Wilson's Disease (WD) is a rare autosomal recessive disorder caused by mutations in the ATP7B gene. These mutations lead to defective copper (Cu) transport and to accumulation of Cu in tissues, primarily in the liver and brain. Current treatment options such as D-penicillamine, trientine, and zinc salts focus on increasing Cu excretion or reducing Cu absorption, but often cause debilitating side effects. N,N'-bis(2-mercaptoethyl)isophthalamide (NBMI) is a lipophilic thiol-based compound originally developed for environmental decontamination. It has been shown to chelate toxic metals such as mercury, lead, and cadmium. This study was designed to evaluate the efficacy of NBMI to mitigate Cu overload using both in vitro and in vivo models of WD. HepG2 cells with the ATP7B gene knocked down had increased sensitivity to copper sulfate (CuSO4) compared to wild-type (WT) cells, validating the cell model for WD. Pretreatment with NBMI (2.5-50 μM) improved cell viability, reduced Cu-induced oxidative stress, decreased metallothionein levels, mitigated resulting DNA damage, and reduced overall levels of free intracellular Cu. In an established toxic milk mouse (tx-J) model of WD, 1% dietary NBMI effectively lowered hepatic, cerebral, and renal Cu levels. Treatment with 1% NBMI also improved liver function, as evidenced by reduced ALT levels and normalized hepatocyte morphology. Tx-J mice displayed higher liver-to-body weight ratios compared to WT mice, and treatment with 1% NBMI effectively reduced this ratio. While NBMI did not impact the elevated white blood cell counts and low platelet levels characteristic of tx-J mice, it also did not cause any detrimental effects on red blood cell, hemoglobin, and hematocrit levels. This dose of NBMI also restored homeostasis of other dysregulated essential metal ions in tx-J mice. These findings suggest that dietary administration of NBMI effectively chelates excess free Cu, ameliorates WD symptoms and offers a promising alternative to existing chelators.

OBJECTIVE To evaluate the effects of aging on phenylbutazone (PBZ) disposition in older horses (>= >= 25 years old) compared to young adults (4 to 10 years old) by characterizing the pharmacokinetic profile of PBZ and its active metabolite, oxyphenbutazone (OPBZ), following a 2.2-mg/kg dose, IV. We hypothesized that the disposition of PBZ will be affected by age. ANIMALS 16 healthy horses (8 young adults aged 4 to 10 years and 8 geriatric horses >= 25 years old). METHODS Horses were administered a single 2.2-mg/kg PBZ dose, IV. Plasma samples were collected at designated time points and frozen at -80 degrees C until assayed using liquid chromatography-tandem mass spectrometry. Pharmacokinetic analyses were performed using Phoenix WinNonlin, version 8.0 (Certara). Both clinical and pharmacokinetic data were compared between age groups using independent samples t tests, with P < .05 considered significant. RESULTS Baseline characteristics did not differ between groups, with the exception of age, weight, and plasma total solids. Plasma concentrations of PBZ were best described by a two-compartment model. The maximum plasma concentration of OPBZ was reached at 5 hours for both age groups, and the metabolite-to-parent-drug area-under-the-curve ratios were approximately 20% for both groups. None of the pharmacokinetic parameters of PBZ or its metabolite, OPBZ, differed significantly between age groups. CLINICAL RELEVANCE The hypothesis was rejected as there was no significant difference in PBZ disposition in young-adult horses com-- pared to geriatric horses. Our data do not support the need for dose adjustments of PBZ in clinically healthy geriatric horses.

BACKGROUND/AIM:We examined the effect of low-intensity focused ultrasound (FUS) on unbinding cisplatin from plasma proteins and enhancing its chemotherapeutic efficacy using a mouse model of xenograft human cervical cancer.MATERIALS AND METHODS:FUS, operating in a pulsed mode, was applied to a dialysis cassette immersed in a normal saline bath containing both bovine serum albumin (BSA) and cisplatin, and the unbound level of cisplatin diffused into the cassette was measured. To assess the in vivo efficacy of the technique, athymic nu/nu mice were inoculated with human cervical cancer cells under four different combinatory conditions, with and without the administration of cisplatin and FUS. FUS was delivered to the tumor mass for 1 h across four separate sessions spanning a period of 10 days, following the intraperitoneal injection of cisplatin.RESULTS:In vitro equilibrium dialysis revealed that non-thermal application of FUS increased the concentration of unbound cisplatin compared to cassettes that were not exposed to sonication, suggesting successful unbinding. Assessment of tumor growth in vivo showed that FUS following cisplatin administration resulted in a significant reduction in tumor growth, whereas the administration of cisplatin alone exhibited plateau growth. Without administration of cisplatin, equivalent rates of aggressive tumor growth were observed regardless of the application of FUS.CONCLUSION:Pulsed application of FUS can unbind cisplatin from albumin and enhance its tumoricidal effects in cervical cancer. Further assessment of intratumoral/systemic cisplatin concentration is required to quantify its selective delivery to the tumor.

The efficacy of many anti-epileptic drugs, including phenytoin (PHT), is reduced by plasma protein binding (PPB) that sequesters therapeutically active drug molecules within the bloodstream. An increase in systemic dose elevates the risk of drug side effects, which demands an alternative technique to increase the unbound concentration of PHT in a region-specific manner. We present a low-intensity focused ultrasound (FUS) technique that locally enhances the efficacy of PHT by transiently disrupting its binding to albumin. We first identified the acoustic parameters that yielded the highest PHT unbinding from albumin among evaluated parameter sets using equilibrium dialysis. Then, rats with chronic mesial temporal lobe epilepsy (mTLE) received four sessions of PHT injection, each followed by 30 min of FUS delivered to the ictal region, across 2 weeks. Two additional groups of mTLE rats underwent the same procedure, but without receiving PHT or FUS. Assessment of electrographic seizure activities revealed that FUS accompanying administration of PHT effectively reduced the number and mean duration of ictal events compared to other conditions, without damaging brain tissue or the blood–brain barrier. Our results demonstrated that the FUS technique enhanced the anti-epileptic efficacy of PHT in a chronic mTLE rodent model by region-specific PPB disruption.

Background Nebulized lidocaine appears promising as a novel corticosteroid-sparing therapeutic for equine asthma, but its safety and pharmacokinetic behavior have yet to be confirmed. Objective To describe the effect of nebulized lidocaine on upper airway sensitivity, lung mechanics, and lower respiratory cellular response of healthy horses, as well as delivery of lidocaine to lower airways, and its subsequent absorption, clearance, and duration of detectability. Animals Six healthy university- and client-owned horses with normal physical examination and serum amyloid A, and no history of respiratory disease within 6 months. Methods Prospective, descriptive study evaluating the immediate effects of 1 mg/kg 4% preservative-free lidocaine following nebulization with the Flexineb®. Prior to and following nebulization, horses were assessed using upper airway endoscopy, bronchoalveolar lavage, and pulmonary function testing with esophageal balloon/pneumotachography and histamine bronchoprovocation. Additionally, blood and urine were collected at predetermined times following single-dose intravenous and nebulized lidocaine administration for pharmacokinetic analysis. Results Upper airway sensitivity was unchanged following lidocaine nebulization, and no laryngospasm or excessive salivation was noted. Lidocaine nebulization (1 mg/kg) resulted in a mean epithelial lining fluid concentration of 9.63 ± 5.05 μg/mL, and a bioavailability of 29.7 ± 7.76%. Lidocaine concentrations were higher in epithelial lining fluid than in systemic circulation (Cmax 149.23 ± 78.74 μg/L, CELF:Cmaxplasma 64.4, range 26.5–136.8). Serum and urine lidocaine levels remained detectable for 24 and 48 h, respectively, following nebulization of a single dose. Baseline spirometry, lung resistance and dynamic compliance, remained normal following lidocaine nebulization, with resistance decreasing post-nebulization. Compared to the pre-nebulization group, two additional horses were hyperresponsive following lidocaine nebulization. There was a significant increase in mean airway responsiveness post-lidocaine nebulization, based on lung resistance, but not dynamic compliance. One horse had BAL cytology consistent with airway inflammation both before and after lidocaine treatment. Conclusions Nebulized lidocaine was not associated with adverse effects on upper airway sensitivity or BAL cytology. While baseline lung resistance was unchanged, increased airway reactivity to histamine bronchoprovocation in the absence of clinical signs was seen in some horses following nebulization. Further research is necessary to evaluate drug delivery, adverse events, and efficacy in asthmatic horses.

Abstract Background High-intensity ultrasound has been used to induce acoustic cavitation in the skin and subsequently enhances skin permeability to deliver hydrophobic topical medications including lidocaine. In contrast, instead of changing skin permeability, pulsed application of low-intensity focused ultrasound (FUS) has shown to non-invasively and temporarily disrupt drug-plasma protein binding, thus has potential to enhance the anesthetic effects of hydrophilic lidocaine hydrochloride through unbinding it from serum/interstitial α1-acid glycoprotein (AAG). Methods FUS, operating at fundamental frequency of 500 kHz, was applied pulse-mode (55-ms pulse duration, 4-Hz pulse repetition frequency) at a spatial-peak pulse-average intensity of 5 W/cm2. In vitro equilibrium dialysis was performed to measure the unbound concentration of lidocaine (lidocaine hydrochloride) from dialysis cassettes, one located at the sonication focus and the other outside the sonication path, all immersed in phosphate-buffered saline solution containing both lidocaine (10 µg/mL) and human AAG (5 mg/mL). In subsequent animal experiments (Sprague-Dawley rats, n = 10), somatosensory evoked potential (SSEP), elicited by electrical stimulations to the unilateral hind leg, was measured under three experimental conditions—applications of FUS to the unilateral thigh area at the site of administered topical lidocaine, FUS only, and lidocaine only. Skin temperature was measured before and after sonication. Passive cavitation detection was also performed during sonication to evaluate the presence of FUS-induced cavitation. Results Sonication increased the unbound lidocaine concentration (8.7 ± 3.3 %) from the dialysis cassette, compared to that measured outside the sonication path (P < 0.001). Application of FUS alone did not alter the SSEP while administration of lidocaine reduced its P23 component (i.e., a positive peak at 23 ms latency). The FUS combined with lidocaine resulted in a further reduction of the P23 component (in a range of 21.8 − 23.4 ms after the electrical stimulations; F(2,27) = 3.2 − 4.0, P < 0.05), indicative of the enhanced anesthetic effect of the lidocaine. Administration of FUS neither induced cavitation nor altered skin conductance or temperature, suggesting that skin permeability was unaffected. Conclusions Unbinding lidocaine from the plasma proteins by exposure to non-thermal low-intensity ultrasound is attributed as the main mechanism behind the observation.

Surface modification of liposomes with a ligand is facilitated by the conjugation of the ligand to a hydrophobic molecule that serves to anchor the ligand to the liposomal bilayer. We describe here a simple protocol to conjugate a triphenylphosphonium group to several commercially available functionalized phospholipids. The resulting triphenylphosphonium-conjugated lipids can be used to prepare liposomes that preferentially associate with mitochondria when exposed to live mammalian cells in culture.

Environmental and occupational exposure to heavy metals remains one of the major concerns in public health. Increased levels of manganese (Mn) pollution are associated with profound neurotoxic effects, including neurobehavioral deficits and disturbances resembling Parkinson’s disease. While Mn absorption is in part mediated by iron transporters, recent studies have shown that the levels of iron transporters are modified by alcohol and that chronic alcohol consumption increases body iron stores. However, it is largely unexplored whether alcohol exposure influences the transport and neurotoxicity of Mn. To address this question, we exposed mice to ethanol (10%; v/v) by drinking water for 4 weeks, during which period MnCl2 (5 mg/kg) or saline solutions were administered daily by intranasal instillation. Ethanol consumption in mice increased brain Mn levels in a dose-dependent manner after Mn instillation, determined by inductively-coupled plasma mass spectrometry, which was accompanied by up-regulation of iron transporters, as assessed by western blotting and qPCR. In addition, alcohol drinking increased hypoxic response and decreased hepcidin expression, providing the molecular mechanism of increased iron transporters and Mn uptake upon alcohol consumption. Moreover, brain dopamine levels, analyzed by HPLC, were decreased after intranasal Mn instillation, which was worsened by alcohol. Likewise, alcohol-Mn co-exposure synergistically altered dopaminergic protein expression. Finally, alcohol binge-drinking, which resembles alcohol drinking manner in humans, increased brain Mn content along with upregulation of iron transporters. Our study suggests that individuals who consume alcohol may have a higher risk of Mn neurotoxicity upon Mn exposure.

Plasma protein binding (PPB) plays an important role in drug pharmacokinetics, particularly for central nervous system drugs, as PPB affects the blood concentration of unbound drug available to cross the blood-brain barrier (BBB). We report the non-invasive, spatially specific disruption of PPB to phenytoin, an anti-epileptic drug with high affinity to albumin, using 250-kHz focused ultrasound (FUS) delivered in a pulsed manner (55-ms tone burst duration, 4-Hz pulse repetitions). Equilibrium dialysis performed on sonicated phosphate-buffered saline solution containing phenytoin and bovine serum albumin revealed a 27.7% elevation in the unbound phenytoin concentration compared with an unsonicated control. Sonication of a unilateral brain hemisphere in rats (n = 10) after intraperitoneal phenytoin injection revealed increased parenchymal phenytoin uptake compared with the unsonicated hemisphere, without evidence of temperature change or BBB disruption. These findings illustrate the use of FUS as a novel technique for spatially selective disruption of PPB, which may be applied to a wide range of drug-plasma protein interactions.

Inflammation is a vital component of the immune system response to injury and infection, and it is linked to autoinflammatory and autoimmune disorders. Toll‐like receptor 4 (TLR4) has been identified as a receptor for lipopolysaccharide (LPS), a bacterial endotoxin which can induce systemic inflammation and sepsis. The LPS/TLR4 pathway triggers downstream signaling cascades, producing proinflammatory cytokines and chemokines. Therefore, the search for novel compounds that can reduce inflammation at this level is of great interest. The plant Lepidium meyenii L., known as maca, has attracted attention in the past several years due to its traditional uses in folk medicine, which include treating anemia and gastritis, preventing cancer, high blood pressure and depression, as well as enhancing fertility and sexual performance. Macamides are secondary metabolites isolated from maca which cause pharmacological inhibition of fatty acid amide hydrolase (FAAH), and this inhibition has been found to lead to anxiolytic, anti‐inflammatory and analgesic effects. However, the anti‐inflammatory mechanisms by which these natural compounds act remain to be elucidated. The purpose of this study was to evaluate the anti‐inflammatory effects of the n‐pentane extract of Lepidium meyenii L. (PELM), N‐(benzyl) linolenamide (compound 1) and N‐(3‐methoxybenzyl) linolenamide (compound 2) utilizing THP‐1 cells. To assess these effects, THP‐1 cells were treated with either 10 μg/mL PELM, 10 μM Cmpd 1, 10 μM Cmpd 2 or 0.5 % DMSO (vehicle); then, inflammation was induced for 24 h with 30 ng/mL LPS. Media supernatants were used to evaluate protein levels of cytokines in a human protein array. From 36 cytokines, maca and macamides reduced IL‐1β and IL‐6 protein levels following a 24 h stimulation with LPS. In order to confirm these results, ELISA was utilized to determine IL‐1β and IL‐6 cytokine levels in LPS‐stimulated THP‐1 cells. After a 24 h treatment with either 10 μg/mL PELM, 10 μM Cmpd 1 or 10 μM Cmpd 2, these treatments reduced protein levels of both cytokines IL‐1β and IL‐6. The transcriptional levels of these cytokines were also monitored by RT‐qPCR; treatment with 10 μM Cmpd 1 and 10 μM Cmpd 2 significantly decreased mRNA levels of IL‐1β. But, interestingly with a 10 μg/mL treatment of PELM the reduction of IL‐1β mRNA levels was not significant. Furthermore, IL‐6 mRNA levels were significantly reduced by the n‐pentane extract and isolated macamides. Taken together, these results demonstrated that Lepidium meyenii L. and isolated macamides reduced LPS‐induced inflammatory response by decreasing pro‐inflammatory cytokine production in THP‐1 cells.Effect of PELM, Cmpd 1 and Cmpd 2 on IL‐1β production in THP‐1 cells.Figure 1Effect of PELM, Cmpd 1 and Cmpd 2 on IL‐1β mRNA levels in THP‐1 cells.Figure 2

Autophagy is a conserved catabolic process which channels intracellular proteins and organelles into lysosomes to be degraded. This process can be triggered either by nutrient deprivation or by metabolic stress. Autophagy malfunction has been linked to a variety of human diseases such as neurodegeneration, myopathies, infectious diseases and different cancer types. Therefore, the search for novel autophagy modulators is of great interest to reveal autophagic mechanisms involved in these disease processes. As many natural product‐derived compounds have been used as traditional medicines, they have great potential as lead autophagy modulators. Macamides are secondary metabolites isolated from the Peruvian plant Lepidium meyenii L. (Maca). These metabolites have exhibited various biological activities, including anti‐oxidative, anti‐fatigue, anti‐cancer, anti‐osteoporotic and neuroprotective effects. The purpose of this study was to evaluate the effect of two macamides: N‐(benzyl)linolenamide (Cmpd 1) and N‐(3‐methoxybenzyl)linolenamide (Cmpd 2) on autophagy in both HepG2 and HeLa cancer cell lines. Cell viability in both cell lines was strongly reduced by both Cmpd 1 (HepG2, IC50 = 75.6 μM; HeLa, IC50 = 116 μM) and Cmpd 2 (HepG2, IC50 = 62.8 μM; HeLa, IC50 = 117 μM) as determined by the MTS assay. Subsequently, it was investigated whether macamides inhibit lysosomal acidification, by monitoring the red fluorescence emitted by cells stained with LysoTracker Red DND‐99. Significantly, both Cmpd 1 and Cmpd 2 (50 μM) decreased the red fluorescence in both cell lines. Furthermore, to evaluate autophagic flux in HepG2 and HeLa cells, protein levels of microtubule‐associated protein 1A/1B‐light chain 3 (LC3) and p62 were measured. No significant differences were observed in LC3 levels. Nonetheless, Cmpd 1 was identified as the most effective compound to induce accumulation of p62 in HepG2 cells. Because p62 is degraded in the lysosome during the last stage of autophagy, these results suggest that macamides inhibit autolysosomal degradation. However, a more in‐depth study is needed to determine the inhibitory mechanism and/or if another type of autophagy is involved.Cmpd 1 and Cmpd 2 decreases acidification of lysosomes in HepG2 cells.Figure 1Cmpd 1 and Cmpd 2 decreases acidification of lysosomes in HeLa cells.Figure 2

Iron deficiency is closely associated with altered GABA metabolism and affective behavior. While mutation in the hemochromatosis (HFE) gene disrupts iron homeostasis and promotes oxidative stress that increases the risk of neurodegeneration, it is largely unknown whether HFE mutation modifies GABAergic homeostasis and emotional behavior. The goal of our study was to investigate the impact of HFE on GABAergic neurochemistry and redox-epigenetic regulation in the brain using H67D HFE-mutant mice that recapitulates the H63D-HFE mutation in humans. H67D mice displayed elevated redox-active iron levels in the brain by 32% compared to age-matched wild-type mice. Moreover, the H67D brain had increased isoprostane and decreased glutathione, indicating elevated oxidative stress. Additionally, the H67D brain had decreased global methylation and attenuated DNA methyltransferase (DNMT) activity. Direct addition of iron to purified DNMT in vitro decreased enzyme activity in a concentration-dependent manner. Last, H67D mice exhibited decreased anxiety-like behavior, which was associated with increased expression of the GABA(A) receptor 2 subunits by 93%, and these changes were also observed in H67D mice fed a low-iron diet. Taken together, our results suggest a putative role of HFE in regulating labile iron status in the brain, and mutation in H67D perturbs redox-methylation status, contributing to GABAergic dysfunction.Ye, Q., Trivedi, M., Zhang, Y., Bohlke, M., Alsulimani, H., Chang, J., Maher, T., Deth, R., Kim, J. Brain iron loading impairs DNA methylation and alters GABAergic function in mice.

Triple negative breast cancer (TNBC) is one of the most aggressive types of breast cancer accounting for 12% of breast cancer cases. It is characterized by the lack of the estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER‐2), which limits treatment options and enhances its ability to metastasize with the risk of recurrence. Patients with TNBC are not responsive to conventional therapies. Capsaicin (CAP) is the most abundant capsaicinoid produced in chili pepper fruits and has been utilized for its analgesic and anti‐inflammatory effects. While several studies have demonstrated that capsaicin has anti‐carcinogenic properties in various types of human cancers, the underlying molecular mechanisms remain to be explored. The aim of this study was to investigate the effects of capsaicin in human TNBC, utilizing a BT‐20 cell model. Capsaicin demonstrated concentration‐ and time‐dependent in the viability of BT‐20 cells, as determined by the MTS assay. Capsaicin caused significant increases in cytochrome C release, caspase 3/7 activity and expression of cleaved poly‐(ADP‐ribose) polymerase (PARP), all of which are markers of apoptotic activation. These effects were accompanied by down‐regulation of cyclin D1 production, an indicator of cell cycle arrest at the G0/G1 phase. Further analyses of signaling mechanisms revealed that capsaicin significantly inhibited EGFR phosphorylation and the phosphorylation of its downstream signaling proteins AKT and MAPK, which may provide some explanation for its mechanism of action in TNBC. In conclusion, capsaicin has demonstrated inhibitory effects on cell growth and cell cycle progression on TNBC cells by enhancing apoptosis. Our data demonstrated a novel mechanism for capsaicin, modulating EGFR signaling by AKT/MAPK pathways in BT‐20 cells. These results provide useful information relevant to the development of a potential new therapy to treat TNBC with capsaicin. Support or Funding Information This work was funded by the College of Medicine, King Saud bin Abdulaziz University for Health Sciences (KSAU‐HS), Jeddah, Saudi Arabia. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic receptor involved in the uptake of a variety of molecules, such as apoE, α2-macroglobulin, and the amyloid β peptide (Aβ), for either transcellular transport, protein trafficking or lysosomal degradation. The LRP1 gene can be transcribed upon activation of peroxisome proliferator receptor activated-γ (PPARγ) by the potent PPARγ agonist, rosiglitazone (RGZ). In previous studies, RGZ was shown to upregulate LRP1 levels in concentrations between 0.1 and 5 μM in HepG2 cells. In this study, we sought to replicate previous studies and to investigate the molecular mechanism by which high concentrations of RGZ reduce LRP1 levels in HepG2 cells. Our data confirmed that transcriptional activation of LRP1 occurred in response to RGZ at 3 and 10 μM, in agreement with the study reported by Moon et al. (2012a). On the other hand, we found that high concentrations of RGZ decreased both mRNA and protein levels of LRP1. Mechanistically, transcriptional dysregulation of LRP1 was affected by the downregulation of PPARγ in a time- and concentration-dependent manner. However, downregulation of PPARγ was responsible for only 40% of the LRP1 reduction and thereby the remaining loss of LRP1 (60%) was found to be through degradation in the lysosomal system. In conclusion, our findings demonstrate the mechanisms by which high concentrations of RGZ caused LRP1 levels to be reduced in HepG2 cells. Taken together, this data will be helpful to better explain the pharmacological modulation of this pivotal membrane receptor by PPARγ agonists.

Macamides are a distinct class of secondary metabolites, benzylamides of long chain fatty acids, which were isolated from the Peruvian plant Lepidium meyenii (Maca). As structural analogues of the endocannabinoid anandamide (AEA), they have demonstrated neuroprotective effects in vitro and in vivo. The purpose of this study was to demonstrate the neuroprotective activity of the macamides: N-(3-methoxybenzyl)oleamide (MAC 18:1), N-(3-methoxybenzyl)linoleamide (MAC 18:2) and N-(3-methoxybenzyl)linolenamide (MAC 18:3) in a neurotoxic environment caused by exposure of U-87 MG glioblastoma cells to manganese chloride (MnCl2). The neuroprotective effects of these macamides were reversed by the CB1 antagonist AM251. The mechanism by which manganese (Mn) induces cell damage was investigated by studying its effects on mitochondria. Reactive oxygen species (ROS) increase intracellular calcium and enhance the opening of mitochondrial permeability transition pores (MPTP), which leads to decreased mitochondrial membrane potential (MMP), to disruption of mitochondria and to neuron death in neurodegenerative disorders. In this study, MnCl2 at 50μM was responsible for mitochondrial disruption, which was attenuated by all three of the macamides tested. Human peroxisome proliferator-activated receptor gamma (PPARγ) has been proposed to be a cannabinoid target, and PPARγ has also been demonstrated to mediate some of the longer-term vascular effects of the plant cannabinoid, ∆9-tetrahydrocannabinol. PPARγ activation was observed in response to exposures of cells to MAC 18:2 and MAC 18:3. These findings suggest that macamides achieve their neuroprotective effects by binding to CB1 receptors to protect against Mn-induced toxicity in U-87 MG glioblastoma cells. Additionally these macamides, in a manner similar to the analogous endocannabinoid AEA, interact with other targets such as PPARγ to regulate metabolism and energy homeostasis, cell differentiation and inflammation.

Lepidium meyenii, known as Maca, is a Peruvian herbaceous biennial plant used by indigenous Andean societies as a source of nourishment and healing. Macamides, a distinct class of secondary metabolites which are structurally similar to the endocannabinoids, have neuroprotective effects. The purpose of this study was to evaluate the neuroprotective effects of some of the most prominent macamides: N‐(3‐methoxybenzyl) oleamide, N‐(3‐methoxybenzyl) linoleamide and N‐(3‐methoxybenzyl) linolenamide, at concentrations of 0.1 to 50uM, on U87MG glioblastoma cells exposed to a neurotoxic concentration (10 μM) of MnCl2. A cell viability assay utilizing 3‐(4, 5‐dimethylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium (MTS) was performed to evaluate the neuroprotective effects. The results demonstrated that the macamides counteracted the toxicity of MnCl2, showing significant increases in cell viability in response to N‐(3‐methoxybenzyl) oleamide and N‐(3‐methoxybenzyl) linoleamide at all concentrations studied (21% and 25% respectively). A dose dependent increase in cell viability was observed for N‐(3‐methoxybenzyl) linolenamide (P≤ 0.001). Moreover, glutathione levels and other markers were measured to evaluate the neuroprotective effects of macamides. Further assays will be performed in order to determine the mechanism of neuroprotective activity demonstrated by these macamides.

Lepidium meyenii (Maca) is a Peruvian plant that has previously been implicated to have effects on the endocannabinoid system. N‐Benzylamides, also known as macamides, are compounds present in the Maca pentane extract (MPE) with a demonstrated inhibitory effect on fatty acid amide hydrolase (FAAH), one of the enzymes that terminates endocannabinoid signaling. The purpose of this project was to evaluate the effect of MPE on monoacyl glycerol lipase (MAGL), another enzyme involved in the endocannabinoid system, and additionally to demonstrate the effect of MPE on the expression of the low density lipoprotein receptor‐related protein 1 (LRP1) in neuroblastoma SH‐SY5Y cells. LRP1 is a receptor involved in the transcellular transport of peptides and proteins, including β‐amyloid.Macamides were quantified by LC‐MS/MS, demonstrating that N‐benzylpalmitamide, N‐benzyloctadeca‐9Z, 12Z‐dienamide and N‐benzyloctadeca‐9Z, 12Z, 15Z‐trienamide were the most predominant compounds in the MPE with concentrations of 27.0, 8.4 and 5.5 mg/g, respectively. The FAAH and MAGL inhibitory activity of MPE was demonstrated with IC50 values of 9.15 and 7.12 μg/mL, respectively, supporting an action via the endocannabinoid system.SH‐SY5Y neuroblastoma cells were treated with MPE at three different concentrations between 0.1 and 10 μg/mL. Cells were lysed and LRP1 levels were analyzed by immunoblotting, demonstrating that Lepidium meyenii was able to significantly increase LRP1 expression.While MPE was previously demonstrated to have neuroprotective effects associated with the inhibition of FAAH, this investigation reports that MPE additionally inhibits MAGL. This dual inhibitory activity on FAAH and MAGL could be responsible for the upregulation of LRP1. It is well known that LRP1 reduction is associated with neurological disorders such as Alzheimer's disease. The increase of LRP1 levels by MPE supports a novel mechanism which might be responsible for some of the biological activity of this plant. Further studies are necessary to better elucidate the activation of transcription factors by Maca constituents.

Fatty acid amide hydrolase (FAAH) is one of the enzymes responsible for terminating endocannabinoid signaling in the CNS and periphery. The objective of this study was to determine and characterize the inhibitory effects of N-(pyridine-3-ylmethyl)oleamide (NPMO) on FAAH. NPMO is a synthetic analog of the macamides present in Lepidium meyenii (Maca), a Peruvian plant with demonstrated neuroprotective effects in vitro and in vivo. Recent studies have also demonstrated that Maca extracts have activity on the endocannabinoid system and inhibitory activity on FAAH. In this study NPMO, from 2 to 100 µM, was tested using an FAAH inhibitor screening assay method. The results demonstrated that NPMO possesses concentration-dependent FAAH inhibitory activity with anapproximate IC50 of 24.4 µM. Michaelis-Menten and Lineweaver-Burk plot analyses revealed that NPMO decreased the enzyme Vmax from 1.57±0.09 to 0.58±0.04 pmol/min with increasing NPMO concentrations, while Km remained constant with Ki equal to 84.2 ±5.1 µM, indicating a non-competitive mechanism of inhibition. However, LC-MS/MS analysis showed that FAAH is hydrolyzed 92 % of NPMO in a 60 minute of reaction, indicating that NPMO is also a substrate for FAAH. These results provide valuable information regarding macamide analogs and derivatives as FAAH inhibitors. NPMO could be used as a lead compound to design promising, selective, and potent FAAH inhibitors that could be used clinically as neuroprotectants targeting the endocannabinoid system.

Lepidium meyenii (Maca), a Peruvian plant, has been used as a folk medicine for centuries. Recent studies have indicated that Maca extracts and their active constituents, macamides, act as inhibitors of fatty acid amide hydrolase (FAAH). FAAH is the enzyme responsible for the degradation of endocannabinoids. The aim of this study was to demonstrate and characterize the FAAH inhibitory effect of the macamide, N-benzylpalmitamide, and its natural analog, N-(3-methoxybenzyl)palmitamide. Each compound was tested at concentrations from 1 to 100 µM, using an FAAH inhibitory activity assay, which is a fluorescence-based method. The results demonstrated that each of the test compounds causes a concentration-dependent inhibition of FAAH. The pre-incubation study revealed that N-benzylpalmitamide and N-(3-methoxybenzyl)palmitamide inhibit FAAH in a time-dependent manner. The % inhibition of FAAH produced by 100 μM N-(3-methoxybenzyl)palmitamide without pre-incubation was comparable to that of N-benzylpalmitamide. However, a 120 min pre-incubation of inhibitor with FAAH caused a significant increase in the % inhibition produced by 100 µM N-(3-methoxybenzyl)palmitamide. The enzyme kinetics study indicated that N-(3-methoxybenzyl)palmitamide is likely an uncompetitive inhibitor of FAAH. LC-MS/MS analysis determined that N-(3-methoxybenzyl)palmitamide is a substrate of FAAH since it undergoes hydrolysis by FAAH. The results of this study indicated that N-(3-methoxybenzyl)palmitamide is a promising inhibitor of FAAH and could potentially offer a good alternative for the treatment of pain, inflammation and CNS degenerative disorders.