( pharsight corp ., mountain view , calif .). c max and t max were the observed values from the mean concentration data . the auc last was calculated using the log - linear trapezoidal method . λ z was determined from the slope of the terminal phase of the concentration - time profile . the terminal half - life ( t 1 / 2 ) was determined by dividing 0 . 693 by λ z . if the r 2 of λ z was & lt ; 0 . 9 , the t 1 / 2 was not reported . relative systemic exposure to mbz was calculated using the auc last : metabolites auc last / mbz auc last . relative systemic exposure in brain or brain tumor compared with plasma was calculated using the auc last : brain or brain tumor auc last / plasma auc last . animal survival data were analyzed by graphpad prism 5 . 0 . the p - values were determined by a mantel - cox test . a p - value under 0 . 05 was accepted as statistically significant . for the pharmacokinetic studies comparing the polymorphs or administration with eld , the method of bailer was used to estimate the variance of auc last given the calculated variance of the mean concentration at each time point ( 17 ). this was then followed by a pairwise comparison using a z - test to determine whether there was a significant difference between mbz exposure as expressed by auc last ( 18 ). comparisons of individual data were conducted using the nonparametric wilcoxon signed rank test with post - hoc analysis using an all pairs tukey - kramer test . the level of significance was p & lt ; 0 . 05 . polymorph c was most effective for treating brain tumors in mice we examined the polymorph content of several commercially available tablets ( janssen , medley and teva ) and two made to order tablets ( aurochem s2015 used the current api that typically has mixed polymorphs and s2017 was specified as pure mbz - c ) by comparing their ir profiles with the individual mbz polymorphs ( fig1 a and b ). based on the ir peaks of — c ═ o and — nh bonds , we determined that the janssen and medley tablets were made of mainly mbz - c as well as the aurochem s2017 . aurochem s2015 and teva tablets that have been stored at rt for 2 years showed mainly the profiles of mbz - a . as a control , polymorph a , b and c were dissolved in dmso and incubated individually with gl261 glioma cells , which showed equal cytotoxicity ( data not shown ). mbz - a appeared to be ineffective in treating intracranial gl261 glioma - bearing mice , while mbz - c displayed the best efficacy ( fig1 c ). although mbz - b showed a similar survival to mbz - c , it caused more toxicity with 1 treatment - related death among 6 treated mice ( fig1 c ). the efficacy data reflected the polymorph composition of mbz tablets well in the sense that s2015 was ineffective and other tablets made of mbz - c all showed significant efficacy by extending the mean survival to 42 - 50 days from 29 days of the control group ( fig1 e ). following an oral dosing of 50 mg / kg , mbz - c achieved a plasma auc 0 - 24 h of 16 , 039 h * ng / ml ( fig2 a and table 1a ). in comparison , mbz - b reached a plasma auc 0 - 24 h of 26 , 474 h * ng / ml , while mbz - a plasma auc 0 - 24 h reached only 3 , 052 h * ng / ml , by far the lowest among all three polymorphs ( p & lt ; 0 . 05 for auc 0 - 24 h with mbz - b & gt ;- c & gt ;- a ; table 1a ). measurements of brain tissues following a thorough perfusion revealed significant presence of mbz - c over a time course , correlating closely with the plasma mbz levels with a brain / plasma ( b / p ) ratio of 0 . 75 on average that remained relatively stable during the 8 hours ( fig2 b - 2c ). comparing the polymorphs at 6 h following oral gavage , we found mbz - c and - b achieved similar brain levels , despite mbz - b &# 39 ; s higher levels and auc 0 - 24 h in the plasma ( fig2 d and fig6 , table 1a ), resulting in a slightly favorable mean b / p ratio of mbz - c over mbz - b ( 0 . 80 for c vs 0 . 64 for b and 0 . 29 for c , p = 0 . 055 ) ( fig2 e ). this corroborates well with the efficacy data in fig1 c , where mbz - b and - c demonstrated similar survival benefit in gl261 model ( mean survival : 45 days of mbz - b vs 48 . 5 days of mbz - c ). however , it is notable that mbz - b displayed greater toxicity , resulting in early death of one mouse among the six treated animals ( fig2 d ). analysis of the gl261 brain tumor and the contralateral brain tissues indicated equal distribution of mbz - c in the brain tumor and the normal brain tissues ( fig2 d ). we determined the plasma levels of the major metabolites mbz - nh 2 and mbz - oh of mbz polymorphs ( p & lt ; 0 . 05 for auc 0 - 24 h of mbz - nh 2 with mbz - b & gt ; c & gt ; a ; p & lt ; 0 . 05 for auc 0 - 24 h of mbz - oh with mbz - b and c & gt ; a ; table 1a ). the levels of mbz - c &# 39 ; s metabolites in plasma and brain generally followed the same pattern of mbz - c &# 39 ; s concentration ( fig3 a and b ). mbz - nh 2 showed higher levels than mbz - oh in the plasma ( fig3 a ), with an auc 0 - 24 h of 10 , 516 h * ng / ml compared to 5 , 781 h * ng / ml of mbz - oh ( table 1a ). notably , in a reversed pattern , mbz - nh 2 was measured at much lower levels than mbz - oh in the brain in terms of c max and auc 0 - 24 h ( fig3 b and table 1a ). interestingly , in gl261 glioma , mbz - nh 2 reached significantly higher levels than in the contralateral brain ( fig3 c ). in order to elucidate the anti - tumor role of mbz metabolites , we compared the ic 50 of mbz , mbz - oh and mbz - nh 2 in gl261 cells and determined mbz - nh 2 is the least cytotoxic derivative of mbz in vitro ( fig3 d ). achieving a sufficient therapeutic concentration in the tumor and the surrounding brain tissue is a critical challenge that is faced by almost all brain cancer therapies . four hours after oral administration , we found mbz - c brain concentration peaked at 2 , 016 ng / g ( equivalent to 7 . 1 μm ) ( table 1a ), which was well above the ic 50 s of cultured glioma and medulloblastoma cells ( 0 . 11 - 1 μm ) and also above mbz &# 39 ; s inhibitory ic 50 with vegfr2 kinase at 4 . 3 μm in vitro ( 3 , 4 ). the relatively high brain concentration might help explain mbz efficacy in brain tumor models . next , we reasoned that a further increase in the brain distribution of mbz would be desirable as it may increase therapeutic efficacy . aside from a pure mechanical barrier , the bbb employs active efflux mechanisms to limit drug entry such as p - glycoprotein ( p - gp ). elacridar ( eld ) is a potent third - generation inhibitor that inhibits p - gp as well as breast cancer resistance protein ( bcrp ) and co - administration of elacridar has increased the brain penetration of several drugs ( 15 , 19 ). we first examined the cytotoxicity of elacridar in gl261 mouse glioma cells and determined the ic 50 to be 5 . 8 μm ( fig4 a ). combining elacridar with 0 . 25 μm mbz only marginally increased the cytotoxicity in vitro ( fig4 b ). oral administration of 50 mg / kg elacridar two hours prior to mbz - c did not significantly change the brain concentration of mbz in terms of auc 0 - 8h , while b / p ratio average of 2 . 5 , 4 and 8 h was shifted slightly higher from 0 . 75 to 1 . 03 , which , however , was not statistically significant ( table 1b and fig4 c ). interestingly , this was accompanied by a significant increase in mbz - nh 2 along with an elevation of the b / p ratio from 0 . 12 to 0 . 30 in the brain when treated with a combination of elacridar and mbz - c ( table 1b ). combination therapy of elacridar and mbz increased the survival benefit in gl261 syngeneic glioma and d425 xenograft medulloblastoma models ( fig5 ). this was achieved by adding 7 or 14 days of 50 mg / kg elacridar treatment to the standard mbz ( mbz - c ) regimen of 50 mg / kg . specifically , in gl261 , combination therapy improved the median survival to 92 . 5 and 110 . 5 days dependent on the treatment length , which is a stark increase from 53 days of mbz alone as well as 29 . 5 days ( control ) and 34 days ( elacridar alone ) ( fig5 b ). similarly , in the orthotopic d425 medulloblastoma xenograft model , the combination of elacridar with mbz increased the median survival to 77 days ( fig5 d ). this is a significant improvement from mbz only treatment with 53 days of survival and elacridar alone , which showed a marginal survival benefit of 9 days in this particular animal model . a prolonged treatment course with elacridar and mbz was attempted , however , increased toxicity such as severe weight loss and mortality limited those studies ( data not shown ). 1 . bai r y , staedtke v , riggins g j . molecular targeting of glioblastoma : drug discovery and therapies . trends mol med 2011 ; 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