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Invest New DrugsDOI 10.1007/s10637-011-9730-5 PRECLINICAL STUDIES Enhancing the anti-lymphoma potential of 3,4-methylenedioxymethamphetamine (‘ecstasy')through iterative chemical redesign: mechanismsand pathways to cell death Agata M. Wasik & Michael N. Gandy & Matthew McIldowie & Michelle J. Holder &Anita Chamba & Anita Challa & Katie D. Lewis & Stephen P. Young &Dagmar Scheel-Toellner & Martin J. Dyer & Nicholas M. Barnes & Matthew J. Piggott &John Gordon Received: 8 June 2011 / Accepted: 1 August 2011 # Springer Science+Business Media, LLC 2011 Summary While 3,4-methylenedioxymethamphetamine ‘best' compounds (containing 1- and 2-naphthyl and para- (MDMA/‘ecstasy') is cytostatic towards lymphoma cells biphenyl substituents) some 100-fold more potent than in vitro, the concentrations required militate against its MDMA versus the BL target. When assessed against translation directly to a therapeutic in vivo. The possibility derived lines from a diversity of B-cell tumors MDMA of ‘redesigning the designer drug', separating desired anti- analogues were seen to impact the broad spectrum of lymphoma activity from unwanted psychoactivity and malignancy. Expressing a BCL2 transgene in BL cells neurotoxicity, was therefore mooted. From an initial afforded only scant protection against the analogues and analysis of MDMA analogues synthesized with a modified across the malignancies no significant correlation between α-substituent, it was found that incorporating a phenyl constitutive Bcl-2 levels and sensitivity to compounds was group increased potency against sensitive, Bcl-2-deplete, observed. Bcl-2-deplete cells displayed hallmarks of apo- Burkitt's lymphoma (BL) cells 10-fold relative to MDMA.
ptotic death in response to the analogues while BCL2 From this lead, related analogs were synthesized with the overexpressing equivalents died in a caspase-3-independentmanner. Despite lymphoma cells expressing monoamine Matthew J. Piggott, Nicholas M. Barnes, and John Gordon are joint transporters, their pharmacological blockade failed to reverse the anti-lymphoma actions of the analogues studied.
A. M. Wasik : M. J. Holder : A. Chamba : A. Challa : Neither did reactive oxygen species account for ensuing S. P. Young : D. Scheel-Toellner : J. Gordon (*) cell death. Enhanced cytotoxic performance did however School of Immunity & Infection, The Medical School, track with predicted lipophilicity amongst the designed Birmingham, University of Birmingham, compounds. In conclusion, MDMA analogues have been Edgbaston,Birmingham B15 2TT, UK discovered with enhanced cytotoxic efficacy against lym- phoma subtypes amongst which high-level Bcl-2—often abarrier to drug performance for this indication—fails to M. N. Gandy : M. McIldowie : K. D. Lewis : M. J. Piggott School of Biomedical, Biomolecular and Chemical Sciences,The University of Western Australia,Crawley, Australia Keywords Apoptosis . Bcl-2 . Cytotoxicity. Lymphoma .
MDMA M. J. DyerMedical Research Council Toxicology Unit,Leicester, UK Activated B-Cell-like Burkitt's lymphoma Cellular and Molecular Neuropharmacology Research Group, Dopamine transporter Clinical and Experimental Medicine, The Medical School,Birmingham, UK Diffuse large B-cell lymphoma Epstein-Barr virus background the impact of its dysregulated, high level Follicular lymphoma expression on the efficacy of promising new drug Germinal B-Cell-like candidates. Within this context we have been investigating compounds which target components of neurotransmitter Non-Hodgkin lymphomas pathways that can be found in immune cells and their Poly (ADP-ribose) polymerase cancers: most notably the transporters for serotonin and dopamine (SERT and DAT, respectively), each expressed in Post-transplant lymphoproliferative disease a broad range of the NHL subtypes and other B-cell Serotonin transporter Amongst such compounds, the amphetamine derivatives fenfluramine and 3,4-methylenedioxymethamphetamine(MDMA, ‘Ecstasy') were found to be anti-proliferative against B-cell lines of diverse malignant B-cell origin. Itwas shown (at least with fenfluramine) that in Bcl-2-deplete The incidence of B-cell lymphomas, constituting around BL cells, growth arrest was accompanied by apoptotic cell 95% of all the non-Hodgkin lymphomas (NHL), is death following activation of caspase-3: these latter features increasing steadily year-on-year. NHL is a heterogeneous being reversed on introducing BCL2 as a transgene group of neoplasia ranging from indolent examples like Unfortunately the concentrations of the amphetamine slow growing follicular lymphoma (FL) to highly aggres- derivatives required to elicit anti-proliferative/pro-apoptotic sive, rapidly proliferating entities exemplified by diffuse activity in vitro were too high for safe translation to a large B-cell lymphoma (DLBCL) - the most common of the cancer therapeutic in vivo. Therefore we mooted for NHL in Europe, Australasia and the US—and Burkitt's MDMA the potential of "redesigning the designer drug" lymphoma (BL): rare in the West but endemic in the to enhance lymphoma killing while reducing neurotoxicity World's malarial belt. The diversity of tumors reflects a and psychoactivity.
composite of factors including the differentiation stage of Research by Shulgin and co-workers suggests that the target B-cell and the mutations/translocations arising extending the α- or N-substituent of MDMA to anything therein. Multiple profiling platforms such as gene array are larger than an ethyl group abolishes the drug's psycho- disclosing additional heterogeneity within previously con- activity. Nash and Nichols, studying acute effects in rats, sidered single clinical entities which can be manifested showed that a simple substitution of the methyl group at the molecularly, cellularly and prognostically. DLBCL for α-C of MDMA with an ethyl substituent, creating MBDB, example is now considered a composite of disease subtypes significantly diminishes the amount of dopamine released comprising primarily ‘Activated B-Cell-like' (ABC) cases in the striatum . The α-substituent was therefore and those that are ‘Germinal B-Cell-like' (GCB): survival deemed a rational plinth for redesign. We now describe rates among the former being substantially worse than the improved cytotoxic performance of MDMA analogues with latter. Moreover, within ABC DLBCL constitutive expres- modified α-substituents against a spectrum of B-cell sion of the pro-survival gene BCL2 further discriminates a malignancies giving attention to the mechanisms and substantially inferior subgroup with regards overall survival pathways to cell death, including the impact of anti- even in the face of intense therapy.
apoptotic Bcl-2. A companion study details in full the Anti-apoptotic BCL2, originally identified as the gene chemistry and synthesis of the analogues while providing translocating to the IGH locus on chromosome 14 in the evidence for diminished neurotoxicity and psychoactivity hallmark t(14;18) of FL, offers a considerable barrier to of selected compounds, together with a brief description of drug efficacy in lymphoma treatment. BL, while extremely their rank potency in targeting a BL cell line ].
aggressive, lacks genetic alterations in BCL2, is deplete inBcl-2 protein and has a high cure rate using combinationchemotherapy. Over the past decade we have adopted BL Materials and methods as a template on which to explore novel therapeuticopportunities for lymphoma: BL offering a sensitive monitor of pro-apoptotic/anti-proliferative activities andat the same time being a tumor that is readily adaptable to MDMA and analogues with modified α-substituents were tissue culture with derived lines remaining ‘biopsy-like' synthesized by reductive amination of the corresponding when maintained in early passage. Transfection of BCL2 piperonyl ketones as described recently All target on a constitutive promoter into these cells allows the amines were converted to their hydrochlorides and were opportunity to model directly on an otherwise isogenic tested as such.
2.1 available online . A plot of average log P versuspIC50 showing the SEM in each variable was constructed Cell lines deriving from different B-cell malignancies and and a curve was fitted by weighted linear regression using variants of the L3055 BL cell line were as described Grafit 4, where the weighting of each point was inversely previously []. EBV-transformed lymphoblastoid cell lines proportional to the respective error in pIC50. Due to the were from the School of Cancer Sciences, University of uniformity in the magnitude of errors of average log P Birmingham U.K. All cell lines were cultured in RPMI 1640 across the dataset, these were ignored when the weighted medium supplemented with 2 mM glutamine, 10% v/v FCS, curve was fitted.
100 U/ml penicillin, 100 U/ml streptomycin under 5% CO2 at37°C and passaged three times weekly.
Pharmacological interpretation and statistics Cellular cytotoxicity Pharmacological interpretation of cytotoxicty assays togenerate the pIC50 and Hill coefficient of a compound's Cellular cytotoxicity/viability was assessed by staining activity against L3055 cells was performed using a four treated cells with propidium iodide (PI, a DNA binding parameter logistic equation with iterative fitting using dye incapable of penetrating intact cell membranes, (Sigma Kaleida Graph []. Regression analysis for cytotoxic Aldrich, Dorset, UK)) at a final concentration of 0.85 μg/ml response vs Bcl-2 expression was calculated as a ratio or 1.15 μg/ml prior to flow cytometric analysis (FACS between remaining cell viability (assessed as in 2.3 above) Calibur BD) of PI+ve versus PI-ve cells. Results were following treatment with MDMA and analogues and the analysed using FlowJo 8 software for Macintosh.
optical density (computed using ImageJ for Macintosh) ofBcl-2 vs calnexin protein bands as determined by Western blot. Graphs were created in OriginPro 8 (OriginLab,Northampton, MA).
Apoptosis was assessed by dual staining of cells with PIand PhiPhiLux (Oncoimmunin, Gaithersburg, MD, USA)an indicator of active caspase-3 followed by analysis on FACS exactly as described previously []. Activation ofcaspase-3 was additionally assessed by staining cells with a Substitutions at the α-carbon in MDMA can augment rabbit antibody specific for the active form of caspase-3 cytotoxic performance against L3055 Burkitt's lymphoma (BD Pharmigen, Oxford, UK), followed by FACS analysis; non-immune rabbit IgG (control) was from Sigma Aldrich.
Cells were pre-treated using the FIX and PERM kit for The first generation of α-substituted MDMA analogues intracellular staining (Caltag, Invitrogen, Paisley, UK) synthesized contain either novel alkyl/cycloalkyl groups according to the manufacturer's instructions. Cleavage of (compounds 1–5) or, in the case of compound 6, a phenyl poly(ADP-ribose) polymerase-1 (PARP-1) as determined by substituent. When assessed for anti-lymphoma potential Western blot and mitochondrial membrane permeability as against L3055, a prototype early-passage BL cell line, assessed by JC-1 staining were performed as detailed compound 6 was the most potent (approximately 10-fold > elsewhere Bcl-2 protein content of cells was deter- MDMA) both in inhibiting 3H-thymidine incorporation mined by Western blot as described previously [].
(data not shown) and in its cytotoxic efficacy (Fig. ):pIC50=4.12±0.03 versus pIC50=3.39±0.09 for MDMA.
Treatment with antioxidants Compound 6 therefore formed the template on which todesign the next generation of compounds in the quest for a Cells were pre-treated with catalase (Sigma Aldrich, Dorset, lymphoma therapeutic based on MDMA.
UK) for 1 h or PEG-catalase (Sigma Aldrich, Dorset, UK)for 1.5 h before seeding cells onto 96-well plates containing Larger aromatic α-substituents enhance cytotoxic potential MDMA/MDMA analogue. Cells at final density at 105/ml towards L3055 cells were incubated with drug for 24 h and cell viability wasassessed by PI uptake analysed by flow cytometry.
The second generation of α-modified MDMA analogues allcontain aromatic rings (two in the case of compounds 16, 17, 18), apart from compound 7, which possesses acyclohexyl group (Fig. ). The substituents in this series Estimates of lipophilicity were obtained from the of MDMA analogues differ from each other with respect to "average log P" value output by the applet ALOGPs three-dimensional structure, rigidity, and electron density:


Hill coefficient ± SEM
3.39 ± 0 09
3.89 ± 1 12
3.10 ± 0.02
3.36 ± 0 06
4.56 ± 0 98
3.15 ± 0 01
3.4 ± 0 93
3.42 ± 0.03
4.79 ± 0.77
3.20 ± 0.07
3.40 ± 0.81
4.12 ± 0.03
4.65 ± 0.31
Fig. 1 Cytotoxic efficacy of MDMA and Series 1 (first generation) b typical concentration-response curves showing cytotoxic perfor- MDMA analogues versus L3055 Burkitt's lymphoma cells.
mance of MDMA and Series 1 analogues against the L3055 BL cell a Chemical structure of MDMA with α-carbon (α-C) indicated (and line. Cells were cultured at 5 x 105/ml with MDMA or indicated in tabulated right hand panel) MDMA analogues with the first analogue at concentrations shown for 48 h prior to measuring iteration of α-C substituents constituting Series 1 compounds 1–6 as cytotoxicity by PI uptake using flow cytometry. Results are repre- shown, together with calculated pIC50 ± SEM and Hill coefficients ± sented as the mean of three independent experiments ± SEM in terms SEM from response curves as generated in (b) with number of of the percentage of cells remaining viable with respect to vehicle (no separate experiments performed with each compound given as ‘n'; the benzene ring possessing all six carbons within one acceptor and therefore adds to the hydrophobicity of the plane (sp2-hybridized) by contrast to the cyclohexyl group, molecule [Metabolic stability is also increased by where the carbon atoms are sp3-hybridized and therefore the inclusion of fluorine.
non-planar and conformationally flexible. Compound 8 has Compounds 16, 17 and 18 have much larger hydrophobic an α-benzyl group and thus an additional sp3-hybridized substituents at the α-position of MDMA, and therefore carbon between the main carbon chain and the aromatic α- increased lipophilicity. The naphthyl group (compounds 16 substituent. This provides additional flexibility compared to and 17) is highly rigid as all the carbon atoms are positioned phenyl substituents, and extends the aromatic ring from the in one plane, whereas the biphenyl group differs from main chain, exploring the depth of a putative hydrophobic compound 6 by the addition of a para-phenyl group and pocket in the target receptor(s).
therefore both of the benzene rings are able to rotate around Compounds 9, 10 and 11 are more polar than their parent the axis of the bond between them.
(6) due to the addition of a methoxy group. The lone pairs From results presented in Fig. it can be noted that from of electrons make the methoxy oxygens hydrogen bond the second generation of MDMA analogues modified at the acceptors, and also increase the electron density in the α-carbon, compounds 16–18 were by far the most potent aromatic ring. Compounds 9–11 differ only in the position regards cytotoxicity towards L3055 cells; compounds 17 of the methoxy group. Similarly, compounds 13, 14 and 15 and 18 being the most efficacious and equipotent with a possess ortho-, meta-, and para-methyl groups, respectively, pIC50=5.18±0.03 and 5.22±0.08; representing a 10-fold exploring steric tolerance within the binding site(s). The and 100-fold improvement over compound 6 and MDMA ortho-substituents in 9 and 13 are also likely to reduce the respectively. Similar rank potency of these analogues was range of low energy conformations available to the side observed when assessed for their capacity to inhibit 3H- thymidine incorporation into L3055 cells (data not shown).
Compound 12 contains fluorine in the para-position It should be noted that all compounds tested for which reduces electron density in the aromatic ring but concentration-dependent cytotoxicity generated steep otherwise is very similar to a hydrogen atom (i.e. an response curves yielding relatively high Hill coefficients isosteric replacement). Although the fluorine atom has three (Figs. and ) suggesting deviation from simple mass lone pairs of electrons, it is a very poor hydrogen bond interaction ].


Hill coefficient ± SEM
4.10 ± 0.04
6.56 ± 0.43
4.16 ± 0 03
5.92 ± 0 58
4.04 ± 0.04
4.82 ± 0.38
4.10 ± 0.01
6.05 ± 0.16
4.16 ± 0.02
5.60 ± 0.73
4.20 ± 0.02
5.89 ± 1.12
4.20 ± 0.05
6.68 ± 073
4.29 ± 0.05
5.05 ± 0.17
4.37 ± 0.03
5.69 ± 0.27
4.90 ± 0.02
4.04 ± 0.74
5.18 ± 0.03
4.38 ± 0.73
5.22 ± 0.08
5.05 ± 0.97
Fig. 2 Cytotoxic efficacy of Series 2 (second generation) analogues versus L3055 Burkitt's lymphoma cells. As in Fig. but here with Series 2compounds 7–18; MDMA again included for comparison Cytotoxic efficacy of selected α-substituted MDMA (primarily detecting active caspase-3) and propidium iodide analogues towards B-cell lines of different malignant (plasma membrane permeability) staining, L3055 BL cells transfected with empty vector showed classic progressionfrom early to late apoptosis over the course of the 6 h The constituent cells of B-cell lines from a diverse monitored (Fig. ). While at the fixed concentration of the range of malignancies were treated with MDMA and six analogues used L3055-Bcl-2 cells again showed a degree of of the α-substituted MDMA analogues (selected accord- resistance to their cytotoxic actions, nevertheless the death ing to their activity versus sensitive L3055 cells and that occurred failed to register an ‘early apoptosis' stage at applied at a concentration at or close to their maximal any time point as indicated by cells staining as PhiPhiLux+/ cytotoxic performance against this cell line) then PI-. While no PhiPhiLux positivity was developed with analysed for remaining viability (Fig. Given the compound 6, compound 18 progressively moved a portion resistance often afforded to therapeutic regimens by of cells to what is conventionally considered a ‘late dysregulated/overexpressed BCL2 in B-cell lymphoma, apoptotic' stage: PhiPhiLux+/PI+. However, assessing cells were simultaneously assessed for Bcl-2 protein engagement of the apoptotic machinery by alternative more content (vs calnexin standard) by Western blotting. The direct methods gave no evidence for compound 18 origin of cells spanned patients additional to those provoking this pathway in L3055-Bcl-2 cells. Thus the diagnosed with BL (L3055 series; KHM2B): precursor specific detection of active caspase-3 by antibody revealed acute lymphoblastic leukemia (LILA), pro-lymphocytic leu- its appearance in response to compounds 6 and 18 in kemia (JVM2), mantle cell lymphoma (Rec-1; NCEB-1), L3055-VC but not in L3055-Bcl-2 cells (Fig. Like- primary mediastinal B-cell lymphoma (K1106), diffuse large wise, the cleavage of poly (ADP-ribose) polymerase B-cell lymphoma (K422; DoHH2), multiple myeloma (PARP) ], as shown occurring in L3055-VC cells with (KMS11; H929). Immortalized B-cell lines generated from the well characterized apoptosis-inducing agent anti-IgM, the peripheral blood of three donors by transformation with was also seen on application of MDMA and here more Epstein-Barr virus (EBV) were also included (HCD1; AT-AY; potently with compounds 16, 17, and 18 whereas L3055- ViWo)—EBV being invariably linked to endemic BL, PTLD Bcl-2 cells revealed little if any PARP cleavage in response (post-transplant lymphoproliferative disease) and a high to any of the agents applied (Fig. JC-1 staining to proportion of HIV-associated lymphoma.
indicate collapse of mitochondrial potential similarly MDMA and its analogues were set at concentrations supported the different routes to cell death by analogs displaying maximal/near-maximal impact on L3055 cell depending on the expression of Bcl-2 in L3055 BL cells viability to serve as a reference. At these concentrations (data not shown).
each of the analogues tested displayed (albeit a varyingdegree of) cytotoxicity against the spectrum of malignan- Mechanisms and pathways to lymphoma cell killing cies included. The best compound (18) showed a consis- by selected α-substituted MDMA analogues tently substantive impact against each of the subtypes. Asreported previously Bcl-2 content showed some degree Depending upon cell type and system studied, MDMA has of correlation with a cell's ability to resist killing from been purported to provoke toxicity via a diverse array of MDMA. With each of the analogues, however, there wasscant correlation between Bcl-2 protein level and extent of Fig. 3 Cytotoxic performance of MDMA and selected analogues b against B-cell lines of different malignant derivation and relation- cytotoxicity observed (Fig. ). To assess the influence of ship to Bcl-2 expression. a Cells from lines as shown plated at 5 x Bcl-2 directly, a detailed concentration-dependent response 105/ml and cultured for 24 h with compounds indicated prior to was established for the cytotoxic efficacy of the analogues assessing (absolute) % viability of population as in Fig.
against L3055 cells transfected with empty vector versus Concentration of drug applied as follows: MDMA, 2000 μM;compound 6, 500 μM; compound 12, 250 μM; compound 15, cells expressing the BCL2 transgene. The latter were only 125 μM; compound 16, 31.25 μM; compound 17, 31.25 μM; marginally more resistant (approximately a single log2 compound 18, 31.25 μM. Below is shown representative western difference) to each of the analogues than cells negative for blot analysis of Bcl-2 protein levels amongst the lines together with Bcl-2 expression (Fig. ). This was consistent for cells calnexin blotting control. Next are shown regression plots with Rvalues generated from remaining % viability in response to plated at relatively low or high starting density.
compound versus relative Bcl-2 content amongst the lines tested; bconcentration-response curves to compounds of L3055 cells carrying Mode of cell death induced by selected first and second empty vector (L3055-VC) or a Bcl-2 transgene (L3055-Bcl2). Cells were generation α-substituted MDMA analogues plated at two different starting densities, 105 cells/ml and 5x105 cells/mlas indicated, and incubated with compound for 24 h prior toassessing viability/cytotoxicity as in Fig. Results represent the When assessing cell integrity in response to compound 6 at mean of three independent experiments ± SEM given as % viability 500 μM and compound 18 at 31.25 μM by dual PhiPhiLux relative to vehicle control







compound 6
compound 18
f respo
o
% o

time of incubation [hours]
compound 6
compound 18
100 101 102 103 104 100 101 102 103 104 100 101 102 103 104 Fig. 4 Mode of cell death in L3055-VC and L3055/Bcl2 cells in generate an estimate of lipophilicity where the output value response to MDMA analogues 6 and 18. a Cells from L3055 variant is known as "average log P" [In brief, the average log lines indicated were cultured with compound 6 (500 μM) or P value is the simple average of log P estimates determined compound 18 (31.25 μM) for 6 h before dual staining with PhiPhiLux(PPL) and PI (upper graph; dot plots) revealing four subpopulations of using eight different models. A plot of average log P versus cells: PIlo/PPLlo = viable (bottom left quadrant), PIlo/PPLhi = early the pIC50 value of cytotoxic performance (including SEM apoptotic (bottom right), PIhi/PPLhi=late apoptotic (top right), and values for both variables) was constructed and a curve was PIhi/PPLhi=necrotic (top left). The lower set of graphs illustrate similar fitted by weighted linear regression as detailed in Fig. analyses arising from exposing cells to the compounds over 1-6 h withthe data represented as the % of cells arising in each quadrant at the When operating at pH values that favour ionisation of the different times of harvest: viable marked in white, early apoptotic compounds under consideration, as in this case (pH=7.4), marked light grey, late apoptotic marked dark grey, necrotic marked log P values should be corrected using the pKa to account black. Data are the mean of three independent experiments (n=3) ± for the increase in aqueous solubility of the ionised form.
SEM with the values shown obtained after subtracting vehicle control;b L3055-VC and L3055-Bcl2 cells at 5x105/ml treated for 2 h with However, uncorrected log P values were used here as the compound 6 at 500 μM and compound 18 at 31.25 μM (black line) or pKa values (dictated by the shared amino group), and vehicle control (shaded) then stained with antibody to active caspase-3 therefore the correction factors, were expected to be very with intensity of staining analysed by FACS. A representative example similar for all compounds, thus, not affecting the rank order of two independent experiments is shown. c Western blot analysis ofPARP cleavage in L3055-VC and L3055-Bcl2 cells plated at 106/ml obtained. Furthermore, the use of uncorrected log P and treated for 6 h with MDMA at 2000 μM or compounds 16, 17 or predictions to estimate lipophilicity has been shown to be 18 at 31.25 μM; upper 117 kDa band = intact PARP, lower 97 kDa more reliable, as it avoids the introduction of a second band = cleaved PARP; anti-IgM (25 μg/ml) is a positive control source of error associated with the calculation of pK treatment known to signal PARP cleavage in L3055-VC cells via cell surface BCR. This experiment was performed twice with a represen- Bearing these considerations in mind, a persuasive correla- tative example shown tion emerges with r2=0.88 as seen in Fig. not necessarily mutually exclusive pathways as reviewedfor example in ]. Here, the possible involvement of monoamine transporters in delivering MDMA and itsanalogues to engage intracellular pathways for lymphoma Analogues of MDMA with modified α-substituents were B-cell killing was first investigated. For this, L3055-VC cells iteratively designed and synthesised, and found to be up to were pre-treated with a range of monoamine transporter 10-fold (first generation) and 100-fold (second generation) (MAT) inhibitors targeting: SERT (fluoxetine), SERTand NET more potent than the parent amphetamine derivative at (clomipramine and imipramine) or DAT (GBR12909); and promoting lymphoma cell death: the goal and driver to this also with cocaine, which blocks all three MATs. From results study. Impressively, forced over-expression or high consti- detailed in Fig. a it can be seen that none of the MAT tutive levels of anti-apoptotic Bcl-2 failed to protect, to any inhibitors afforded protection against lymphoma cell toxicity significant degree, the anti-lymphoma actions of the induced by MDMA or two of its more potent analogues analogues; this despite their ability to promote apoptotic indicating that they are unlikely to be serving as conduits to cell death in Bcl-2-deplete cells. Thus, in the face of high- the compounds' actions in this regard.
level Bcl-2, death still occurred but in a caspase-3-, PARP- Since MDMA has been widely reported to mediate independent fashion that was similarly independent from a toxicity via direct or indirect production of reactive oxygen collapse in mitochondrial membrane potential. It should be species (ROS), L3055-WT and -VC cells were pre-treated noted, however, that while analogues of MDMA efficiently with enzymes that either degrade superoxide (O •– generated active caspase-3 within 4–6 h of exposure in the oxide dismutase, SOD) or H2O2 (catalase). However, bulk of native L3055 BL cells, a majority of their Bcl-2- neither SOD (data not detailed) nor catalase were seen to overexpressing counterparts were still alive at 6 h. Thus, at protect tumor B cells from the detrimental effect of the least for BL, if translated to an in vivo therapeutic, these MDMA analogues studied, whereas catalase efficiently compounds show potential to reduce tumor burden through reversed cell killing provoked by H2O2 (Fig. ). Cells efficient apoptotic clearance without the attendant inflam- pre-treated with these enzymes but now conjugated to matory side effects of necrotic death.
polyethylene glycol (PEG) to facilitate cell uptake , Importantly, improved cytotoxic performance against similarly failed to protect (Fig. and data not detailed).
lymphoma cells does not simply reflect a generally Finally, we examined the possibility that increased enhanced, non-specific toxicity profile of the compounds.
lipophilicity may be associated with the enhanced anti- A companion study shows that the most active compound lymphoma performance of the more potent analogues in versus lymphoma cells from Series 1 (compound 6) and this study. To explore this, we used the online program, two of the even more active ones from Series 2 (compounds ALOGPs 2.1, which accepts a structural formula to 16 and 17) are in fact less toxic than MDMA to SH-SY5Y: compound 15
compound 16
at 10µMa
catalase at 1500U/ml
catalase at 1000U/ml
PEG-catalase
at 100U/ml

catalase at 500U/ml
ability %
at 62.5µ
Fig. 5 Investigation of potential pathways through which MDMA H2O2 or compounds indicated (compound 6, 500 μM; compound 12, analogues elicit cytotoxicity in L3055 cells. a Impact of monoamine 250 μM; compound 15, 125 μM; compounds 16, 17 and 18, transporter (MAT) inhibitors. L3055 cells at 105/ml were pre-incubated 31.25 μM) and then culturing for 20 h prior to assessing viability as with MAT inhibitors for 1 h before adding MDMA or compounds 15 above; c Influence of scavenging intracellular ROS with PEG- and 16 at 125 μM and 31.25 μM respectively then culturing for 20 h catalase. L3055-VC cells at 105/ml were pre-treated with PEG- prior to assessing cell viability as in Fig. b Influence of scavenging catalase for 1.5 h before adding H2O2, MDMA, or compound 6 at extracellular ROS with catalase. L3055 cells at 105/ml were pre- concentrations indicated and then culturing for 24 h prior to assessing treated with catalase at concentrations shown for 1 h before adding a catecholaminergic neuroblastoma cell line that is used to The literature around MDMA and the mechanisms model MDMA neurotoxicity. The same study also shows underlying its toxicity is large, varied and occasionally compound 6 having diminished psychoactivity when contradictory –the cell system, cellular origin, compared with MDMA in the prepulse inhibition of the animal species, drug concentration and other elements all acoustic startle reflex test in Wistar rats []. Further- contributing confounding factors. Here we scrutinized more, in the present study, while constituent cells of several of the major candidate pathways proposed for derived lines from all B-cell malignancies proved suscep- MDMA for their potential contribution to the toxic action tible to one or more of the analogues tested, the relative of the analogues versus B-lymphoma cells. The current level of sensitivity to a given compound could be quite study was predicated on the discovery that B lymphoma different depending upon the cell line targeted indicating a cells express both SERT and DAT, the transporters for degree of selectivity in the compounds' actions against serotonin and dopamine, respectively, and to which MDMA lymphoma cell subtypes.
binds in the human with high affinity as it also does to NET, MDMA and its analogues in this respect. Similar failure ofPEGylated SOD and catalase to inhibit death deliveredfrom the compounds under study equally argued againstintracellular ROS formation contributing to the lymphomacell killing observed.
If not through ROS generation or from entering via monoamine transporters, how are MDMA and its rede-signed analogues attacking the lymphoma cells? Screeningagainst the sensitive L3055 cell line revealed no significantdifference in the cells' response to compounds containing α-subsituents with either different steric (13–15, 16–17) orstereoelectronic (9–12) properties. Instead, the addition offurther aromatic rings, thereby increasing the size ofsubstituents at the α-carbon of MDMA, appeared aunifying factor to increasing potency: i.e. compound 6 inSeries 1 with a single aromatic ring and compounds 18, 17and 16 in Series 2 with two aromatic rings being the mostpotent from each iteration. That said, the non-aromaticcyclohexyl substituent confers equipotency to phenyl. Size Fig. 6 Calculated liphophilicities of MDMA and analogues versus of the α-subsituent and overall lipophilicity of the com- cytotoxic performance. Relationship between average log P ± SEM pound may therefore be primary determinants of potency. In and pIC50 ± SEM for MDMA (□), alkyl α-substituted analogues 1–5 an earlier study we noted from a seemingly otherwise (◊), monocyclic aromatic α-substituted analogues 6–15 (Δ) and disparate set of compounds capable of killing lymphoma polycyclic aromatic α-substituted analogues 16–18 (○). The curvewas fitted to all data points shown using weighted linear regression cells the shared feature of being cationic amphiphiles that gave an r2 value of 0.88 This class of compounds has the capacity to disrupt cellularmembranes, as do amphiphilic molecules generally. Greater the norepinephrine transporter [Against serotoner- lipophilicity also enhances entry into cells, thereby increasing gic JAR cells for example, MDMA's cytotoxicity is the effective intracellular concentration, and entropically delivered via SERT: being inhibited by imipramine, a favours complex formation (the hydrophobic effect) and thus, monoamine transporter blocker with highest affinity for potentially, affinity of drug for intracellular receptors/targets.
SERT [The capacity of serotonin to drive apoptosis in Numerous studies indicate a selectivity of lipophilic com- BL cells is reversed by SERT blockade with e.g. the pounds for impacting rapidly proliferating cancer cells over selective serotonin reuptake inhibitor, fluoxetine []. How- normal cells ] and others show, amongst related series ever, adopting the approach of pharmacological transporter of compounds, a clear correlation between anti-proliferative blockade in this work, neither MDMA nor two of its more activity/cytotoxicity and degree of lipophilicity potent redesigned analogues were seen to be delivering When this relationship was examined for the newly their toxic hit to lymphoma cells via any of the three synthesized analogues of MDMA, a strong correlation was monoamine transporters probed. Moreover, Montgomery indeed observed with anti-lymphoma potency closely track- and colleagues [examining the action of MDMA and ing calculated lipophilicity, at least for those compounds with several MDMA analogues on 5-HT and NA uptake in cells aromatic α-substituents. We are currently exploring pre- transfected with SERT or NET reported a Hill coefficient for cisely how this physiochemical property of the compounds inhibition by MBDB (our compound 3) of 1 for both translates mechanistically to improved lymphoma killing HEK-SERT and PC12-NET compared to that generated in order to assist further rational design of MDMA from its anti-lymphoma action in this study of >3. Others analogues as anti-neoplastics.
have shown that MDMA is capable of promoting cell death Irrespective of relative anti-lymphoma potency all com- independently of SERT expression or activity [A pounds including MDMA generated steep inhibition curves second major mechanism for MDMA's cellular toxicity in with Hill coefficients >3 indicating a high degree of other systems was similarly ruled out here for both the lead cooperativity in their action. Similar behaviour has been compound and the more (anti-lymphoma) potent synthe- observed from SSRIs and tricyclic antidepressants (Sera- sized analogues: namely the, direct or indirect, production feim Blood 2003; Meredith FASEB J 2005) and at least of reactive oxygen species. Inhibitors of extracellular ROS with the former class of compound we know that cell death which have previously been shown to reverse the anti- is preceded by the stimulation of Ca2+ entry. Preliminary lymphoma actions of dopamine ] did not protect against data (unpublished) indicate similarly altered Ca2+ flux in L3055 BL cells on exposure to MDMA and analogues 8. Nichols DE, Hoffman AJ, Oberlender RA, Jacob P 3rd, Shulgin AT (1986) Derivatives of 1-(1,3-benzodioxol-5-yl)-2-butanamine: studied here. As an alternative to cooperative binding at a representatives of a novel therapeutic class. J Med Chem defined molecular target, a possibility under consideration is that the lipophilic compounds undergo aggregate forma- 9. Shulgin A, Shulgin A (1991) Phenethylamines I have known and tion dependent upon a critical association concentration— loved: A chemical love story transform Pr Berkeley, California 10. Nash JF, Nichols DE (1991) Microdialysis studies on 3,4- perhaps established in situ within the lipid bilayer of the methylenedioxyamphetamine and structurally related analogues.
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atypical 5-HT3 receptor ligand, [3H]-BRL46470, labels additional5-HT3 binding sites compared to [3H]-granisetron. Br J Pharmacol This work was supported in part by Leukaemia and Lymphoma Research, UK, and the Ada Bartholomew Medical 14. Bohm HJ, Banner D, Bendels S, Kansy M, Kuhn B, Muller K, Research Trust, W.A. MNG and KDL were recipients of a UWA Obst-Sander U, Stahl M (2004) Fluorine in medicinal chemistry.
postgraduate scholarship and Australian Postgraduate Award, respec- Chembiochem 5:637–643 tively. DS-T was supported by an Arthritis Research UK Career 15. Smart BE (2001) Fluorine substutuent effects (on bioactivity). J Progression Fellowship. JG was in receipt of a Raine Visiting Fluorine Chem 109:3–11 Professorship at the University of Western Australia while writing 16. Cornish-Bowden A, Koshland DE Jr (1975) Diagnostic uses of the paper. The authors declare that they have no conflict of interest.
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Toxicology 192 (2003) 249–261 Reducing acute poisoning in developing countries—options for restricting the availability of pesticides Flemming Konradsen , Wim van der Hoek , Donald C. Cole , Gerard Hutchinson , Hubert Daisley , Surjit Singh , Michael Eddleston a Department of International Health, Institute of Public Health, University of Copenhagen, Panum, Blegdamsvej 3,

Microsoft word - februarypaperbcco gmmmg nts summary paper

North Manchester CCG Board Meeting – 11 February 2015 Dr Martin Whiting Paper prepared by: Dr Martin Whiting Dr Martin Whiting Sub-Committee consideration Chief Clinical Officer's Report Background papers and links to priorities/objectives: To provide an update to the board on strategic Purpose of the paper: developments within Greater Manchester.