Structurally, the vulnerability of DCC-2036 to P-loop mutations suggests subtle nearby alterations in the ATP binding web-site may well properly destabilize the inactive conformation, as for imatinib . A complete explanation pertaining to DCC-2036 will demand additional crystallographic, dynamic , and in silico analysis1 . Impressively, resistant outgrowth was totally suppressed mTOR inhibitors selleckchem by DCC-2036 at 750 nmol/L . As clinically achievable plasma levels of DCC-2036 haven’t but been reported and select P-loop mutants confer partial resistance to DCC-2036, nilotinib , and dasatinib , we evaluated dual-combinations of DCC-2036 with each and every clinical ABL inhibitor in resistance screens. When the mixture of DCC-2036 with imatinib decreased the fraction of wells with outgrowth, P-loop vulnerabilities at residues G250, Y253, and E255 have been detected . No resistant subclones had been recovered with dual-combinations of DCC-2036 and clinically achievable concentrations of nilotinib or dasatinib . These findings are similar to individuals from research with one more ABLT315I inhibitor, SGX393 , and propose that ABL inhibitor cocktails that involve an ABLT315I inhibitor for example DCC-2036 may perhaps signify a rational therapeutic strategy to mitigating resistance.
Because the instant clinical application of an ABLT315I inhibitor is in refractory CML patients harboring this mutation, we carried out resistance screens starting from Ba/F3 cells expressing BCR-ABLT315I to identify BCR-ABL compound mutations conferring improved resistance to DCC-2036. Such mutations Romidepsin are already reported in clinical failures to dasatinib or nilotinib salvage therapy, suggesting possible for assortment on sequential remedy with ABL inhibitors .
The compound mutationbased resistance profile for DCC-2036 narrowed predominantly to BCR-ABLE255V / T315I . An additional mutant featuring substitution of your baseline isoleucine at residue 315 for methionine was also recovered. To our awareness, mutation from the gatekeeper residue to methionine hasn’t been observed in resistance to other ABL tyrosine kinase inhibitors. DCC-2036 forms hydrogen bonds with the nearby ATP hinge web site residue M318 as well as the K271-E286 salt bridge, enabling for accommodation of the bulky isoleucine substitution in BCR-ABLT315I. Electrostatic interaction with E282 aids in stabilizing the E282-R386 switch control pair interaction and, consequently, the inactive kinase conformation1 . 1 explanation for your resistance in the mutant featuring methionine at residue 315 might possibly be that the methionine sidechain impinges on DCC-2036 binding. Alternatively, introducing methionine at the gatekeeper position could induce the ABL kinase domain to adopt an active conformation.

Consequently, AP24534 has the capability to do away with compound mutations involving T315I and E255V predicted to get extremely resistant to all other inhibitors. Currently, the amount of clinically documented compound mutations inside the kinase domain of BCR-ABL linked with treatment failure is very low . Nonetheless, they signify a formidable difficulty for PF 477736 selleckchem these sufferers harboring them, and incidence may grow using the prolonged survival of CML patients and with a lot more patients undergoing sequential ABL kinase inhibitor therapy . Overall, whilst no mutagenesis screen is usually totally exhaustive, our information indicate AP24534 has the possible to deal with this at present unmet clinical problem. Our pre-clinical profiling signifies that AP24534 has possible as a significant selection for controlling resistance in CML. The combined outcomes of our biochemical, cell-based, and in vivo research recommend that AP24534 exhibits adequate action towards native BCR-ABL and all tested BCR-ABL mutants to warrant consideration for single-agent use as being a pan-BCR-ABL inhibitor. Moreover, our success indicate that AP24534 holds promise for controlling compound mutants involving T315I, whereas raising awareness that it is advantageous to wipe out resistant subclones in the single-mutation stage.
In the longer term, this may perhaps advocate for your possible future utilization of a pan-BCR-ABL inhibitor this kind of as AP24534 in the first-line therapeutic capacity. Clinical utilization of a pan-BCR-ABL inhibitor lively towards T315I could make long-term remissions an achievable goal a minimum of for some individuals with state-of-the-art CML. A phase one clinical trial evaluating oral AP24534 SNX-5422 in patients with refractory CML as well as other hematologic malignancies is ongoing . EXPERIMENTAL PROCEDURES Inhibitors AP24534, 3- -4-methyl-N- methyl)-3- phenyl)benzamide was synthesized at ARIAD Pharmaceuticals. Imatinib, dasatinib, and nilotinib were obtained from the Oregon Wellness & Science University pharmacy or made at ARIAD. All inhibitors have been prepared as 10.0 mM stock solutions and stored at ?20?C. Serial dilutions of 10.0 mM stock solutions were carried out just prior to use in each experiment. Crystallization and Structural Determination of ABLT315I:AP24534 Complex The kinase domain of murine ABLT315I was co-expressed with YopH protein tyrosine phosphatase in E. coli as described and purified in the presence of AP24534 to near homogeneity using metal affinity, Mono Q, and size exclusion chromatography. The typical yield of purified ABLT315I bound with AP24534 was about one mg/L. Co-crystals of ABLT315I and AP24534 had been grown by the hanging drop vapor diffusion method at 4oC by mixing equal volumes of the AP24534:ABLT315I complex and well solution .

In vitro cell proliferation was measured by tritiated thymidine cell incorporation assays as previously described. KAS-6/1-MIP1? Mouse Model?We utilized an animal model of a variety of myeloma in which systemic administration of KAS-6/1-MIP1? myeloma cells into immunocompromised female mice generates progressive diffuse bone reduction measured by BMD. In this animal model, initiation of bone loss occurs inside of two weeks immediately after tumor cell injection, hind-limb paralyses arise inside of 7 to eight weeks post-injection, and death GW9662 kinase inhibitor generally happens inside eight to nine weeks post-injection. Human KAS-6/1 myeloma cells had been genetically engineered to carry the osteoclast activating aspect, MIP1? , which induces the observed bone density reduction in these mice. No BMD reduction was observed in SCID mice injected with mother or father KAS-6/1 cells along with the observed BMD increase was much like animals not injected with any tumor cells. In contrast, a progressive decrease in BMD was observed during the femurs and lumbar spines of your animals that had been injected with the KAS-6/1-MIP1? cells starting up inside two weeks just after cell injection. This model does not cause localized bone destruction but diffuse osteoporosis thanks to diffuse marrow infiltration from the cells, similar to precisely what is frequently observed in a number of myeloma.
Various Myeloma Efficacy Research?About 4 week previous female CB17 SCID mice were obtained from Harlan-Sprague Dawley Industries and housed in the barrier facility, beneath a twelve hour light/dark cycle, with ad libitum entry Imiquimod to foods and water. Roughly twenty-four hrs prior to tumor cell injection, un-anesthetized mice had been irradiated with 250 cG Cesium making use of a Mark one?25 gamma irradiator. KAS-6/1-MIP1? myeloma cells had been injected to the tail vein of effectively restrained mice at day 0. The compounds and dosages were depending on the outcomes from our tolerability and breast cancer efficacy research and included 0.04, 0.4, or four.0 ?g/day of MBC-29, -11, and -1, 0.04 or 4.0 ?g/day of etidronate, AraC, AraC+etidronate, FUR, FU, and zoledronate. PBS was integrated because the vehicle management. As the control compounds weren’t examined at the mid-dose degree, the results of your 0.40 ?g/day dose are presented in Supplemental Table S3. Each day s.c. administration with the compounds resuspended in sterile PBS was begun two weeks after cell injection at the time of original bone loss right up until the required sacrifice from the animals. Mice have been sacrificed whenever they could no longer acquire foods and water on their particular, lost their righting reflex, lost extreme weight , or if persistent paralysis occurred. Bone density scans have been routinely carried out on anesthetized mice every two weeks commencing on the time of cell injection until the required sacrifice date using dual-energy x-ray absorptiometry measured by a Lunar-PIXImus instrument.

The reasons for this lack of action include issues with regards to the toxicity of nucleoside analogues and also the necessary goal of developing new drugs with significantly less toxicity than the classical agents and concerns that maybe new nucleoside analogues would not be sufficiently distinct from individuals MEK Inhibitors by now known and authorized for human use, and as a result no more advances have been possible. Though toxicity is still a problem and is a problem that is definitely difficult to circumvent with antimetabolites , the information provided in the preceding pages clearly indicates that compact structural improvements can have profound effects within the biological exercise of nucleoside analogues and suggests that new agents with practical activities can still be identified. A significant element within the design and style of purine and pyrimidine antimetabolites is the fact that the drug design operation is largely empirical in nature. Compounds are created which are structurally just like existing agents depending on a thorough understanding of the previous job within this area, and they’re tested in many biological assays. As indicated in this analysis, a considerable quantity of structure activity romance data is available from your a lot of many years of do the job with this particular class of compounds that aids guide the style of new compounds.
Despite the fact that this evaluate has centered around the results stories, you can find countless far more examples of antimetabolites which have been constructed and synthesized that have not been flourishing, as well as a thorough comprehending of each the successes and failures is vital for the rational ZD-1839 development of new agents of this class. The analysis from the present FDA-approved anticancer nucleosides indicates a clear and hassle-free guideline that must be deemed inside the style of new agents on this class. The new compounds should certainly include things like structural adjustments which are as smaller as you possibly can, and as couple of adjustments as is possible need to be produced to the purely natural molecule, with 1?three changes getting the most desirable quantity. Because all of the purine and pyrimidine analogues utilized in the therapy of cancer are prodrugs , their mechanism of action is extremely complex and consists of interaction with a variety of anabolic and catabolic enzymes. Therefore, it is going to not be simple to replace this empirical operation having a much more ?rational? drug design and style operation. Whilst the empirical approach used in the style of new nucleosides can be a ?rational? way to design and style new medicines, the newer ?rational? drug style concepts optimally involve using the three-dimensional construction with the protein target coupled with biochemical effects and in silico modeling methodology. This strategy is most handy when a drug is envisioned to act largely by affecting a single enzymatic target. Though structural information using the different enzymes is more and more staying utilized to assist while in the design of new antimetabolites, the style of new antimetabolites isn’t driven through the wish to interact with only one enzyme.

In summary, quite possibly the most very important getting in our examine was that higher meTIMP concentrations are associated with all the risk of creating myelotoxicity, and that individuals who have meTIMP concentrations exceeding a stipulated lower off level of 11 450 pmol/86108 RBC early in regular state possess a substantially improved possibility of establishing bone marrow toxicity. As myelotoxicity tended to develop quite a few weeks after maximum meTIMP concentrations were reached it may well be attainable to recognize patients at risk early inside the program of therapy, therefore preventing the advancement of myelotoxicity and Secretase inhibitors the associated threat of infections. We conclude that metabolite measurements in the time point when regular state concentrations are reached are of worth for identifying sufferers in danger of producing myelotoxicity. A bigger potential examine is now required to confirm this observation. The Tenets on the Declaration of Helsinki have been followed and institutional ethics committee approval was granted. Informed consent was obtained in advance of surgical treatment for all sufferers. Laboratory studies Major human Tenon?s capsule fibroblasts had been propagated from explanted subconjunctival Tenon?s capsule isolated during glaucoma filtration surgical treatment.
Explanted tissue was anchored onto the bottom of the six properly plate that has a sterile cover slip and overlaid with RPMI culture medium. Culture media were supplemented with Tivozanib L-glutamine 2MM and penicillin 100 000 units/l and fetal calf serum. When the monolayers had reached confluence , the fibroblasts have been passaged and cultured in 175 cm2 tissue culture flasks.
For experimentation, fibroblasts have been trypsinised, seeded into 96 effectively tissue culture plates, and incubated overnight to allow attachment. MMC and 5-FU had been applied at 0.four mg/ ml and 25 mg/ml respectively. Trypan blue was additional to a ultimate concentration of 0.05%. All antimetabolites had been reconstituted in serum-free culture medium. Tenon?s fibroblasts have been seeded at a concentration of 5000 fibroblasts per well into 96 properly plates and incubated overnight. The fibroblast monolayers have been then washed to clear away serum and covered with a single application of MMC as described previously.17 Unless otherwise stated, the remedy time for all experiments was five minutes. Manage fibroblasts were treated using a five minute application of serum-free RPMI with or without 0.05% trypan blue. Following therapy the monolayers were washed quickly 3 times and incubated in serum-free RPMI. A lactate dehydrogenase release assay was made use of to quantify fibroblast death as previously described.18 LDH is actually a steady cytoplasmic enzyme existing in all cells. It really is quickly launched in dying cells right after damage to the plasma membrane. Lactate dehydrogenase catalyses the reduction of a colourless tetrazolium salt to coloured formazan that absorbs a broad spectrum of light with maximum absorbance about 492 nm. Lactate dehydrogenase exercise is existing in serum.

Amongst the agents at the moment implemented for NB remedy,doxorubicin,topotecan and vincristine had been active against NB cell lines SB 271046 at each the substantial and also the minimal concentrations.Etoposide was only active in our assay in the high concentration although cisplatin,cyclophosphamide and iphosphamide didn’t present action against the 2 NB cell lines in our assays.Cyclophosphamide and iphosphamide are pro-drugs that demand metabolic activation in liver 16; therefore,they should certainly not show activity in our in vitro assay.Cisplatin brought on roughly 20% reduction of cell amount in SK-N-AS and 97% reduction in SH-SY5Y.In addition,our screen identified bortezomib and 1 of its structural congener are a lot more lively than those at the moment put to use within the regular NB therapies.Bortezomib,a synthetic tripeptide,may be a selective proteasome inhibitor at the moment implemented in treatment method of relapsed various myeloma 17.It’s proven to become productive against a lot of malignancies such as lung 18,breast 19,pancreatic cancers twenty,glioblastoma multiforme 21,and neuroblastoma 22 in pre-clinical scientific studies; likewise asin the murine NB model 23.Bortezomib and its congener had been efficient at lowering cell quantity by better than 90% at 72 hrs immediately after drug treatment.
Furthermore,bortezomib and its structural congener had been capable to induce apoptosis by better than 15-fold.These outcomes,in conjunction with other pre-clinical and clinical information,recommend that proteasome inhibition represents a novel therapeutic target for NB therapy.Between the active compounds,CDDO-Me,helenalin and cucurbitacin I belong to a class of compounds recognized as terpenoids that incorporate isoprene units.CDDO-Me can be a synthetic triterpenoid which has proven efficacy above a wide array of malignancies this kind of as prostate 24,ovarian 25,and lung cancers 26,and neuroblastoma 27.Alabran Salbutamol et al.reported many NB cell lines sensitive to CDDO-Me at concentrations under 150 nM 27.A variety of molecular pathways like p53 28,STAT eleven,and NF?B 29 are linked using the viability,metastasis and chemotherapy resistance in NB.CDDO-Me,which targets numerous molecular pathways like Akt,mTOR 24,JNK 30,NF?B 31,STAT 25,and Notch1 32 in other malignancies,could show to get a lot more effective against NB than focusing on just one pathway.N-Allylamino-17-demethoxygeldanamycin showed 70% growth inhibition in both NB cell lines.17-AAG inhibits the molecular chaperone heat shock protein 90 that maintains stability and conformation of necessary proteins like p53,AKT,and ERBB2.Consequently,inhibiting Hsp90 leads to blocking of many pathways crucial for that survival of cancer cells.In recent pre-clinical studies,17-AAG has been reported to inhibit growth of the broad array of malignancies ranging from NB 33 to pancreatic cancer 34.

A single-agent phase I review in refractory AML sufferers is evaluating single-agent activity like a prelude to blend therapy trials.Even more examine of ON 01910.Na is warranted Romidepsin cost to greater define biological activity and appropriate target populations and to define mechanism of action.Outlook and Summary The major improvements in AML treatment method through the final two decades haven’t been the introduction of new therapeutic agents but rather the even more optimal use of well-known drugs.For younger patients with poor-risk cytogenetics and an readily available donor,HSCT gives you the most beneficial chance for a cure.For older individuals and for relapsed and refractory patients,there is an apparent should produce far better strategies with helpful regimens.The limit of acceptable toxicity for typical chemotherapeutic medicines utilized in AML treatment has been reached.New therapeutic tactics are therefore essential.Although a number of deregulated proteins and genes have been identified,they are so varied amongst AML situations that acquiring a substance with prospective exercise against all of them is difficult.Not long ago,various new agents are explored and have shown guarantee in treating AML.
However,it is actually unlikely that these agents will likely be curative when administered as monotherapy; it is actually even more possible they will likely be applied in mixture with other new agents or with standard treatment.It has long been appreciated that AML is actually a clinically heterogeneous sickness with marked differences in survival following intensive chemotherapy based on age,blast cell morphology,cytogenetic abnormalities,and gene mutations.
As described above,in many circumstances,1 within the partners in a gene arrangement codes PD98059 selleck chemicals for a transcription issue.As being a consequence,AML-associated fusion proteins typically perform as aberrant transcriptional regulators and in the end interfere using the system of myeloid differentiation regardless of variations in gene expression modifications induced by them.25 Similarly,class I mutations that activate signal transduction pathways and class II mutations that have an effect on transcription factors or components with the cell cycle machinery also influence blast cell differentiation and elicit AML phenotype.These success recommend that mutation or upregulation in one particular pathway will not account for AML transformation.Blasts depend on several dysregulated pathways to emerge and survive and also to in the end build resistance to therapy.For this reason,pursuing many molecular lesions in the concurrent or serial trend could possibly be a promising technique to targeted treatment.Although many of the breakpoints associated with exact chromosomal translocations are actually cloned and novel ones are still getting found,typically,the molecular mechanisms as well as central gamers resulting in tumorigenesis are not elucidated.

Somnolence was the sole DLT and no responses were witnessed with any dose level.A second dose-finding examine was performed in 43 individuals with sophisticated tumors evaluating each day doses from 10mg to 80mg orally daily in divided doses.38 The DLTs recognized have been grade three reversible somnolence and liver perform check elevations.It was evident that somnolence and liver peptide synthesis selleckchem toxicity restricted dose escalations to level demanded to adequately inhibit aurora kinase A.Based mostly upon these benefits,MLN8054 development was abandoned in favor of MLN8237.two.1.four MLN8237?MLN8237 shares structural homology to MLN8054,but has four-fold better inhibitory potency for aurora A kinase and decreased tendency to trigger somnolence.In vitro and in vivo testing making use of murine models investigated MLN8237 within a range of malignancies popular to pediatrics,each sound and hematologic.39,40 Additional preclinical research in models of lymphoma41,42,Philadelphia chromosome constructive leukemias 43,several myeloma44,acute myeloid leukemia as single agent and in combination45,breast and prostate cancer 46,have constantly proven anti-tumor results by direct and surrogate marker evaluation.
Importantly,in designs of chronic myelogenous PF-562271 leukemia and Ph+ acute lymphoblastic leukemia ,MLN8237 showed equivalent effects irrespective of p53 exercise standing.42 A phase I examine of 43 sufferers with innovative tumors demonstrated antiproliferative results at a dose amount of 80mg/day orally and DLTs at 150mg/day orally for 7 consecutive days every single 21 days.47 The side result profile differed substantially from MLN8054 as only grade I somnolence,grade 3 neutropenia and mucositis have been observed.Two similar phase I research in innovative strong tumors established MLN8237 50mg orally twice everyday for 7 days just about every 21 days for being most promising routine in grownups,with DLT of febrile neutropenia and myelotoxicity.48,49 Other adverse occasions,just like mild somnolence,nausea,and diarrhea was dose-related and reversible.A secondary analysis of 117 sufferers enrolled within the phase I trials confirmed 50mg orally twice everyday for 7 days each and every 21 days to provide steady-state normal serum concentrations roughly 1.7?M,just about double the serum concentration established in preclinical models to maximize anti-tumor effects.50 A phase I examine in 37 pediatric patients located elevated dose-related toxicities of myelosuppression and dermatologic toxicity with numerous day by day dosing and established a phase two dose in pediatric sufferers to be 80mg/m2/day orally.51

In contrast, a 47-kDa immunoreactive band corresponding towards the predicted molecular bodyweight for CB2 receptors was recognized by the CB2 receptor antibody in membranes ready from CHO?CB2 cells kinase inhibitors , but not from mouse cortex.In spinal cord membranes ready from WT-OE and G93A mice , selective antibodies identified immunoreactive bands with all the predicted molecular fat for CB2 or CB1 receptors.In addition, the band acknowledged by each antibodies was eradicated upon pre-incubation of antibodies with an extra on the acceptable blocking peptide.Though very little CB2 receptor immunoreactivity is current in spinal cords of 120-day-old WT-OE mice , somewhere around fourfold better CB2 receptor density is observed in end-stage G93A animals.In contrast, CB1 receptor immunoreactivity is decreased essentially fourfold in spinal cord membranes of 120-day-old G93A , relative to WT-OE control mice.Cannabinoid receptor binding experiments had been carried out to verify the outcomes observed from western analysis.Equivalent to final results reported for mRNA and western examination, predominantly CB1 and a good deal much less CB2 receptors are existing in spinal cord membranes of 120-day-old WT-OE control mice.
In agreement with elevated CB2 mRNA and immunoreactivity, CB2 receptor density also is elevated over 13-fold from the spinal cords of 120-day-old G93A mice , relative to that observed in age-matched WT-OE controls.Equivalent to decreased immunoreactivity, CB1 receptor density also is lowered somewhat, despite the fact that not considerably, by 20% in 120-day-old G93A relative to age-matched WTOE manage mice.
To identify regardless if the up-regulated CB2 receptors in G93A spinal cord membranes are functional, G-protein activation assays Temsirolimus selleck had been carried out.We at first attempted to assess CB1 and CB2 receptor activation of G-proteins among WT-OE and G93A spinal cord membranes by conducting GTP?S binding assays during the presence of selective agonists.Even so, after considerable energy, we had been not able to show constant, measurable G-protein activation together with the selective CB1 agonist ACEA or even the CB2 agonists GW-405833 and AM-1241 in mouse spinal cord membranes.Hence, G-protein activation developed by CB1 and CB2 receptors was rather quantified by selectively antagonizing the GTP?S binding created through the CB1/CB2 full agonist HU-210 using the CB1 antagonist 0?2050 or even the CB2 antagonist SR-144528.In WT-OE spinal cord membranes , stimulation of CB1/CB2 receptors by HU-210 produces thirty.7 ? six.two fmol/mg protein of GTP?S binding to G-proteins.Co-incubation with all the CB1 selective antagonist O-2050 just about fully blocks G-protein stimulation by HU-210.Interestingly, the CB2 selective antagonist SR-144528 also appreciably lowers HU-210 stimulation by roughly 50%.

Over the day of testing, A-836339 or AM1241 was injected thirty min just before testing for thermal hyperalgesic results.Thermal hyperalgesia was established using a commercially on the market thermal paw stimulator as described jak2 inhibitors by Hargreaves et al..Rats had been placed into person plastic cubicles mounted on a glass surface maintained at 30?C, and allowed a 20 min habituation period.A thermal stimulus, from the form of radiant heat emitted from a focused projection bulb, was then utilized towards the plantar surface of each hind paw.The stimulus present was maintained at four.50 _ 0.05 amp, and also the greatest time of publicity was set at twenty.48 s to limit probable tissue harm.The latency to a brisk withdrawal in the hind paw from your thermal stimulus was recorded instantly employing photodiode motion sensors.The proper and left hind paws of each rat have been examined in three sequential trials at about 5 min intervals.Paw withdrawal latency was calculated because the mean on the two shortest latencies.PWL have been measured 30 min post-A-836339 or AM1241 administration in each the CFA-inflamed and un-injected paws.Rat SNL model of neuropathic discomfort As previously described in detail by Kim and Chung , rats had been positioned under isoflurane anaesthesia and also a one.
5 cm incision was made dorsal to your lumbosacral plexus.The paraspinal muscles had been separated from your spinous processes, the L5 and L6 spinal nerves isolated, and tightly ligated with 5-0 silk suture distal to your dorsal root ganglion.Care was taken in order to avoid ligating the L4 spinal nerve.Following spinal nerve ligation, cetirizine a minimum of seven days of recovery and no greater than two weeks was allowed just before the behavioural testing.Only rats with threshold scores _4.5g have been regarded as allodynic and utilized in pharmacological experiments.Mechanical allodynia was measured employing calibrated von Frey filaments.Paw withdrawal threshold was determined through the use of the Dixon?s up?down process.Rats have been positioned into inverted personal plastic containers on best of a suspended wire mesh using a 1 cm2 grid to supply access on the ventral side with the hind paws, and acclimated on the check chambers for 20 min.The von Frey filaments had been presented perpendicularly towards the plantar surface within the picked hind paw, after which held in this place for about eight s with enough force to bring about a slight bend from the filament.Constructive responses included an abrupt withdrawal of your hind paw from the stimulus, or flinching behaviour quickly following elimination in the stimulus.A 50% withdrawal threshold was established employing an up?down process.The strength of your highest filament applied for von Frey testing was 15.0g.A per cent maximal conceivable result of testing compound was calculated according on the formula:100%, exactly where the maximum threshold was equal to 15g.