AGI-134: a fully synthetic α-Gal glycolipid that converts tumors into in situ autologous vaccines, induces anti-tumor immunity and is synergistic with an anti-PD-1 antibody in mouse melanoma models.

BACKGROUND: Treatments that generate T cell-mediated immunity to a patient's unique neoantigens are the current holy grail of cancer immunotherapy. In particular, treatments that do not require cumbersome and individualized ex vivo processing or manufacturing processes are especially sought after. Here we report that AGI-134, a glycolipid-like small molecule, can be used for coating tumor cells with the xenoantigen Galα1-3Galß1-4GlcNAc (α-Gal) in situ leading to opsonization with pre-existing natural anti-α-Gal antibodies (in short anti-Gal), which triggers immune cascades resulting in T cell mediated anti-tumor immunity. METHODS: Various immunological effects of coating tumor cells with α-Gal via AGI-134 in vitro were measured by flow cytometry: (1) opsonization with anti-Gal and complement, (2) antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells, and (3) phagocytosis and antigen cross-presentation by antigen presenting cells (APCs). A viability kit was used to test AGI-134 mediated complement dependent cytotoxicity (CDC) in cancer cells. The anti-tumoral activity of AGI-134 alone or in combination with an anti-programmed death-1 (anti-PD-1) antibody was tested in melanoma models in anti-Gal expressing galactosyltransferase knockout (α1,3GT-/-) mice. CDC and phagocytosis data were analyzed by one-way ANOVA, ADCC results by paired t-test, distal tumor growth by Mantel-Cox test, C5a data by Mann-Whitney test, and single tumor regression by repeated measures analysis. RESULTS: In vitro, α-Gal labelling of tumor cells via AGI-134 incorporation into the cell membrane leads to anti-Gal binding and complement activation. Through the effects of complement and ADCC, tumor cells are lysed and tumor antigen uptake by APCs increased. Antigen associated with lysed cells is cross-presented by CD8α+ dendritic cells leading to activation of antigen-specific CD8+ T cells. In B16-F10 or JB/RH melanoma models in α1,3GT-/- mice, intratumoral AGI-134 administration leads to primary tumor regression and has a robust abscopal effect, i.e., it protects from the development of distal, uninjected lesions. Combinations of AGI-134 and anti-PD-1 antibody shows a synergistic benefit in protection from secondary tumor growth. CONCLUSIONS: We have identified AGI-134 as an immunotherapeutic drug candidate, which could be an excellent combination partner for anti-PD-1 therapy, by facilitating tumor antigen processing and increasing the repertoire of tumor-specific T cells prior to anti-PD-1 treatment.

A pegylated leptin antagonist ameliorates CKD-associated cachexia in mice.

Elevated serum leptin levels correlate with inflammation and predict changes in lean body mass in patients with CKD, and activation of the melanocortin system by leptin signaling mediates the pathophysiology of CKD-associated cachexia. We tested whether treatment with a pegylated leptin receptor antagonist (PLA) attenuates cachexia in mice with CKD. CKD and Sham mice received vehicle or PLA (2 or 7 mg/kg per day). At these doses, PLA did not influence serum leptin levels in mice. Treatment with 7 mg/kg per day PLA stimulated appetite and weight gain, improved lean mass and muscle function, reduced energy expenditure, and normalized the levels of hepatic TNF-α and IL-6 mRNA in mice with CKD. Furthermore, treatment with 7 mg/kg per day PLA attenuated the CKD-associated increase in the transcriptional and protein abundance of uncoupling proteins that mediates thermogenesis, and it normalized the molecular signatures of processes associated with muscle wasting in CKD, including proteolysis, myogenesis and muscle regeneration, and expression of proinflammatory muscle cytokines, such as IL-1α, -1ß, and -6 and TNF-α. Our results suggest that leptin antagonism may represent a viable therapeutic strategy for cachexia in CKD.

DSP107 combines inhibition of CD47/SIRPα axis with activation of 4-1BB to trigger anticancer immunity.

BACKGROUND: Treatment of Diffuse Large B Cell Lymphoma (DLBCL) patients with rituximab and the CHOP treatment regimen is associated with frequent intrinsic and acquired resistance. However, treatment with a CD47 monoclonal antibody in combination with rituximab yielded high objective response rates in patients with relapsed/refractory DLBCL in a phase I trial. Here, we report on a new bispecific and fully human fusion protein comprising the extracellular domains of SIRPα and 4-1BBL, termed DSP107, for the treatment of DLBCL. DSP107 blocks the CD47:SIRPα 'don't eat me' signaling axis on phagocytes and promotes innate anticancer immunity. At the same time, CD47-specific binding of DSP107 enables activation of the costimulatory receptor 4-1BB on activated T cells, thereby, augmenting anticancer T cell immunity. METHODS: Using macrophages, polymorphonuclear neutrophils (PMNs), and T cells of healthy donors and DLBCL patients, DSP107-mediated reactivation of immune cells against B cell lymphoma cell lines and primary patient-derived blasts was studied with phagocytosis assays, T cell activation and cytotoxicity assays. DSP107 anticancer activity was further evaluated in a DLBCL xenograft mouse model and safety was evaluated in cynomolgus monkey. RESULTS: Treatment with DSP107 alone or in combination with rituximab significantly increased macrophage- and PMN-mediated phagocytosis and trogocytosis, respectively, of DLBCL cell lines and primary patient-derived blasts. Further, prolonged treatment of in vitro macrophage/cancer cell co-cultures with DSP107 and rituximab decreased cancer cell number by up to 85%. DSP107 treatment activated 4-1BB-mediated costimulatory signaling by HT1080.4-1BB reporter cells, which was strictly dependent on the SIRPα-mediated binding of DSP107 to CD47. In mixed cultures with CD47-expressing cancer cells, DSP107 augmented T cell cytotoxicity in vitro in an effector-to-target ratio-dependent manner. In mice with established SUDHL6 xenografts, the treatment with human PBMCs and DSP107 strongly reduced tumor size compared to treatment with PBMCs alone and increased the number of tumor-infiltrated T cells. Finally, DSP107 had an excellent safety profile in cynomolgus monkeys. CONCLUSIONS: DSP107 effectively (re)activated innate and adaptive anticancer immune responses and may be of therapeutic use alone and in combination with rituximab for the treatment of DLBCL patients.

The induction of tuftelin expression in PC12 cell line during hypoxia and NGF-induced differentiation.

The tuftelin protein isoforms undergo post-translation modifications, and are ubiquitously expressed in various tissues in embryos, adults, and tumors. Developmental and pathological studies suggested an apparent correlation between oxygen deprivation and tuftelin expression. The aim of the study was therefore to investigate the effect of a pathological insult (hypoxia) and a physiological growth factor (NGF), which antagonistically regulate HIF1 expression, on tuftelin expression using the neuronal PC12 cell model. In the present study, we first demonstrated the expression of tuftelin in PC12 cells, providing an experimental system to investigate the pathophysiological role of tuftelin. Furthermore, we demonstrated the induction of tuftelin during hypoxia by oxygen deprivation and during chemical hypoxia by cobalt chloride. Down-regulation of HIF1α mRNA blocked hypoxia-induced HIF1α expression, and reduced by 89% hypoxia-induced tuftelin expression. In mice, intraperitoneal injection of cobalt chloride significantly induced tuftelin mRNA and protein expression in the brain. During NGF-mediated PC12 differentiation, tuftelin expression was significantly induced in correlation with neurite outgrowth. This induction was partially blocked by K252a, a selective antagonist of the NGF receptor TrkA, indicating the involvement of the TrkA-signaling pathways in tuftelin induction by NGF. Revealing the physiological role of tuftelin will clarify mechanisms related to the "hypoxic genome," and NGF-induced neurotrophic and angiogenic effects.

In vivo administration of BL-3050: highly stable engineered PON1-HDL complexes.

BACKGROUND: Serum paraoxonase (PON1) is a high density lipoprotein (HDL)-associated enzyme involved in organophosphate (OP) degradation and prevention of atherosclerosis. PON1 comprises a potential candidate for in vivo therapeutics, as an anti-atherogenic agent, and for detoxification of pesticides and nerve agents. Because human PON1 exhibits limited stability, engineered, recombinant PON1 (rePON1) variants that were designed for higher reactivity, solubility, stability, and bacterial expression, are candidates for treatment. This work addresses the feasibility of in vivo administration of rePON1, and its HDL complex, as a potentially therapeutic agent dubbed BL-3050. METHODS: For stability studies we applied different challenges related to the in vivo disfunctionalization of HDL and PON1 and tested for inactivation of PON1's activity. We applied acute, repetitive administrations of BL-3050 in mice to assess its toxicity and adverse immune responses. The in vivo efficacy of recombinant PON1 and BL-3050 were tested with an animal model of chlorpyrifos-oxon poisoning. RESULTS: Inactivation studies show significantly improved in vitro lifespan of the engineered rePON1 relative to human PON1. Significant sequence changes relative to human PON1 might hamper the in vivo applicability of BL-3050 due to adverse immune responses. However, we observed no toxic effects in mice subjected to repetitive administration of BL-3050, suggesting that BL-3050 could be safely used. To further evaluate the activity of BL-3050 in vivo, we applied an animal model that mimics human organophosphate poisoning. In these studies, a significant advantages of rePON1 and BL-3050 (>87.5% survival versus <37.5% in the control groups) was observed. Furthermore, BL-3050 and rePON1 were superior to the conventional treatment of atropine-2-PAM as a prophylactic treatment for OP poisoning. CONCLUSION: In vitro and in vivo data described here demonstrate the potential advantages of rePON1 and BL-3050 for treatment of OP toxicity and chronic cardiovascular diseases like atherosclerosis. The in vivo data also suggest that rePON1 and BL-3050 are stable and safe, and could be used for acute, and possibly repeated treatments, with no adverse effects.

Neuroprotective effects of the stable nitroxide compound Tempol on 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in the Nerve Growth Factor-differentiated model of pheochromocytoma PC12 cells.

Nerve growth factor (NGF) differentiated pheochromocytoma PC12 cells exposed to 1-methyl-4-phenylpyridinium (MPP+) toxin were used as an in vitro pharmacological model of Parkinson's disease to examine the neuroprotective effects of 4-hydroxy-2,2,6,6-tetramethyl piperidine-n-oxyl (Tempol), a free radical scavenger and a superoxide dismutase-mimetic compound. MPP+-induced PC12 cell death was measured 72 h after exposure to 1.5 mM MPP+ by the release of lactate dehydrogenease, caspase-3 activation and stimulation of survival and stress mitogen-activated protein kinases. Exposure of PC12 cells to MPP+ activated ERK1 and ERK2 (forty-fold over control after 72 h), JNK1 and JNK2 (fourfold after 48 h) and p-38alpha (tenfold after 24 h). Pretreatment of PC12 cells with 500 microM Tempol, 1 h before induction of the MPP+ insult, reduced by 70% the release of LDH into the medium, inhibited caspase-3 activity by 30% and improved by 33% mitochondrial function, effects correlated with a 70% reduction in ERK1 and ERK2 phosphorylation activity. These findings support the neuroprotective effect of Tempol in the MPP+-induced PC12 cell death model and its use as a potential drug for treatment of Parkinson's disease.

Neuroprotection by NGF in the PC12 in vitro OGD model: involvement of mitogen-activated protein kinases and gene expression.

Neurodegenerative disorders and chronic disability due to stroke in the brain or spinal cord afflict a large sector of the population. To investigate the mechanism involved in ischemic stroke and to develop neuroprotective drugs/therapies, in vivo and in vitro, pharmacological models are needed. To investigate the cellular and molecular neuroprotective mechanisms of nerve growth factor (NGF), a member of the nervous system neurotrophin family of growth factors, under ischemia, we used an oxygen-glucose-deprivation (OGD) device and pheochromocytoma PC12 cells exposed to a paradigm of ischemic insult. Pretreatment of the cultures with 50 ng/mL of NGF, 18 h prior to OGD insult, conferred 30% of neuroprotection. Time-course experiments showed marked activation of the ERK, JNK, and p-38 MAPK isoforms during the OGD phase, but not during OGD reperfusion. Pretreatment of the cultures with 50 ng/mL of NGF, 18 h prior to OGD insult, resulted in 50% attenuation of OGD-induced activation of JNK 1, and 20% and 50% attenuation of OGD-induced activation of p-38 alpha and beta, respectively. The effect of NGF on gene expression in the PC12 ischemic model using Affymatrix Rat DNA-Microarray technology indicates that only 6% of the genes are differentially regulated (induced/suppressed) by OGD insult and/or NGF. These findings support the notion that pretreatment with NGF confers neuroprotection from OGD insult, a phenomenon coincidentally related to differential inhibition of MAPK stress kinase isoforms and differential gene expression. This ischemic model may be useful to investigate molecular mechanisms of OGD-induced neurotoxicity and NGF-induced neuroprotection, and to generate novel therapeutic concepts for stroke treatment.

Nerve growth factor pretreatment attenuates oxygen and glucose deprivation-induced c-Jun amino-terminal kinase 1 and stress-activated kinases p38alpha and p38beta activation and confers neuroprotection in the pheochromocytoma PC12 Model.

Neurotrophins such as nerve growth factor (NGF) are considered putative neuroprotective compounds in the central nervous system. To investigate the cellular and molecular neuroprotective mechanisms of NGF under ischemia, we used a unique oxygen and glucose deprivation (OGD) device. In this system we used pheochromocytoma PC12 cells to elucidate NGF neuroprotective effect. PC12 cells were exposed to OGD, followed by addition of glucose and oxygen (OGD reperfusion). Neuronal cell death induced in this model was measured by the release of lactate dehydrogenase (LDH), activation of caspase-3 and mitogen-activated protein kinases (MAPKs), measured with specific anti-phospho-antibodies. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, conferred 30% neuroprotection. However, treatment of the cultures with NGF concomitantly with the OGD insult did not result in neuroprotection. Time-course experiments showed marked activation of extracellular signal-regulated protein kinase, c-Jun N-terminal kinase (JNK), and p38 MAPK isoforms during the OGD phase but not during OGD reperfusion. Pretreatment of the cultures with 50 ng/mL NGF, 18 h prior to OGD insult, resulted in 50% attenuation of OGD-induced activation of JNK1, and 20% and 50% attenuation of OGD-induced activation of p38alpha and beta, respectively. These findings support the notion that NGF confers neuroprotection from OGD insult, a phenomenon coincidentally related to differential inhibition of MAPK stress kinase isoforms, and provide the PC12 model as an in vitro OGD system to investigate molecular mechanisms of neurotoxicity and neuroprotection.

Interactions between the cells of the immune and nervous system: neurotrophins as neuroprotection mediators in CNS injury.

Inflammatory processes in the central nervous system (CNS) are considered neurotoxic, although recent studies suggest that they also can be beneficial and confer neuroprotection (neuroprotective autoimmunity). Cells from the immune system have been detected in CNS injury and found to produce and secrete a variety of neurotrophins such as NGF, BDNF, NT-3 and NT-4/5, and to express (similarly to neuronal cells), members of the tyrosine kinase (Trk) receptor family such as TrkA, TrkB and TrkC. Indeed, autocrine and paracrine interactions are observed at the site of CNS injury, resulting in a variety of homologic-heterologic modulations of immune and neuronal cell function. The end result of the inflammatory process, neurotoxicity and/or neuroprotection, is a function of the fine balance between the two cellular systems, i.e., of the complex signaling relationships between anti-inflammatory neuroprotective factors (neurotrophins and other chemical mediators) and proinflammatory neurotoxic factors (TNF, free radicals, certain cytokines, etc.). Autoimmune neuroprotection is a novel therapeutic approach aimed at shifting the balance between the immune and neuronal cells towards survival pathways in a variety of CNS injuries. This review focuses on data supporting this concept and its future therapeutical implications for optic nerve injury and multiple sclerosis.

Neuroprotective and neurotoxic effects of monoamine oxidase-B inhibitors and derived metabolites under ischemia in PC12 cells.

Selegiline and rasagiline are selective and irreversible monoamine oxidase-B inhibitors that exert neuroprotective effects in various preclinical models. The aim of the present study was to examine the effect of selegiline and its major metabolite, L-methamphetamine in comparison to rasagiline and its major metabolite, 1-R-aminoindan on oxygen-glucose deprivation induced cell death in nerve growth factor (NGF)-differentiated pheochromocytoma (PC12) cells. Our results show that selegiline reduces oxygen-glucose deprivation induced cell death by 30%. When the cultures were treated with rasagiline at similar concentrations, cell death induced by oxygen-glucose deprivation was reduced by 45-55%. L-methamphetamine, a major selegiline metabolite, but not 1-R-aminoindan, the major rasagiline metabolite, enhanced oxygen-glucose deprivation-induced cell death by 70%. Under normoxic conditions, both metabolites lack neurotoxicity. Concomitant exposure of the cultures under oxygen-glucose deprivation, to a combination of either selegiline and L-methamphetamine or rasagiline and 1-R-aminoindan, indicated that L-methamphetamine, but not 1-R-aminoindan, blocked the neuroprotective effect of the parental drug. These results suggest there may be a neuroprotective advantage of rasagiline over selegiline.

BL-7010 demonstrates specific binding to gliadin and reduces gluten-associated pathology in a chronic mouse model of gliadin sensitivity.

Celiac disease (CD) is an autoimmune disorder in individuals that carry DQ2 or DQ8 MHC class II haplotypes, triggered by the ingestion of gluten. There is no current treatment other than a gluten-free diet (GFD). We have previously shown that the BL-7010 copolymer poly(hydroxyethyl methacrylate-co-styrene sulfonate) (P(HEMA-co-SS)) binds with higher efficiency to gliadin than to other proteins present in the small intestine, ameliorating gliadin-induced pathology in the HLA-HCD4/DQ8 model of gluten sensitivity. The aim of this study was to investigate the efficiency of two batches of BL-7010 to interact with gliadin, essential vitamins and digestive enzymes not previously tested, and to assess the ability of the copolymer to reduce gluten-associated pathology using the NOD-DQ8 mouse model, which exhibits more significant small intestinal damage when challenged with gluten than HCD4/DQ8 mice. In addition, the safety and systemic exposure of BL-7010 was evaluated in vivo (in rats) and in vitro (genetic toxicity studies). In vitro binding data showed that BL-7010 interacted with high affinity with gliadin and that BL-7010 had no interaction with the tested vitamins and digestive enzymes. BL-7010 was effective at preventing gluten-induced decreases in villus-to-crypt ratios, intraepithelial lymphocytosis and alterations in paracellular permeability and putative anion transporter-1 mRNA expression in the small intestine. In rats, BL-7010 was well-tolerated and safe following 14 days of daily repeated administration of 3000 mg/kg. BL-7010 did not exhibit any mutagenic effect in the genetic toxicity studies. Using complementary animal models and chronic gluten exposure the results demonstrate that administration of BL-7010 is effective and safe and that it is able to decrease pathology associated with gliadin sensitization warranting the progression to Phase I trials in humans.

Neuroprotective effects of nimodipine and nifedipine in the NGF-differentiated PC12 cells exposed to oxygen-glucose deprivation or trophic withdrawal.

The goal of this study was to compare the neuroprotective properties of the L-type Ca²âº channel blockers, nimodipine and nifedipine, using nerve growth factor (NGF)-differentiated PC12 neuronal cultures exposed to oxygen-glucose deprivation (OGD) and trophic withdrawal-induced cell death. Nimodipine (1-100 µM) conferred 65±13% neuroprotection upon exposure to OGD and 35±6% neuroprotection towards different trophic withdrawal-induced cell death measured by lactate dehydrogenase and caspase 3 activities. The time window of nimodipine conferred neuroprotection was detected during the first 5h but not at longer OGD exposures. Nifedipine (1-100 µM), to a lower potency than nimodipine, conferred 30-55±8% neuroprotection towards OGD in PC12 cells and 29±5% in rat hypocampal slices, and 10±3% neuroprotection at 100 µM towards trophic withdrawal-induced PC12 cell death. The ability to demonstrate that nimodipine conferred neuroprotection in a narrow therapeutic time-window indicates that the OGD PC12 model mimics the in vivo models and therefore suitable for neuroprotective drug discovery and development.

Pharmacokinetic and efficacy study of cisplatin and paclitaxel formulated in a new injectable poly(sebacic-co-ricinoleic acid) polymer.

Injectable biodegradable polymer poly(sebacic-co-ricinoleic acid), P(SA-RA) is currently under development for intratumoral (IT) delivery of drugs for treating solid tumors. This study presents formulation development, pharmacokinetic and efficacy studies of two anticancer drugs (cisplatin and paclitaxel) formulated with P(SA-RA) polymer. In pharmacokinetic study, systemic exposure and pharmacokinetic parameters of cisplatin/paclitaxel following single intravenous (IV) or subcutaneous (SC) doses of cisplatin/paclitaxel was compared with intramuscular (IM) or SC doses of cisplatin/paclitaxel formulated with P(SA-RA) polymer in male CD rat. Simultaneously, the tumor reduction effect and toxicity for these formulations were evaluated in human FaDu head and neck tumor xenograft subcutaneous nude mouse model. Pharmacokinetic data reflect the lower maximal concentrations and sustained release of polymer-cisplatin/paclitaxel formulations compared to standard cisplatin/paclitaxel administration. Regarding efficacy study, a single IT or near the tumor injection (NT) of polymer-paclitaxel or polymer-cisplatin formulation significantly reduced the tumor size, compared to the standard paclitaxel or cisplatin treatments. No death or toxicity and no effect on body weight as well as macroscopic and/or microscopic changes in or near the injected area were observed, proving biocompatibility and acceptability of polymer-formulations. In conclusion, the developed formulation demonstrated controlled release and significant efficacy in delivering these agents and exhibit potential for further clinical development.

Improved muscle strength and mobility in the dy(2J)/dy(2J) mouse with merosin deficient congenital muscular dystrophy treated with Glatiramer acetate.

The therapeutic effect of Glatiramer acetate, an immune modulating agent, was evaluated in the dy(2J)/dy(2J) mouse with merosin deficient congenital muscular dystrophy, which is a milder variant of the dy/dy mouse. The treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter and in motor performance quantified by video detection software. Glatiramer acetate treatment was associated with significantly increased expression of regeneration transcription factors MyoD and myogenin, and attenuation of the fibrosis markers vimentin and fibronectin. No effective treatment is currently available in congenital muscular dystrophy and Glatiramer acetate may present a new potential treatment for this disorder.

Neuroprotection by cord blood neural progenitors involves antioxidants, neurotrophic and angiogenic factors.

Human umbilical cord blood (HUCB) is a valuable source for cell therapy since it confers neuroprotection in stroke animal models. However, the responsible sub-populations remain to be established and the mechanisms involved are unknown. To explore HUCB neuroprotective properties in a PC12 cell-based ischemic neuronal model, we used an HUCB mononuclear-enriched population of collagen-adherent cells, which can be differentiated in vitro into a neuronal phenotype (HUCBNP). Upon co-culture with insulted-PC12 cells, HUCBNP conferred approximately 30% neuroprotection, as evaluated by decreased lactate dehydrogenase and caspase-3 activities. HUCBNP decreased by 95% the level of free radicals in the insulted-PC12 cells, in correlation with the appearance of antioxidants, as measured by changes in the oxidation-reduction potential of the medium using cyclic-voltammetry. An increased level of nerve growth factor (NGF), vascular endothelial growth factor and basic fibroblast growth factor in the co-culture medium was temporally correlated with a -medium neuroprotection effect, which was partially abolished by heat denaturation. HUCBNP-induced neuroprotection was correlated with changes in gene expression of these neurotrophic factors, while blocked by K252a, an antagonist of the TrkA/NGF receptor. These findings indicate that HUCBNP-induced neuroprotection involves antioxidant(s) and neurotrophic factors, which, by paracrine and/or autocrine interactions between the insulted-PC12 and the HUCBNP cells, conferred neuroprotection.

The antioxidant tempamine: in vitro antitumor and neuroprotective effects and optimization of liposomal encapsulation and release.

The piperidine nitroxide tempamine (TMN) is a cell-permeable, stable radical having antioxidant, anticancer, and proapoptotic and/or pronecrotic activities, as was demonstrated by us in cell cultures. We also demonstrated synergism between TMN and doxorubicin in doxorubicin-sensitive and doxorubicin-resistant cell lines. Treatment of the C26 mouse colon carcinoma model in vivo also demonstrated synergism between TMN and doxorubicin in sterically stabilized liposomes (SSLs) containing TMN (SSL-TMN) and those containing doxorubicin. The above effects of TMN and SSL-TMN motivated us to develop and optimize the SSL-TMN formulation so that it will be able to reach the disease site with a sufficiently high TMN level and a release rate needed to achieve a therapeutic effect. Because TMN is an amphipathic weak base, it was remote loaded by an intraliposome high/extraliposome low transmembrane ammonium sulfate gradient. The kinetics and level of TMN loading were monitored by cyclic voltammetry (CV) and electron paramagnetic resonance (EPR); the latter also indicates TMN precipitation in the intraliposomal aqueous phase. The regeneration of the original CV and EPR signals by the ionophore nigericin indicates that TMN remained fully intact during loading and release. The cardinal role of the transmembrane ammonium ion gradient in the loading process was proven by the use of the selective ionophores nonactin (for NH4+) and nigericin (for H+). The anion of the ammonium salts affects loading stability and the rate of TMN release, both mediated through the TMN state of aggregation in the intraliposomal aqueous phase. The greater the TMN salt precipitation, the slower the TMN release rate. This was supported by measurement of osmolality, which is inversely related to TMN salt precipitate. Precipitation is in the order SO4(-2)>Cl-1>glucuronate-1. Liposome lipid composition, magnitude of the transmembrane ammonium ion gradient, and type of anion of the ammonium salt determine the amount of TMN loaded and its release rate.

Neuroprotective effects of carnosine and homocarnosine on pheochromocytoma PC12 cells exposed to ischemia.

The development of neuroprotective drugs against ischemic insults is hampered by the lack of pharmacological in vitro models. We developed an ischemic model using PC12 cell cultures exposed to oxygen-glucose-deprivation (OGD) followed by reoxygenation (18 hr) under regular atmospheric oxygen level. The toxicity induced in this model, that is partially caused by generation of reactive oxygen species (ROS), was measured morphologically as well as by the release of lactate dehydrogenase (LDH) and the prostaglandin PGE(2) from the cells. Carnosine and homocarnosine, histidine dipeptides antioxidants, found in high concentration in the brain, have been suggested to provide neuroprotection. Using the OGD model we found that 5 mM carnosine and 1 mM homocarnosine provided maximal neuroprotection of about 50% against OGD insult. This neuroprotective effect was similar to that of a known antioxidant, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol), and was not observed in a serum-deprivation toxicity model of PC12 cells, indicating that carnosine and homocarnosine may act as antioxidant-neuroprotective agents in the brain. Our ischemic model may provide a useful tool for investigating the mechanisms involved in the neuroprotection afforded by histidine dipeptides.

Neuroprotection by monoamine oxidase B inhibitors: a therapeutic strategy for Parkinson's disease?

Parkinsonism (PD) is a neurodegenerative disorder of the brain resulting in dopamine deficiency caused by the progressive death of dopaminergic neurons. PD is characterized by a combination of rigidity, poverty of movement, tremor and postural instability. Selegiline is a selective and irreversible propargylamine type B monoamine oxidase (MAO-B) inhibitor. This drug, which inhibits dopamine metabolism, has been effectively used in the treatment of PD. However, its therapeutic effects are compromised by its many neurotoxic metabolites. To circumvent this obstacle, a novel MAO-B inhibitor, rasagiline, was developed. Paradoxically, the neuroprotective mechanism of propargylamines in different neuronal models appears to be independent of MAO-B inhibition. Recent investigations into the neuroprotective mechanism of propargylamines indicate that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), MAO-B and/or other unknown proteins may represent pivotal proteins in the survival of the injured neurons. Delineation of the mechanism(s) involved in the neuroprotective effects exerted by MAO-B inhibitors may provide the key to preventive novel therapeutic modalities.

Apoptotic characteristics of cell death and the neuroprotective effect of homocarnosine on pheochromocytoma PC12 cells exposed to ischemia.

We recently improved an in vitro ischemic model, using PC12 neuronal cultures exposed to oxygen-glucose deprivation (OGD) for 3 hr in a special device, followed by 18 hr of reoxygenation. The cell death induced in this ischemic model was evaluated by a series of markers: lactate dehydrogenase (LDH) release, caspase-3 activation, presence of cyclin D1, cytochrome c leakage from the mitochondria, BAX cellular redistribution, cleavage of poly (ADP-ribose) polymerase (PARP) to an 85-kDa apoptotic fragment, and DNA fragmentation. The OGD insult, in the absence of reoxygenation, caused a strong activation of the mitogen-activated protein kinase (MAPK) isoforms extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and stress-activated protein kinase (SAPK), also known as p-38. The detection of apoptotic markers and activation of MAPKs during the ischemic insult strongly suggest that apoptosis plays an important role in the PC12 cell death. Homocarnosine, a neuroprotective histidine dipeptide, present in high concentrations in the brain, was found to provide neuroprotection, as expressed by a 40% reduction in LDH release and caspase-3 activity at 1 mM. Homocarnosine reduced OGD activation of ERK 1, ERK 2, JNK 1, and JNK 2 by 40%, 46%, 55%, and 30%, respectively. These results suggest that apoptosis is an important characteristic of OGD-induced neuronal death and that antioxidants, such as homocarnosine, may prevent OGD-induced neuronal death by inhibiting the apoptotic process and/or in relation to the differential attenuation of activity of MAPKs.