Novel Synthetic PEGylated Conjugate of Î±-Lipoic Acid and Tempol

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A Novel Synthetic PEGylated Conjugate of #-Lipoic Acid and Tempol Reduces Cell Death in a Neuronal PC12 Clonal Line Subjected to Ischemia Adi Lahiani, Adel Hidmi, Jehoshua Katzhendler, Ephraim Yavin, and Philip Lazarovici ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.6b00211 • Publication Date (Web): 08 Aug 2016 Downloaded from http://pubs.acs.org on August 16, 2016

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ACS Chemical Neuroscience

A Novel Synthetic PEGylated Conjugate of α-Lipoic Acid and Tempol Reduces Cell Death in a Neuronal PC12 Clonal Line Subjected to Ischemia

Adi Lahiani 1, Adel Hidmi 1, Jehoshua Katzhendler 1 ,Ephraim Yavin 2 and Philip Lazarovici 1*

1

School of Pharmacy Institute for Drug Research, The Hebrew University of Jerusalem,P.O.Box

12065, Jerusalem 91120, Israel

2

Department of Neurobiology, The Weizmann Institute of

Science, Rehovot 76100, Israel

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ABSTRACT

α-Lipoic acid (α-LA), a natural thiol antioxidant and Tempol, a synthetic free radical scavenger are known to confer neuroprotection following ischemic insults both in in vivo and in vitro models. The aim of this study was to synthesize and characterize a conjugate of α-LA and Tempol linked by polyethylene glycol (PEG) in order to generate a more efficacious neuroprotectant molecule. AD3 (α-Tempol ester-ω-lipo ester PEG) was synthesized, purified and characterized by flash chromatography, reverse phase high pressure liquid chromatography and by 1H nuclear magnetic resonance, infrared spectroscopy and mass spectrometry. AD3 conferred neuroprotection in a PC12 pheochromocytoma cell line of dopaminergic origin, exposed to oxygen and glucose deprivation (OGD) insult measured by LDH release. AD3 exhibited EC50 at 10 µM and showed a 2-3 fold higher efficacy compared to the precursor moieties, indicating an intrinsic potent neuroprotective activity. AD3 attenuated by 25% the intracellular redox potential, by 54% lipid peroxidation and prevented phosphorylation of ERK, JNK and p38 by 57%, 22% and 21%, respectively. Cumulatively, these findings indicate that AD3 is a novel conjugate that confers neuroprotection by attenuation of MAPK phosphorylation and by modulation of the redox potential of the cells.

KEYWORDS: Ischemia; Antioxidants; Neuroprotection; PC12 pheochromocytoma cells; Lipid Peroxidation; MAP Kinases.


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INTRODUCTION

Diminished blood flow after stroke or traumatic injury to the brain is a leading cause for mental impairment and high morbidity in humans1,2. Restoration of circulation (reperfusion) accompanied by oxygen and glucose replenishment at the injured site, an area usually known as penumbra, may paradoxically accelerate the damage because of excessive reactive oxygen species (ROS) generated in the reperfused areas3. The latter generated from peroxidation of macromolecules such as DNA4 or certain membrane lipids5 result in activation of cellular signaling pathways, each with a potential to exacerbate ischemia-induced

apoptosis6 and

accelerated necrotic cell death7. Concomitant to these events , there is an activation of the cellular antioxidant machinery which should confer neuroprotection8, but the signals are usually below the required levels9. Therefore, a search for alternative routes to enhance neuroprotection is of great importance10. For example inhibition of mitogen activated protein kinases such as ERK1/211, JNK 1/212 and p-3813 by a variety of agents, including low molecular weight, natural and/or synthetic antioxidants have been suggested to possess beneficial effects following ischemic brain injury14. Acute administration of such agents or cocktails containing complementary antioxidants15 may constitute an additional route for therapy. One such potential antioxidant is α-lipoic acid (α-LA, 1, 2-dithiolane-3-pentanoic acid). α-LA is a low molecular weight natural antioxidant that is absorbed from the diet and is known by its ability to cross the blood brain barrier (BBB). α-LA is taken up and reduced by brain cells to dihydrolipoic acid (DHLA)16 which is subsequently also exported to the extracellular medium; hence, protection is provided to both intracellular and extracellular compartments. α-LA has been shown to be a key antioxidant, to regenerate through redox cycling of other antioxidants such as vitamin C, and to raise intracellular glutathione levels. The latter cannot be directly administered since it does not cross the BBB17. Given its clinical safety18 α-LA is considered a potential molecule for cerebral oxidative insults treatment. Early studies in both humans and experimental animal models have found that α-LA could decrease redox potential markers of oxidative stress following stroke or traumatic brain damage19. Administration of α-LA to rodents has been also demonstrated to reduce the damage that occurs after ischemia–reperfusion injuries in the central20 , and peripheral nervous system21. α-LA has been found to prevent hydrogen peroxide-induced neuronal damage22, protect neurons towards neurotoxicity in Parkinson's23 and reduce oxidative damage following stroke through enhancing the levels of superoxide dismutase type 2 (SOD2)20. α-LA is approved as a drug against diabetes comorbidities and since 1966 is available by prescription24. 3 Environment ACS Paragon Plus


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Tempol (4-hydroxy 2,2,6,6,-tetramethylpiperidine-1-oxyl) on the other hand, is a nontoxic25, synthetic nitroxide compound with antioxidant properties, originally used in clinic for imaging procedures26. In contrast to other antioxidants that are generally active against only one species of radicals, Tempol possesses superoxide dismutase (SOD)-like activity and was shown to also modulate nitric oxide (NO) levels, reduce metal levels, and react with peroxyl and carbon-centered radicals, as well as dismutate oxygen radicals27. Most striking, it can restore its own antioxidant capacity, in contrast to other antioxidants, which ultimately exhaust their potency27. Tempol can cross the cell membrane28 where it exerts important intracellular antioxidant action in contrast to other antioxidants, which do not penetrate effectively cell membranes and thus have only limited intracellular potency. In neuronal cell cultures such as PC12 cells, Tempol has been shown to confer neuroprotection towards 1-methyl-4phenylpyridinium ion-induced neurotoxicity (Parkinson's model)29 and attenuate cocaineinduced cell death through decreased oxidative damage30. In a mouse model of traumatic brain injury, Tempol protected brain tissue from ischemic damage31 and was also effective in a rat model of stroke and transient focal ischemia32. Currently, Tempol is used in the clinic as a topical drug to prevent radiation-induced alopecia33. The aim of this study was to generate a bifunctional conjugate composed of α-LA and Tempol antioxidants and examine the outcome of the drug in a model of experimental ischemia insult. To this end, we have used polyethylene glycol (PEG), one of the better biocompatible, FDA-approved, polymers. PEG conjugation is well known for the improved biological properties of many bioactive substances in terms of extended circulating life time, increased resistance to degradation and lack of immunogenicity and toxicity34. To generate drug conjugate, monofunctional PEG with only one reactive terminal group, such as hydroxy, amine, thiol, aldehyde or carboxylic acid, have been used. Attachment of PEG to antioxidant compounds has been widely used in clinical research and a variety of biomedical applications35. In our laboratory, we screened and characterized the neuroprotective effect of three different groups of compounds: (i) NO donors; (ii) antioxidants based on α-LA linked to alkyl or PEG carrier of several sizes, as esters or amides and (iii) bifunctional compounds, based on αLA linking to NO donors36. In the present study, we have taken it further and synthesized a bifunctional molecule termed AD3 (α-Tempol ester-ω-lipo ester PEG) and characterized its neuroprotective activities in an established ischemic model of pheochromocytoma PC12 cells exposed to oxygen and glucose deprivation (OGD) followed by reperfusion37. We found a significant increase in the efficacy of AD3 compared to the individual moieties and we now demonstrate that its neuroprotective effect is causally correlated with a decrease in cell redox potential, reduced lipid peroxidation and an attenuation of the MAPK phosphorylation activity. 4 Environment ACS Paragon Plus

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This study proposes AD3 as a lead compound towards development of antioxidantneuroprotective drugs for therapy of ischemic disorders.

RESULTS Synthesis and Chemical Characterization of AD3. AD3 was synthesized through a 5 step reaction as illustrated in Figure 1. Each intermediate product was purified by silica gel chromatography and reverse phase high pressure liquid chromatography (HPLC) and analyzed by 1H NMR and ESI-MS or MALDI-TOF-MS. In the first step, polyethylene glycol (4 kDa) (Fig 1, 1a) was converted into α-hydroxy-ωDMT-PEG (Fig 1, 1b) (yield = 71 %), 1H NMR (300 MHz, DMSO-d6): δ (ppm) 7.1-7.65 (m,13 H,Ar(DMT)), 4.6(t,2H,-CH2-OH), 3.92(s,6H,OCH3(DMT)),3.44-3.91(m,354H, (OCH2CH2)), 3.06(m,2H,-CH2OH. Calcd for C202H382O93 4302.9 , found 4302.7 g/mol. In the second step, 1b was subseqently carboxylated to α-carboxyl-ω-DMT-PEG (Fig 1, 1c) (yield= 83%), 1H NMR (300 MHz, DMSO-d6): δ (ppm) 8.1 (s, 1 H, COOH), 7.0-7.6 (m, 13 H, Ar (DMT)), 4.1(s, 2 H,-CH2-COOH), 3.92 (s, 6 H, OCH3 (DMT)), 3.4-3.9 (m, 350 H, (OCH2CH2)). Calcd for C202H380O94 4316.9 , found 4316.9 g/mol. In the third step, Tempol was conjugated to 1c to generate α-DMT-ω-Tempol PEG intermediate, named AD5 (yield=87%), 1H NMR (DMSO-d6): δ (ppm) 7.0-7.6 (m, 13 H, Ar (DMT)), 3.95 (s, 2H,-CH2-OC-O-), 3.85 (s, 6 H, OCH3 (DMT)), 3.4-3.8 (m, 350 H, -(CH2-CH2O)n and 1H, Tempol)), 1.8-1.65 (m, 4H, Tempol) , 1.21 (s, 12H, Tempol). Calcd for C211H396NO95 4455.1 , found 4455.1 g/mol. In the fourth step, DMT was removed from AD5,generating α-Hydroxy-ω-Tempol-PEG named AD4 (yield=90%), 1H NMR (DMSO-d6): δ (ppm) 4.6 (t,1 H,-CH2-OH), 3.95 (s, 2H,CH2-OC-O-), 3.85 (s, 6H, OCH3 (DMT)), 3.4-3.8 (m, 350H, -(CH2-CH2-O)n and 1H, Tempol)), 1.8-1.65 (m, 4H, Tempol) , 1.19 (s, 12H, Tempol). Calcd for C190H377NO93 4153.0 , found 4153.0 g/mol. In the fifth and last step, AD4 was coupled to α-LA with the cross linker DCC generating the final compund α-Tempol ester-ω-lipo ester PEG named AD3 (yield=76%), 1H NMR (DMSO-d6): δ (ppm) 4.1 (t, 2H, (-OC-O-CH2-CH2)), 3.45-3.87 (m, 350H, -(CH2-CH2-O)n , 1H, Tempol, (1H, H-S-S-) and (2H, -O-OC-CH2-CH2-O)), 3.17 (m, 2H,-S-S-2H), 2.39 ( m, 1 H, CH2-CH-S-), 2.31 (t, 2H, -CH2-CO-), 1.9-1.45 (m, 7H, -(CH2)3, CH2-CH-S-) and ( 4H, Tempol)), 1.22 (s, 12H, (Tempol)). Calcd for C198H391NO94S2 4342.0 , found 4342.0 g/mol.



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Synthesis and Chemical Characterization of AD3 Intermediates. AD3 is a PEG conjugate of two different active drugs each bearing discrete chemical structures, and therefore can be considered as a co-drug that possess pharmacological activity of its own. To clarify their relative contribution, each of the individual intermediates ,PEG-Tempol (AD4) and PEG-Lipoic acid (AD6),have been synthesized. The latter was synthesized in a 4-steps reaction as illustrated in Figure 2. First, compound 1b from Fig 1, was brominated to α-Bromo-ω-DMT-PEG (Fig 2 , 1d) (yield= 73%), 1H NMR (300 MHz, DMSO-d6): δ (ppm) 7.1-7.6 (m, 13 H, Ar (DMT)), 4.08 (t, 4H, O-CH2-CH2-Br, 3.90 (s, 6H, OCH3 (DMT)), 3.4-3.88 (m, 354H, (OCH2CH2)).FTIR (cm1

,KBr): 2881 (C-H), 1111 (CH2OCH2), 687 (C-Br). Calcd for C202H381BrO92 4396.8 , found

4396.4 g/mol. In the second step compound 1d was aminated to generate α-Amino-ω-DMT-PEG (Fig 2, 1e) (yield=80%), 1H NMR (300 MHz, DMSO-d6): δ (ppm) 7.0-7.6 (m,13H, Ar (DMT)), 3.92 (s, 6H, OCH3 (DMT)), 3.4-3.9 (m, 354H, (O-CH2-CH2)), 2.79 (t, 2H,-CH2-NH2). FTIR (cm-1, KBr): 3440 (N-H), 2891 (C-H), 1111 (CH2OCH2). Calcd for C202H383NO92 4332.9, found 4332.6 g/mol. In the third step, α-LA was conjugated to 1e to produce α-DMT-ω-lipoamide PEG (Fig 2, 1f) (yield=87%), 1H NMR (DMSO-d6): δ (ppm) 7.0-7.6(m,13H, Ar(DMT)), 3.92 (s,6H, OCH3(DMT)),3.70(t,2H,-NH-CH2-CH2-O),3.62(m,3H,-NH-CH2-CH2-O,1H,H-S-S-),3.4-3.7 (m,354H,(OCH2CH2)), 3.20(m,2 H,-S-S-2H),2.42(m,1H,CH2-CH-S-),2.38(t,2H,-CH2-CO-),1.901.45(m,7H,-(CH2)3,CH2-CH-S-). Calcd for C210H395NO93S2 4520.9 , found 4520.7 g/mol. In the last step, the final compound α-hydroxy-ω-lipoamide PEG named AD6 (yield=90%), 1H NMR (DMSO-d6): δ (ppm) 4.6 (t,1H,-CH2-OH), 3.70 (t,2H,-NH-CH2-CH2-O), 3.62 (m,3H,-NH-CH2-CH2-O,1H, H-S-S-), 3.4-3.7 (m, 354H,(OCH2CH2)), 3.20 (m,2H, -S-S2H), 3.05(m,2H,-CH2OH), 2.42 (m,1H,CH2-CH-S-), 2.38(t,2H,-CH2-CO-), 1.90-1.45(m,7H,(CH2)3,CH2-CH-S-).Calcd for C189H376NO91S2 4217.8 , found 4217.8 g/mol. Safety of AD3 and its Intermediates in PC12 Cells. The safety of the new synthetic molecules was verified after incubation for 24 h with PC12 cells under normoxic conditions and cell death estimated by the LDH release assay. Table 1 indicates that all tested compounds had a negligible impact on PC12 cells death (range of 3.8-5.1%) similar to the non-supplemented, negative control group. Therefore, these compounds were considered safe at the added doses.

AD3 Rescues PC12 Cells After ischemia. The ability of AD3 to exert its neuroprotective effect was tested in PC12 cells model exposed to oxygen and glucose deprivation (OGD) followed by reoxygenation (ischemia model). Briefly, monolayer cultures of PC12 cells were exposed to 4 h 6 Environment ACS Paragon Plus

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OGD followed by 18 h reoxygenation in the presence of either AD3 (10 µM), intermediate synthetic derivatives such as AD4, AD5 (10 µM), or the parental compounds Tempol at 1500 µM, a concentration previously found most effective as neuroprotective37 and α-LA (10 µM), before or after the OGD insult (Fig. 3 panel A). It is evident that addition of 10 µM AD3 decreased by 35% cell death as measured by LDH release compared to control OGD-exposed but untreated cells. Tempol or α-LA were each significantly less effective compared to AD3. Addition of the three antioxidants either before or after OGD did not change significantly the outcome, indicating that the damage of the cells commenced mainly during reperfusion. In contrast, AD4 and AD5, both intermediary derivatives of AD3, were ineffective. The neuroprotection index (NP-index) shown in Fig 3 (panel A’) a measure of rescue efficacy37, indicated a robust neuroprotection by AD3 with an NP-index of about 0.4, independent of the time of treatment. This neuroprotective effect was 1.5-3 and 1.5-2.5 fold higher compared to the NP-index values of α-LA and Tempol, respectively (Fig. 3, panel A'). In a different set of experiments, we have investigated the relationship between AD3-induced neuroprotection and duration time of the OGD insult, arbitrarily divided into 3 subgroups depending on the duration of OGD insult, to low (10-30%), medium (30-60%) and high (60-90 %) cell death attained by 2, 4 and 6 h of OGD insult, respectively. As illustrated in Fig 3 (panel B), AD3 acted very effectively to provide relative protection to medium and highly-insulted cultures as attested by the high NP-index (Fig. 3 panel B’). Notable, at 4 h OGD, 10 µM AD3 conferred 2 and 2.3 fold higher neuroprotection compared to Tempol and α-LA, respectively. At the highest insult group (6 h OGD), AD3 was 2.6 fold more protective than 1500 µM Tempol. Cumulatively, these findings strongly support the hypothesis that AD3 is a better neuroprotectant compared to its parental precursor compounds.

Concentration Dependency of Neuroprotective Effects of AD3 and its related Intermediates. Since AD3 is a conjugate composed of three molecular entities at non equal concentrations of α-LA, Tempol and PEG, we first clarified the potential contribution of the intermediaries PEG-Tempol (AD4), PEG-α-Lipoic acid (AD6) and PEG alone to neuroprotection. Figure 4 (panel A) shows that either 1500 µM Tempol or 10 µM α-LA addition resulted in a significant decrease of LDH release and a increase in NP-index value of 0.24 (Fig 4, panel A’). PEG alone did not confer neuroprotection to PC12 cells during the OGD insult. The Tempol-PEG-α-LA conjugate, AD3, was by far a better rescue agent than PEG-Tempol (AD4) and PEG-α-LA (AD6) conjugates at 10 µM each (Fig 4 panel B). Thus, the NP-index in the presence of (10 µM) is by far better than either α-LA or Tempol. Interestingly a mixture of conjugate consisting of AD4 and AD6 improved the NP-index substantially (Panel B’). 7 Environment ACS Paragon Plus


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Secondly, we evaluated the neuroprotective effect of individual moieties composing AD3. Therefore, the appropriate molecular ratios of the moieties have been calculated (4% Tempol; 5% α-LA and 91% PEG) and their relative amounts administered directly alone or as a mixture to the OGD-subjected PC12 cells. As noticed in Figure 4 (panel C) none of the individual components added i.e., α-LA (0.5 µM), Tempol (0.4 µM) and PEG (9.1µM) nor their combined, unconjugated mixture (10 µM) have had any rescue activity (Fig 4, panel C’). These findings highly suggest that the newly synthesized conjugate Tempol-PEG-α-LA (AD3) is endowed with intrinsic neuroprotective activity whereas its separate components are not effective at their actual molar concentration represented in 10 µM AD3. To further support this conclusion, we characterized in greater details the concentration dependency of the individual antioxidants on the NP-index of the PC12 cells. As illustrated by the dose response plotted on a semi-log scale (Fig 5 panel A), AD3 conferred the strongest neuroprotection effect (efficacy) with an EC50 of 10 µM. Notable at 0.1 µM, AD3 was the only effective neuroprotectant among the three antioxidants (panels A and B). While the apparent EC50 (potency) of Tempol and α-LA were similar to AD3, the NP-index of the two compounds remained low and as shown by the data, above 10 µM, both may have attained a relative plateau in comparison to AD3. PEG the third moiety composing the conjugate remained ineffective at all concentrations studied. The most plausible explanation for the higher efficacy of AD3 is the synergistic effect between Tempol, α-LA and PEG moieties within the conjugate. A dose response of AD3 in comparison with a mixture of individual moieties at appropriate molar ratio (4% Tempol; 5% α-LA and 91% PEG) was studied in details. As shown in Figure 5 (panel B), AD3 is by far a better neuroprotectant in spite of increasing levels of α-LA and Tempol in the mixture, amplifying the intrinsic activity of AD3 compared to the individual moieties.

Characterization of AD3 Effect on Cell Redox Potential, Lipid Peroxidation and MAPK's Phosphorylation after Ischemia. For this set of experiments, PC12 cells were loaded with the fluorescent redox sensitive dye DCHF-DA and exposed for 4 h to OGD followed by 0.5 h reoxygenation in the absence or presence of 10 µM AD3 (Fig. 6 panel A). Notable, the ischemic insult caused a remarkable increase of 94% in the fluorescence intensity above the normoxic levels. Treatment with 10 µM AD3 reduced fluorescence intensity by approximately 50%, indicative of a clear protection from oxidative stress38. The appearance of lipid peroxidation products was estimated by measuring TBARS, a marker known to be increased in PC12 cells after ischemia-induced oxidative stress37. Under normoxic conditions, the basal levels of TBARS secreted in the medium reached a level of 0.04 µM per cellular mg protein (Fig 6 panel B). These 8 Environment ACS Paragon Plus

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values were nearly doubled after 4 h OGD followed by 0.5 h reperfusion. Addition of AD3 decreased significantly the medium secreted-TBARS by 28%. Under the same, conditions. hydrogen peroxide (0.5 mM H2O2) caused as expected an increase of 2.2 fold in the mediumsecreted TBARS compared to non-supplemented normoxic cultures. Generation of ROS has been reported in the past to activate several MAPK kinases such as ERK, JNK and p-3839. Figure 7 illustrates the consequences of the ischemic insult on activation of MAPKs in PC12 cells and the outcome after addition of AD3. As previously described40 , the ischemic insult differentially stimulated phosphorylation of MAPK members in PC12 cells exposed to ischemia40. Treatment of cells with 10 µM AD3 reduced by 60%, 21% and 23% ischemia-induced ERK1/2, p-38 α/β and JNK1/2, phosphorylation respectively (Fig. 7). Ischemia-induced ERK1/2 phosphorylation was robustly reduced upon treatment with the powerful MEK1/2 inhibitor, UO-126, confirming the involvement of ERK1/2 activation during ischemia. A small decrease of ERK1/2 activation was noticed in the presence of α-LA (15%) while Tempol at either 10 (Fig 7- Tempol-L) or 1500 (Fig 7-Tempol H) µM, reduced ischemiainduced ERK1/2 phosphorylation by 50% and 60% respectively. These findings may indicate that the Tempol moiety of AD3 is the active site, presumably involved in the inhibitory effect of AD3 on ERK1/2 phosphorylation. 10µM AD3 also showed a significant inhibitory effect on JNK (23%) and p-38 (21%) phosphorylation, respectively.

DISSCUSION This study provides compelling evidence that a synthetic conjugate composed of two bifunctional antioxidant molecules provides rescue from cell damage after brief insult of OGD followed by reperfusion in monolayer cultures of adrenergic pheochromocytoma PC12, clonal cells. The natural thiol α-Lipoic acid (α -LA) and the synthetic nitroxide compound Tempol, both comprising the functional feature of the conjugate, have each been shown previously as potential low molecular weight antioxidants to alleviate the outcome of oxidative stress in a number of animal models20, 21, 31 and cell culture systems22, 30. The goal of the present experiments was to investigate the potential consequences resulting from bridging the two parental antioxidants into a new conjugate in providing a more effective and possibly synergistic protection. Biochemical characterization of the AD3 conjugate unambiguously demonstrated that the new molecule AD3 was endowed with synergistic neuroprotective activities which cannot be explained by the additive contribution of the individual α-LA and Tempol moieties alone. This is best illustrated in Figure 5, where the rates of neuroprotection show a dose dependency which in the case of AD3 is already evident at 0.1 9 Environment ACS Paragon Plus


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µM. At this concentration none of the other antioxidants exhibit protection. A best fit analysis of the data shows relative increasing slope values of 0.0034log(x); 0.0375log(x); 0.0570log(x) and 0.0967log(x) for PEG, α-LA, Tempol and AD3, respectively. However as evident from the plot at 100 µM while both α-LA and Tempol appear to attain a relative plateau, the AD3 conjugate still provides concentration-dependent neuroprotection and a steady slope. This is a unique property of AD3 and not of any of its intermediates (Figs 3, 4) suggesting that the crucial synergistic activity of AD3 deemed at the last step of synthesis (Fig 1). The question as to whether the PEGylation of α-LA could alone substitute for AD3 was also of relevance. However as clearly demonstrated (Fig 4 panel B) the resulting AD6 conjugate was equally inactive as other intermediates. In contrast, when a mixture of PEGylated Tempol (AD4) and PEGylated αLA (AD6) was added to the cells, the neuroprotection index slightly increased indicative that PEGylation may be an important feature of the conjugate. The PEG bridging used in the synthesis of AD3 was 4 kDa in molecular weight size and has been entirely inactive while in its unabridged form (Fig 5 panel A). One of the important properties of PEG remains its structural simplicity since it possesses an inert polyether backbone and two functional end-groups. Although, biological activities variations are occasionally encountered with PEGylated molecules (bond stability, surface charge, coupling conditions)41, in this study it did not affect the overall conjugate neuroprotective effect. In contrast, it appears that the molecular contribution of the PEG bridge in the conjugate may relate to enhanced cell membrane permeability, and provision of micro domains for improved interactions of evolving ROS and NOS with free radical targets all of which remains to be determined. Thus, it would appear that the lack or low neuroprotective effect of all other ester and amide derivatives of α-LA linked to NO donor that have been previously synthesized by us36 and examined in the PC12 ischemic model may be due to steric hindrances related to the presence of PEG or an alkyl linker containing more than six methylene groups that produce compounds resistant to esterases or amidases enzymes. In previous studies it has been suggested that mixing various doses of complementary antioxidants15 may attain a greater neuroprotection efficacy. In contrast, the present proof of concept indicates that a synthetic bi-functional conjugate containing two moieties with different antioxidants may represent a more suitable approach than a mixture of these antioxidants. Such bi-functional strategies were employed to generate successful co-drugs using amides or diamide linkage L-Dopa-Glutathione42 , L-Dopa-Carnosine43, LA-Ibuprofen44 and Tempo-NSAIDS45, These co-drugs exhibited bi-functional free radical scavenging activity and turned very effective in treatment of Parkinson or inflammatory diseases. Interestingly some of Tempo-NSAIDS have 10 Environment ACS Paragon Plus

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been proven to possess intrinsic pharmacological activity that was not intended in the original design of the co-drug, similar to present findings with AD346. An important issue remains as to whether AD3 falls in the co-drug or prodrug categories. A prodrug is usually defined as a chemical derivative of a drug molecule that upon enzymatic or chemical transformation in a biological system, will release the active parental drug, resulting in a pharmacological effect of therapeutic importance47. In the most common cases, a prodrug is synthesized from a parental drug by covalently linking with or without carrier, to a pharmacological inert moiety, which can be cleaved upon administration to release the parental drug48. When the moiety for coupling is not pharmacologically inert, i.e. another drug molecule is used; a co-drug (mutual prodrug) is synthesized49. Many of the co-drugs combining two different pharmacophores with similar or different pharmacological activity elicit synergistic action of help to target the parental drug to specific sites49. There are many prodrugs which possess pharmacological activity on their own without the need to liberate the parental drug and therefore, their action is characterized as intrinsic pharmacological activity46. With this background and based on the current experiments, mostly demonstrated via the reconstitution of the single components (Figs 4 and 5), we suggest that AD3 is a co-drug for all practical purposes. A second part of this study addresses the issue as to the possible signaling that may be affected in the ischemic cascade leading to PC12 cell ultimate death. In this study we provided evidences that addition of AD3 to ischemic PC12 cells, induced neuroprotection, an effect causally correlated in one hand with a decrease in the redox potential (Fig 6) and lipid peroxidation of the cells (Fig 6) and on the other hand, with strong inhibition of ERK1/2 (Fig 7) and differential attenuation of JNK1/2 and p-38 α/β phosphorylation activity (Figure 7). Interestingly, the addition of AD3 after the OGD insult (Fig 3) was still effective in reducing the damage outcome which may suggest that ROS may damage most during the reperfusion period. The

precise

cellular

and

molecular

mechanisms,

by

which

AD3

reduced

oxidative/nitrosative damage thus conferring neuroprotection, remain obscure and deserve further investigation. Since ROS/NOS are known to be responsible for the activation of MAPK's39, it is tempting to propose that AD3 by scavenging ROS/NOS (decrease in redox potential and lipid peroxidation) indirectly attenuated the hyper-activation of MAPK's responsible for the ischemic cell death measured resulting with neuroprotection. Clarification of biochemical pathways by which AD3 confers neuroprotection to PC12 cell exposed to ischemic insults, should provide a better understanding of the neuroprotective mechanisms involved in ischemia and may ultimately facilitate further development of AD3 neuroprotective lead compound. 11 Environment ACS Paragon Plus


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METHODS Chemicals, Antibodies and Reagents. Polyethylene glycol (HO-PEG-OH), sodium nitrite, Griess reagent, tert-butyl nitrite, H2SO4, HNO3, HCl, Na2SO4, methanol, ethanol, acetonitrile, silica gel, DL-α-α-LA, N-N’-dicyclohexyl carbodiimide (DCC),N-hydroxy succinimide (NHS), 1,3-propane diol, 1,3-propane diamine, 1,6-hexane diol,1,8-ocatane diol, 1,10-Decane diol were purchased from Aldrich-Sigma Chemical Co, (St. Louis, MO, USA) and used as received. Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, donor horse serum, penicillin, streptomycin, and collagen type 1 were all purchased from Beit Ha'emek (Afula, Israel). Anti-Phospho ERK, JNK and p-38 and anti-pan ERK, JNK and p-38 antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). MAPK inhibitor, UO-126, was purchased from Promega (Madison, WI, USA).

Synthesis of AD Compounds. Synthesis of AD3, AD4, AD5. 1. Synthesis of α-hydroxy-ω-DMT-PEG (1b). Polyethylene glycol (HO-PEG-OH 4kDa) (Fig 11a) (10.0 g, 2.50 mmol) was dissolved in toluene and refluxed overnight under Dean-Stark apparatus to remove traces of moisture. The toluene was evaporated, HO-PEG-OH dissolved in CH2Cl2 under N2 atmosphere, triethyl amine (0.916 ml, 12.5 mmol) and a small portion of 4dimethyl amino pyridine were added to the solution. 4,4’-Dimethoxytrityl chloride (1.02 g, 3.00 mmol) was added to the reaction mixture and stirred for 4 h. The completion of the reaction was followed by thin layer chromatography (TLC) using a solvent system containing 10 % MeOH in CHCl3. The solvent was evaporated and the oily residue was dissolved in hot ethanol, precipitated by cold ether, filtered and washed with ether. 2. Synthesis of α- carboxyl-ω-DMT-PEG (1c). Compound 1b (5.0 g, 1.162 mmol) was dissolved in a solution of KOH: H2O (1:10) (0.078 g, 1.4 mmol). To this stirred mixture, solution of KMnO4 : H2O (1:100) (0.55 g, 3.486 mmol) was added slowly, the reaction mixture was stirred at room temperature for 48 h. The solution was then filtered to remove MnO2 and then concentrated by lyophilization and acidified with HCl to pH = 2.0. NaCl was added to saturation, followed by extraction 3 times with dichloromethane (DCM). The combined extracts were dried over MgSO4, filtered, and evaporated under vacuum. The resulting oil was dissolved in hot ethanol and precipitate as a white solid at room temperature after the addition of ether. 3. Synthesis of α-DMT-ω-Tempol PEG AD5. Compound 1c (5.0 g, 1.162 mmol) was dissolved in CH2Cl2 (20 ml), a solution of DCC (0.475 g, 2.3 mmol) and 4-hydroxy-Tempo (0.403 g, 3.5 mmol) in CH2Cl2 (5 ml) was added, the mixture solution was stirred for 48 h at room temperature. The completion of the reaction was inferred by TLC (5 % MeOH: CHCl3). The 12 Environment ACS Paragon Plus

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precipitated N,N'-dicyclohexylurea (DCU) was filtered and the solvent was removed, the crude product was dissolved in a minimum amount of hot ethanol and cooled overnight. 4. Synthesis of α-Hydroxy-ω-Tempol-PEG -AD4. AD5 (3.0 g, 0.672 mmol) was dissolved in 2% dichloroacetic acid (DCA) in DCM (5 ml). The solution was stirred for 20 min at room temperature. The completion of the reaction was followed by TLC (5 % MeOH:CHCl3) and the solvent was removed under reduced pressure, the residue was dissolved in hot ethanol, precipitated by cold ether, filtered, washed with ether and lyophilized and white crystals were obtained. 5. Synthesis of α-Tempol ester-ω-lipo ester PEG (AD3). A solution mixture of DCC (2.5 g, 12.1 mmol) and α-LA (2.5 g, 12.1 mmol) in CH2Cl2 (10ml) was added to solution of AD4 (5.0 g, 1.203mmol) in CH2Cl2 (20ml); the reaction mixture was stirred for 48 h at room temperture. The precipitated DCU was removed by vacuum filtration and the solvent was evaporated under reduced pressure. The solid was dissolved in minimum volume of hot ethanol and the solution was left at 4 °C for 24 h. Synthesis of AD6. 1. Synthesis of α-Bromo-ω-DMT-PEG (1d). Compound 1b (Fig 1) (10 g, 2.32 mmol) was coevaporated with toluene and dissolved in dry toluene, carbon tetrabromide (1.54 g, 4.64 mmol). Tri-phenyl phosphine (1.26 g, 4.64 mmol) was then added to the solution, diethyl azodicarboxylate (DEAD) (0.938 g, 4.64 mmol) was added drop-wise, the reaction mixture was heated at 60 °C during 3 days after that toluene was evaporated. The oily yellow residue was dissolved in hot ethanol, precipitated by cold ether, filtered and washed with ether. 2. Synthesis of α-Amino-ω-DMT-PEG (1e). Compound 1d (5 g, 1.16 mmol) was dissolved in 25% NH4OH solution (100 ml). The solution mixture was stirred for 24 h at room temperature. The NH4OH solution was then evaporated and the residue was dissolved in hot ethanol and precipitated by cold ether. 3. Synthesis of α-DMT-ω-lipoamide PEG (1f).Compound 1e (5 g, 1.16 mmol), was dissolved in 15% Na2CO3 in water and dioxane (1:1) (50 ml), a solution of LA-NHS (0.55 g, 1.82 mmol) in dioxane was then added to the reaction mixture and stirred for overnight at room temperture. The completion of the reaction was followed by TLC (5% MeOH: CHCl3), the solution was then filtered, and the solvent was evaporated. The solid residue was dissolved in 10 ml of a hot solution of (EtOH:CH2Cl2 1:1) and kept at 4 °C overnight, the solution was filtered again and the solvent was evaporated. 4. Synthesis of α-hydroxy-ω-lipoamide PEG (AD6). Compound 1f (5 g, 1.11 mmol) was dissolved in 2% DCA in DCM (5 ml). The solution mixture was stirred for 20 min at room temperature. 13 Environment ACS Paragon Plus


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Purification For all compunds, unless otherwise mentioned, the completion of the reaction was followed by TLC (5 % MeOH: CHCl3) and the solvent was removed, the residue was dissolved in hot ethanol, precipitated by cold ether, filtered, washed with ether and purified by silica gel flash chromatography performed on Merck silica gel 60 (particle size 230–400 mesh). Thereafter the compounds were purified on reversed phase HPLC (Gilson, Middleton, WI, USA) with C18 preparative and semi-preparative columns, using acetonitrile and double distilled water at different ratios as eluent solvent. Analytical TLC was performed on silica gel 60 F254-precoated plates purchased from Merck (Darmstadt, Germany). The compounds were visualized by using UV light or I2-vapor. Fourier transform infrared (FTIR) spectroscopy. Infrared (IR) spectra of compounds were recorded on Smart iTR NICOLET iS10, FT-IR Thermo scientific spectrophotometer (Waltham, MA ,USA).

Nuclear Magnetic Resonance (NMR).The compounds were characterised using a Varian VXR300 (300MHz) spectrometer equipped with a 5-mm switchable probe and data was processed using the VNMR software. Chloroform-d and dimethyl sulfoxide-d6 were used as solvents while tetramethylsilane Me4Si (δ 0.00 ppm) was added as an internal standard. The splitting pattern abbreviations are as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, unresolved multiplet due to the field strength of the instrument; and dd, doublet of doublet.

Electrospray Ionization Mass Spectrometry (ESI-MS). Electrospray ionization mass spectrometry was measured as previously described 50 on a Thermo Quest Finnigan LCQ-Duo in the positive ion mode. In most cases, elution was in 20:79:1 water/methanol/acetic acid at a flow rate of 15 µL/min. Data were processed using ThermoQuest Finnigan's Xcalibur™ Biomass Calculation and Deconvolution software.

MALDI-TOF MS. Samples for MS analysis were prepared by dissolving the dry compound in chloroform containing 1% trifluoroacetic acid. One microliter of this solution was deposited on top of a matrix deposit (dithranol containing NaI) on the instrument holder. The MALDI-TOF positive ion mass spectra were obtained in reflector mode of Bruker Reflex II MALDI-TOF mass spectrometer (Bruker, Bremen, Germany), equipped with a 337 nm nitrogen laser and with the SCOUT source (delayed extraction and reflector). Each mass spectrum was generated from the signal average of 300 laser shots. 14 Environment ACS Paragon Plus

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Data Analysis. The molecular weight of the PEG and PEGylated compounds used for MS analysis is 4 kDa. Masses obtained are all average mass. The error associated with average mass obtained by MS is generally within 0.01%, which is a specification of the LCT-TOF premier mass spectrometer for average mass measurement. Mass spectra and deconvoluted mass spectra were obtained using Waters MassLynx Mass Spectrometry Software. PC12 Cell Cultures. Rat pheochromocytoma, PC12 cells, were propagated in 25 cm2 flasks in growth medium composed of Dulbecco's modified Eagle's medium (DMEM) supplemented with 7% fetal calf serum (FCS), 7% horse serum (HS), 10,000 U/ml pencillin and 100 µg/ml streptomycin, as previously described37 . The medium was replaced every second day and cells were grown at 37°C, in a humidified atmosphere of 6% CO2. Ischemic Insult Protocol Using PC12 Cells. PC12 cells (1.2 x106 cells/well) were seeded onto 12-wells plates, pre-coated with 200µg/ml collagen type-Ι, and grown for 2 days. On the day of the experiment, cell medium was replaced to glucose-free DMEM (hypoglycemic insult) and cultures introduced into an ischemic chamber with oxygen level below 1% (anoxic insult) for 4 h at 37°C under oxygen and glucose deprivation (OGD) as previously described37 .In order, to mimic in vivo reperfusion conditions due to renewal of blood supply, at the end of the OGD insult, 4.5 mg/ml glucose was added and cultures were incubated for additional 18 h under normoxic conditions (reperfusion/reoxygenation) to complete the ischemic insult. Operationally, ischemic insult represents therefore a combination of both OGD and reperfusion phases. Control cultures were maintained under regular atmospheric conditions (normoxia) in the presence of 6% CO2 with high glucose DMEM. Addition of AD3 and analogs was performed prior to OGD or whenever stated after OGD, before reperfusion. At the end of the reperfusion phase, cell death was measured as detailed below. In selected experiments cell death values using various times of OGD followed by 18 h reoxygenation were grouped into 3 categories of: low insult (2.5 h, 10-30 % cell death); mild insult (4 h, 30-60% cell death) and strong insult (6 h, 60-90% cell death). Comparison of the relative damage according to these categories enabled estimation of the relationship between the neuroprotective effect of the compound and the degree of the insult. All experiments (n=3-5) were carried out under good laboratory practice conditions using a clean room, regulated according to ISO7 requirements (10,000 particles /m3).

Determination of Cell Death by Lactate Dehydrogenase (LDH) Release. Cell death was evaluated by measuring the leakage of LDH into the medium as previously described

37

. LDH

activity was determined at 340 nm using a spectrofluorimeter (TECAN, SPECTRA Fluor PLUS, 15 Environment ACS Paragon Plus


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Salzburg, Austria). Basal LDH release was measured in PC12 monolayer cultures maintained under normoxic conditions. Under OGD insult, LDH release, representing cell death, was expressed as percent of total LDH released into the medium upon subtracting the basal values of LDH release. Total LDH (extracellular + intracellular) was obtained by freezing and thawing the cultures. The neuroprotective effect, defined as the percent decrease in LDH release in the presence of AD3 or analogs was normalized to untreated ischemic cultures and is depicted below: Cell death (%) = (LDH (ischemia – basal) / (LDH total) x 100 Additionally, a neuroprotection index defined as the fractional ratio of cell death in treated versus control PC12 cells was calculated as noted below. NP-Index =1-(% cell death in treated well / % cell death in control well). The rescue capacity on a scale of 0-1 is maximal at 1.

Lipid Peroxidation by Measurement of TBARS. Malondialdehyde-like metabolites released into the culture medium were collected and reacted with thiobarbituric acid (TBA) reagent as previously described

37

.Briefly, the TBA reagent was prepared by dissolving 0.67% TBA

(wt/vol) and 0.01% (wt/vol) butylated hydroxytoluene (BHT) in 50% acetic acid. Aliquots of 0.5 ml culture medium were collected after ischemic insult or normoxic conditions and TBA-reagent (0.5ml) was added. The solution was heated in boiling water for 15 min. The samples were cooled with tap water and the developed color read at 535 nm excitation and 553 nm emission using spectrofluorimetry. For quantification of TBARS, a standard curve was prepared by using a 100 µM 1, 1, 3, 3-tetraethoxypropane (TEP) stock solution diluted in PBS.

Measurement of Cellular Oxidative Stress. Dichlorodihydro-fluorescein diacetate (DCHFDA) was used as a redox indicator probe to measure changes in intracellular redox status of cells 51

as described previously38. DCHF-DA was dissolved in DMSO and diluted with calcium-free

PBS buffer to a final concentration of 5 µM. Two days before the experiment, 1.2×106 cells were plated in 12- well plates coated with 200 µg/ml type-I rat tail collagen. Before the OGD insult, the PC12 cells were loaded with the oxidant-sensitive dye DCHF-DA in PBS for 30 min, and the solution was replaced with calcium and glucose-free PBS containing 0.5 mM magnesium. The cells were then exposed to OGD insult, followed by reoxygenation for 30 min, to allow maximal probe oxidation detection, with or without addition of AD3. At the end of this short reoxygenation period, the PC12 cells were washed twice with PBS and placed on a fluorescence microplate reader. In parallel, control normoxic cultures were loaded similarly with DCHF-DA and identically processed. Probe fluorescence was then determined at 485 nm excitation and 525 16 Environment ACS Paragon Plus

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nm emission using Cytation3 scanning fluorescent plate reader (BioTek Instruments, Winooski, VT, USA) and the fluorescence intensity was measured in arbitrary units.

Measurement of MAPKs Phosphorylation Activity. At the end of the ischemic insult, the cellular proteins were extracted from the cells and the concentration was determined according to the Bradford assay. Samples containing 30 µg of cell protein were boiled for 5 min in SDS sample buffer and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).The protein bands were subsequently transferred onto Immobilon membranes. Following blocking with 5% nonfat milk, the blots were probed with specific anti-phospho primary antibodies for MAPKs (dilution 1:1000) overnight and detected by secondary antibody– HRP conjugate. Specific antibody binding enhanced by chemiluminescence (ECL) and visualized by Image Lab™ Software 5.1 (Bio-Rad Laboratories, Hercules, CA, USA). The ration between MAPK phosphorylated kinase to Pan kinase was calculated as previously described 40

Statistical Analysis. Each experiment was performed 3-6 times in sixplicate wells. The results are presented as mean ± S.E. Statistical comparisons between experimental groups were performed by using analysis of variance program (ANOVA) followed by Dunnett's multiple comparison test. p value of 0.05 or less was considered significant for all comparisons.



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AUTHOR INFORMATION *Corresponding Author: Prof. Philip Lazarovici, School of Pharmacy Institute for Drug Research, The Hebrew University of Jerusalem Rehovot 76100, Israel Phone : +972-2-6758729 Fax : +972-2-6757490 E-mail: [email protected] Author Contributions Adi Lahiani and Adel Hidmi performed the experiments Jehoshua Katzhendler, Ephraim Yavin and Philip Lazarovici designed the study. Adi Lahiani, Jehoshua Katzhendler, Ephraim Yavin and Philip Lazarovici wrote the manuscript Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS PL holds The Jacob Gitlin Chair in Physiology and is affiliated and partially supported by Grass Center for Drug Design and Synthesis of Novel Therapeutics and The Adolph and Klara Berettler Medical Research Center at The Hebrew University of Jerusalem, Israel. EY greatly appreciate the financial support of the Gulton Foundation NY. AL acknowledged a fellowship award from Dalia and Eli Hurvitz Foundation, Israel. ABBREVIATIONS α- LA, α-Lipoic acid; AD3, α-Tempol ester-ω-lipo ester PEG; AD4, α-Hydroxy-ω-Tempol-PEG; AD5, α-DMT-ω-Tempol PEG; AD6, α-hydroxy-ω-lipoamide PEG; α- LA, α-Lipoic acid; DCA, dichloroacetic acid; BBB, blood brain barrier; DCC, DL-α-α-LA, N-N’-dicyclohexyl carbodiimide; DCM, dichloromethane; DEAD, diethyl azodicarboxylate; EC50, effective concentration 50%; ERK, extracellular-signal-regulated kinases; PEG, polyethylene glycol; JNK, c-Jun N-terminal kinases; LDH, lactate dehydrogenase; LPO, lipid peroxidation; MAPK, mitogen-activated protein kinases; NMR, nuclear magnetic resonance; NO, nitric oxide; NPIndex, neuroprotective index; OGD, oxygen and glucose deprivation; PC12, pheochromocytoma cells; p-38, p-38 mitogen-activated protein kinases; ROS, reactive oxygen species; TBARS, thiobarbituric acid reactive substances; Tempol, 4-hydroxy -2,2,6,6-tetramethylpiperidine-1oxyl; TLC, thin layer chromatography; TPP, tri-phenyl phosphine. 18 Environment ACS Paragon Plus

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FIGURE LEGENDS

Figure 1. Synthesis of AD3 and intermediate derivatives Illustration of the five-stage chemical synthesis of AD3 (α-Tempol ester-ω-lipo ester PEG) and its intermediates AD5 (α-DMT-ω-Tempol PEG) and AD4 (α-Hydroxy-ω-Tempol-PEG).

Figure 2. Synthesis of AD6 Illustration of the four-stage chemical synthesis of AD6 (α-hydroxy-ω-lipoamide PEG).

Figure 3. Characterization of AD3 and intermediate derivatives on PC12 cell survival after ischemia. PC12 cells (1.2 x106 cells/well) were seeded in 12-well plates and grown for 2 days. At the start of the experiment (panel A) cells were administered with Tempol (1500 µM), α-LA (10 µM) or 10 µM of AD3, AD4, AD5 before (

bar) or at the end (

bar) of the OGD insult.

The OGD insult was carried out for 4 h followed by 18 h reperfusion. Aliquots from the culture media were taken for LDH release measurements as described in Methods. For panel B cells were subjected to OGD for 2 h (10-30% cell death); 4 h (30-60% cell death,) and 6 h (60-90% cell death) in the absence ( ) or presence of Tempol ( ), α-LA ( ) and AD3 ( ) at concentrations as above and reperfusion time of 18 h. Cell death is expressed as % LDH release out of total LDH (panels A, B) and the corresponding NP-index (panel A’) are mean ± SE (n=12). *p

Novel Synthetic PEGylated Conjugate of Î±-Lipoic Acid and Tempol

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