Therapeutic Effects of Geranium Oil in MPTP-Induced Parkinsonian Mouse Model.

Parkinson's disease (PD) is an incurable neurodegenerative disease characterized by motor and non-motor disabilities resulting from neuronal cell death in the substantia nigra and striatum. Microglial activation and oxidative stress are two of the primary mechanisms driving that neuronal death. Here, we evaluated the effects of geranium oil on 1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine (MPTP) mouse model for PD, on microglial activation, and oxidative stress. We demonstrate that oral treatment with geranium oil improved motor performance in this model. The therapeutic effects of geranium oil were observed as a significant increase in rotarod latency and distance among the mice treated with geranium oil, as compared to vehicle-treated MPTP mice. Geranium oil also prevented dopaminergic neuron death in the substantia nigra of the treated mice. These therapeutic effects can be partially attributed to the antioxidant and anti-inflammatory properties of geranium oil, which were observed as attenuated accumulation of reactive oxygen species and inhibition of the secretion of proinflammatory cytokines from geranium oil-treated activated microglial cells. A repeated-dose oral toxicity study showed that geranium oil is not toxic to mice. In light of that finding and since geranium oil is defined by the FDA as generally recognized as safe (GRAS), we do not foresee any toxicity problems in the future and suggest that geranium oil may be a safe and effective oral treatment for PD. Since the MPTP model is only one of the preclinical models for PD, further studies are needed to confirm that geranium oil can be used to prevent or treat PD.

Neuroprotective Effects of Pulicaria incisa Infusion on Human Neuroblastoma Cells and Hippocampal Neurons.

Reactive oxygen species (ROS) and oxidative stress increase susceptibility to neurodegeneration and other age-related pathologies. We have previously demonstrated that an infusion prepared from Pulicaria incisa (Pi) has protective, anti-inflammatory, and antioxidative effects in glial cells. However, the neuroprotective activities of Pi infusion in cultured neurons and aging mice have never been studied. In the following study, the effects of Pi infusion were explored in a hydrogen peroxide (H2O2)-induced oxidative stress model in SH-SY5Y human neuroblastoma cells. Profiling of the infusion by gas chromatography-mass spectrometry identified chlorogenic acid, quercetin, and aucubin as some of its main constituents. H2O2-induced ROS accumulation and caspase 3 activity decreased SH-SY5Y viability and were prevented upon the pretreatment of cells with Pi infusion. Additionally, the Pi infusion upregulated cellular levels and the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) as well as the phosphorylation of cyclic AMP response element-binding protein (CREB). Aging mice treated daily for 18 months with Pi infusion exhibited reduced neuronal cell death in the hippocampus as compared to age-matched controls. We, therefore, propose Pi infusion as a candidate regulator of oxidative stress in the brain.

Phytochemicals from Achillea fragrantissima are Modulators of AßPP Metabolism.

Plant derivatives offer a novel and natural source of therapeutics. The desert plant Achillea fragrantissima (Forssk) Sch. Bip (Af) is characterized by protective antioxidative and anti-inflammatory properties. Here, we examined the effect of two Af-derived phytochemicals on learning and memory, amyloid-ß protein precursor (AßPP) metabolism, and tau phosphorylation in the familial Alzheimer's disease-linked APPswe/PS1ΔE9 mouse model. We observed that mice that were injected with the phytochemicals showed a trend of improvement, albeit statistically insignificant, in the Novel Object Recognition task. However, we did not observe improvement in contextual fear conditioning, suggesting that the benefits of treatment may be either indirect or task-specific. In addition, we observed an increase in the full-length form of AßPP in the brains of mice treated with Af-derived phytochemicals. Interestingly, both in vivo and in vitro, there was no change in levels of soluble Aß, oligomeric Aß, or the carboxyl terminus fragments of AßPP (APP-CTFs), suggesting that the increase in full length AßPP does not exacerbate AßPP pathology, but may stabilize the full-length form of the molecule. Together, our data suggest that phytochemicals present in Af may have a modest positive impact on the progression of Alzheimer's disease.

ß-amyloid cytotoxicity is prevented by natural achillolide A.

Alzheimer's disease (AD) is the most prevalent cause of dementia in adults. Current available drugs for AD transiently alleviate some of the symptoms, but do not modify the disease mechanism or cure it. Therefore, new drugs are desperately needed. Key contributors to AD are amyloid beta (Aß)- and reactive oxygen species (ROS)-induced cytotoxicities. Plant-derived substances have been shown to affect various potential targets in various diseases including AD. Therefore, phytochemicals which can protect neuronal cells against these insults might help in preventing and treating this disease. In the following research, we have isolated the sesquiterpene lactone achillolide A from the plant Achillea fragrantissima and, for the first time, characterized its effects on Aß-treated neuroblastoma cells. Aß is a peptide derived from the sequential cleavage of amyloid precursor protein, and is part of the pathogenesis of AD. Our current study aimed to determine whether achillolide A can interfere with Aß-induced processes in Neuro2a cells, and protect them from its toxicity. Our results show that achillolide A decreased Aß-induced death and enhanced the viability of Neuro2a cells. In addition, achillolide A reduced the accumulation of Aß-induced ROS and inhibited the phosphorylation of stress-activated protein kinase/c-Jun N-terminal kinase and p44/42 mitogen-activated protein kinase in these cells. We therefore suggest that achillolide A may have therapeutic potential for the treatment of AD.

3,5,4'-trihydroxy-6,7,3'-trimethoxyflavone protects against beta amyloid-induced neurotoxicity through antioxidative activity and interference with cell signaling.

BACKGROUND: Alzheimer's disease is a neurodegenerative disease, characterized by progressive decline in memory and cognitive functions, that results from loss of neurons in the brain. Amyloid beta (Aß) protein and oxidative stress are major contributors to Alzheimer's disease, therefore, protecting neuronal cells against Aß-induced toxicity and oxidative stress might form an effective approach for treatment of this disease. 3,5,4'-trihydroxy-6,7,3'-trimethoxyflavone (TTF) is a flavonoid we have purified from the plant Achillea fragrantissima; and the present study examined, for the first time, the effects of this compound on Aß-toxicity to neuronal cells. METHODS: Various chromatographic techniques were used to isolate TTF from the plant Achillea fragrantissima, and an N2a neuroblastoma cell line was used to study its activities. The cellular levels of total and phosphorylated stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and of total and phosphorylated extracellular signal-regulated kinase (ERK 1/2) were determined by enzyme-linked immunosorbent assay (ELISA). Intracellular reactive oxygen species (ROS) levels were measured by using 2',7'-dichlorofluorescein diacetate (DCF-DA). Cytotoxicity and cell viability were assessed by using lactate dehydrogenase (LDH) activity in cell-conditioned media, or by crystal violet cell staining, respectively. RESULTS: TTF prevented the Aß-induced death of neurons and attenuated the intracellular accumulation of ROS following treatment of these cells with Aß. TTF also inhibited the Aß-induced phosphorylation of the signaling proteins SAPK/JNK and ERK 1/2, which belong to the mitogen-activated protein kinase (MAPK) family. CONCLUSION: TTF should be studied further as a potential therapeutic means for the treatment of Alzheimer's disease.

Glutamate Toxicity to Differentiated Neuroblastoma N2a Cells Is Prevented by the Sesquiterpene Lactone Achillolide A and the Flavonoid 3,5,4'-Trihydroxy-6,7,3'-Trimethoxyflavone from Achillea fragrantissima.

Glutamate toxicity is a major contributor to the pathophysiology of numerous neurodegenerative diseases including amyotrophic lateral sclerosis and Alzheimer's disease. Therefore, protecting neuronal cells against glutamate-induced cytotoxicity might be an effective approach for the treatment of these diseases. We have previously purified from the medicinal plant Achillea fragrantissima two bioactive compounds which were not studied before: the sesquiterpene lactone achillolide A and the flavonoid 3,5,4'-trihydroxy-6,7,3'-trimethoxyflavone (TTF). We have shown that these compounds protect astrocytes from oxidative stress-induced cell death and inhibit microglial activation. The current study examined for the first time their effects on differentiated mouse neuroblastoma N2a cells and on glutamate toxicity. We have found that, although these compounds belong to different chemical families, they protect neuronal cells from glutamate toxicity. We further demonstrate that this protective effect might be, at least partially, due to inhibitory effects of these compounds on the levels of reactive oxygen species produced following treatment with glutamate.

Achillolide A Protects Astrocytes against Oxidative Stress by Reducing Intracellular Reactive Oxygen Species and Interfering with Cell Signaling.

Achillolide A is a natural sesquiterpene lactone that we have previously shown can inhibit microglial activation. In this study we present evidence for its beneficial effects on astrocytes under oxidative stress, a situation relevant to neurodegenerative diseases and brain injuries. Viability of brain astrocytes (primary cultures) was determined by lactate dehydrogenase (LDH) activity, intracellular ROS levels were detected using 2',7'-dichlorofluorescein diacetate, in vitro antioxidant activity was measured by differential pulse voltammetry, and protein phosphorylation was determined using specific ELISA kits. We have found that achillolide A prevented the H2O2-induced death of astrocytes, and attenuated the induced intracellular accumulation of reactive oxygen species (ROS). These activities could be attributed to the inhibition of the H2O2-induced phosphorylation of MAP/ERK kinase 1 (MEK1) and p44/42 mitogen-activated protein kinases (MAPK), and to the antioxidant activity of achillolide A, but not to H2O2 scavenging. This is the first study that demonstrates its protective effects on brain astrocytes, and its ability to interfere with MAPK activation. We propose that achillolide A deserves further evaluation for its potential to be developed as a drug for the prevention/treatment of neurodegenerative diseases and brain injuries where oxidative stress is part of the pathophysiology.

Downregulation of microglial activation by achillolide A.

Chronic inflammation has been implicated in the pathogenesis of various neurodegenerative diseases. During the neuroinflammatory process, microglial cells release neurotoxic and proinflammatory mediators. In the present study, using activity-guided fractionation, we have purified an anti-inflammatory compound determined by spectroscopic methods to be a sesquiterpene lactone named achillolide A from Achillea fragrantissima (Forsk.) Sch. Bip. In primary cultures of lipopolysaccharide-activated microglial cells, achillolide A inhibited the lipopolysaccharide-induced levels of proinflammatory and toxic mediators including glutamate, nitric oxide, matrix metalloproteinase-9, cyclooxygenase-2, induced nitric oxide synthase, interleukin-1ß, and tumor necrosis factor-α. Achillolide A also exhibited an antioxidant capacity, as was shown in a cell free system as well as by its ability to reduce intracellular reactive oxygen species levels in microglial cells. Thus, achillolide A might have therapeutic potential for treatment of neurodegenerative diseases and deserves further studies.

3,5,4'-Trihydroxy-6,7,3'-trimethoxyflavone protects astrocytes against oxidative stress via interference with cell signaling and by reducing the levels of intracellular reactive oxygen species.

Oxidative stress is tightly involved in various neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, and conditions such as ischemia. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species (ROS) and provide them with trophic support. Therefore, any damage to astrocytes will affect neuronal survival. In a previous study we have demonstrated that an extract prepared from the plant Achillea fragrantissima (Af) prevented the oxidative stress-induced death of astrocytes and attenuated the intracellular accumulation of ROS in astrocytes under oxidative stress. In the present study, using activity guided fractionation, we have purified from this plant the active compound, determined to be a flavonoid named 3,5,4'-trihydroxy-6,7,3'-trimethoxyflavone (TTF). The effects of TTF in any biological system have not been studied previously, and this is the first study to characterize the anti-oxidant and protective effects of this compound in the context of neurodegenerative diseases. Using primary cultures of astrocytes we have found that TTF prevented the hydrogen peroxide (H2O2)-induced death of astrocytes, and attenuated the intracellular accumulation of ROS following treatment of these cells with H2O2 or the peroxyl radicals generating molecule 2,2'-Azobis(amidinopropane) (ABAP). TTF also interfered with cell signaling events and inhibited the phosphorylation of the signaling proteins stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), extracellular signal regulated kinase (ERK 1/2) and mitogen activated protein kinase kinase (MEK1) and the phosphorylation of the transcription factor cyclic AMP response element-binding protein (CREB). The mechanism of the protective effect of TTF against H2O2-cytotoxicity could not be attributed to a direct H2O2 scavenging but rather to the scavenging of free radicals as was shown in cell free systems. Thus, TTF might be a therapeutic candidate for the prevention/treatment of neurodegenerative diseases where oxidative stress is part of the pathophysiology.

Protective and antioxidant effects of a chalconoid from Pulicaria incisa on brain astrocytes.

Oxidative stress is involved in the pathogenesis of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species (ROS) and provide them with trophic support, such as glial-derived neurotrophic factor (GDNF). Thus, any damage to astrocytes will affect neuronal survival. In the present study, by activity-guided fractionation, we have purified from the desert plant Pulicaria incisa two protective compounds and determined their structures by spectroscopic methods. The compounds were found to be new chalcones-pulichalconoid B and pulichalconoid C. This is the first study to characterize the antioxidant and protective effects of these compounds in any biological system. Using primary cultures of astrocytes, we have found that pulichalconoid B attenuated the accumulation of ROS following treatment of these cells with hydrogen peroxide by 89% and prevented 89% of the H2O2-induced death of astrocytes. Pulichalconoid B exhibited an antioxidant effect both in vitro and in the cellular antioxidant assay in astrocytes and microglial cells. Pulichalconoid B also caused a fourfold increase in GDNF transcription in these cells. Thus, this chalcone deserves further studies in order to evaluate if beneficial therapeutic effect exists.

Antioxidant and astroprotective effects of a Pulicaria incisa infusion.

Oxidative stress is involved in the pathogenesis of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. Astrocytes, the most abundant glial cells in the brain, protect neurons from reactive oxygen species (ROS) and provide them with trophic support, such as glial-derived neurotrophic factor (GDNF). Thus, any damage to astrocytes will affect neuronal survival. In the present study, an infusion prepared from the desert plant Pulicaria incisa (Pi) was tested for its protective and antioxidant effects on astrocytes subjected to oxidative stress. The Pi infusion attenuated the intracellular accumulation of ROS following treatment with hydrogen peroxide and zinc and prevented the H(2)O(2)-induced death of astrocytes. The Pi infusion also exhibited an antioxidant effect in vitro and induced GDNF transcription in astrocytes. It is proposed that this Pi infusion be further evaluated for use as a functional beverage for the prevention and/or treatment of brain injuries and neurodegenerative diseases in which oxidative stress plays a role.

Polyphenols activate Nrf2 in astrocytes via H2O2, semiquinones, and quinones.

Polyphenols, which occur both in edible plants and in foodstuff, have been reported to exert a wide range of health effects; however, the mechanism of action of these molecules is not fully understood. One important cellular pathway affected by polyphenols is the activation of the transcription factor Nrf2 via the electrophile response element, which mediates generation of phase 2 detoxifying enzymes. Our study found that Nrf2 nuclear translocation and the activity of NAD(P)H quinone oxidoreductase (NQO1) were increased significantly after treatment of astrocytes with tert-butylhydroquinone (tBHQ), resveratrol, or curcumin, at 20-50µM. Incubation of tBHQ, resveratrol, and curcumin in the growth medium in the absence of astrocytes caused the accumulation of H(2)O(2). Treatment of cells with either glutathione or metmyoglobin was found to decrease Nrf2 translocation and NQO1 activity induced by polyphenols by up to 40 and 60%, respectively. Addition of both glutathione and metmyoglobin to growth medium decreased Nrf2 translocation and NQO1 activity by up to 100 and 80%, respectively. In conclusion, because metmyoglobin, in the presence of polyphenols and glutathione, is known to interact with H(2)O(2), semiquinones, and quinones, the up-regulation of the antioxidant defense of the cells through activation of the Nrf2 transcription factor, paradoxically, occurs via the generation of H(2)O(2) and polyphenol-oxidized species generated from the exogenous microenvironment of the cells.

Anti-neuroinflammatory effects of the extract of Achillea fragrantissima.

BACKGROUND: The neuroinflammatory process plays a central role in the initiation and progression of neurodegenerative diseases such as Parkinson's and Alzheimer's diseases, and involves the activation of brain microglial cells. During the neuroinflammatory process, microglial cells release proinflammatory mediators such as cytokines, matrix metalloproteinases (MMP), Reactive oxygen species (ROS) and nitric oxide (NO). In the present study, extracts from 66 different desert plants were tested for their effect on lipopolysaccharide (LPS) - induced production of NO by primary microglial cells. The extract of Achillea fragrantissima (Af), which is a desert plant that has been used for many years in traditional medicine for the treatment of various diseases, was the most efficient extract, and was further studied for additional anti-neuroinflammatory effects in these cells. METHODS: In the present study, the ethanolic extract prepared from Af was tested for its anti-inflammatory effects on lipopolysaccharide (LPS)-activated primary cultures of brain microglial cells. The levels of the proinflammatory cytokines interleukin1ß (IL-1ß) and tumor necrosis factor-α (TNFα) secreted by the cells were determined by reverse transcriptase-PCR and Enzyme-linked immunosorbent assay (ELISA), respectively. NO levels secreted by the activate cells were measured using Griess reagent, ROS levels were measured by 2'7'-dichlorofluorescein diacetate (DCF-DA), MMP-9 activity was measured using gel zymography, and the protein levels of the proinflammatory enzymes cyclooxygenase-2 (COX-2) and induced nitric oxide synthase (iNOS) were measured by Western blot analysis. Cell viability was assessed using Lactate dehydrogenase (LDH) activity in the media conditioned by the cells or by the crystal violet cell staining. RESULTS: We have found that out of the 66 desert plants tested, the extract of Af was the most efficient extract and inhibited ~70% of the NO produced by the LPS-activated microglial cells, without affecting cell viability. In addition, this extract inhibited the LPS - elicited expression of the proinflammatory mediators IL-1ß, TNFα, MMP-9, COX-2 and iNOS in these cells. CONCLUSIONS: Thus, phytochemicals present in the Af extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology.

Amelioration of brain pathology and behavioral dysfunction in mice with lupus following treatment with a tolerogenic peptide.

OBJECTIVE: Central nervous system (CNS) involvement in systemic lupus erythematosus (SLE) is manifested by neurologic deficits and psychiatric disorders. The aim of this study was to examine SLE-associated CNS pathology in lupus-prone (NZBxNZW)F1 (NZB/NZW) mice, and to evaluate the ameliorating effects of treatment with a tolerogenic peptide, hCDR1 (human first complementarity-determining region), on these manifestations. METHODS: Histopathologic analyses of brains from lupus-prone NZB/NZW mice treated with vehicle, hCDR1, or a control scrambled peptide were performed. The messenger RNA expression of SLE-associated cytokines and apoptosis-related molecules from the hippocampi was determined. Anxiety-like behavior was assessed by open-field tests and dark/light transfer tests, and memory deficit was assessed using a novel object recognition test. RESULTS: Infiltration was evident in the hippocampi of the lupus-afflicted mice, and the presence of CD3+ T cells as well as IgG and complement C3 complex deposition was observed. Furthermore, elevated levels of gliosis and loss of neuronal nuclei immunoreactivity were also observed in the hippocampi of the mice with lupus. Treatment with hCDR1 ameliorated the histopathologic changes. Treatment with hCDR1 down-regulated the high expression of interleukin-1beta (IL-1beta), IL-6, IL-10, interferon-gamma, transforming growth factor beta, and the proapoptotic molecule caspase 8 in the hippocampi of the mice with lupus, and up-regulated expression of the antiapoptotic bcl-xL gene. Diseased mice exhibited increased anxiety-like behavior and memory deficit. Treatment with hCDR1 improved these parameters, as assessed by behavior tests. CONCLUSION: Treatment with hCDR1 ameliorated CNS pathology and improved the tested cognitive and mood-related behavior of the mice with lupus. Thus, hCDR1 is a novel candidate for the treatment of CNS lupus.

Protective Effects of the Essential Oil of Salvia fruticosa and Its Constituents on Astrocytic Susceptibility to Hydrogen Peroxide-Induced Cell Death.

Oxidative stress has been implicated in pathologic processes associated with neurodegenerative diseases. Astrocytes, the most abundant glial cell type in the brain, protect neurons from reactive oxygen species (ROS), and any damage to them will affect neuronal survival. This study compares the ability of essential oils prepared from different herbs and spices to protect cultured primary brain astrocytes from H2O2-induced death. The results show that the essential oil of Salvia fruticosa (Sf) among the tested essential oils demonstrated remarkable protective activity. The protective effect of Sf could be attributed to alpha-humulene and alpha-pinene. Following incubation, alpha-humulene and trans-beta-caryophyllene could be found in the cytosol of astrocytes. It is proposed that Sf, by attenuating H2O2-induced cell death, might be used as a functional food or may be offered as a means of therapy in the treatment of neurodegenerative diseases.

The oral absorption of phospholipid prodrugs: in vivo and in vitro mechanistic investigation of trafficking of a lecithin-valproic acid conjugate following oral administration.

The purpose of this study was to evaluate the oral absorption characteristics of a phospholipid-drug conjugate, comprising direct conjugation between the lecithin and the drug moiety through the sn-2 position. We investigated the mechanisms involved with the trafficking of this conjugate following oral administration in the gastrointestinal (GI) lumen, within the enterocyte and further. A phospholipid-valproic acid conjugate (DP-VPA) was utilized as a model molecule. The oral absorption of this conjugate in rats was investigated following administration in long (LCT) vs. medium (MCT) chain triglyceride formulations, and in the postprandial vs. fasted state. Oral administration within the LCT solution caused more than a 3-fold increase in DP-VPA bioavailability in comparison to the MCT solution. Moreover, a significant food effect was evident for DP-VPA. Hence, we evaluated the lymphatic transport of DP-VPA in mesenteric lymph duct cannulated freely moving rats. Sixty percent of the absorbed DP-VPA was associated with lymphatic transport. Similar DP-VPA absorption was obtained in secretory type II PLA(2) knockout mice (C57BL/6) and in control mice (BALB/c). Moreover, nil DP-VPA degradation in serum and very low (4.8%) degradation by bee venom PLA(2)in vitro were obtained. In conclusion, direct conjugation between the drug and the phospholipid produces a complex having unique absorption properties that include: (1) a stable complex that does not undergo degradation in the GI tract; (2) permeation through the gut wall and entering intact to the enterocyte; and (3) association with chylomicron in the enterocyte and reaching the systemic circulation via the lymphatic route. These unique properties may be of interest in drug delivery.

Altered gene expression in mice with lupus treated with edratide, a peptide that ameliorates the disease manifestations.

OBJECTIVE: To identify genes that are differently expressed in (NZB x NZW)F(1) mice with established lupus compared with healthy controls, and to determine how gene expression is affected by treatment with hCDR1 (Edratide), a peptide synthesized on the basis of the sequence of the first complementarity-determining region (CDR1) of an autoantibody. METHODS: RNA was extracted from spleen cells of young, disease-free mice and of older mice with systemic lupus erythematosus (SLE) that were treated with hCDR1 or with vehicle alone. Gene expression was assessed using the DNA microarray technique and verified by real-time reverse transcriptase-polymerase chain reaction (RT-PCR). RESULTS: In mice with SLE, numerous genes showed increased or decreased expression relative to that in the disease-free controls. Treatment with hCDR1 restored the expression of many of these genes to control levels. Real-time RT-PCR verified that in diseased mice RNA transcripts of Tnfsf4, Il5ra, Zbtb20, and Nid1 were up-regulated, while transcripts of Tfpi and S100a8 were down-regulated, and confirmed the effects of hCDR1 on the expression of those genes. Kidney immunostaining demonstrated that the up-regulated expression of OX40 ligand, which is a protein product of the gene tumor necrosis factor (ligand) superfamily member 4, in diseased mice was reduced by hCDR1. CONCLUSION: Expression of numerous genes in mice with SLE differs from that in young, disease-free control mice. Treatment with hCDR1 restores the expression of 22% of these genes to levels similar to those in controls. Thus, one of the mechanisms by which hCDR1 exerts its beneficial effects on the clinical symptoms of SLE is through regulation of gene expression.

A novel mechanism for oral controlled release of drugs by continuous degradation of a phospholipid prodrug along the intestine: in-vivo and in-vitro evaluation of an indomethacin-lecithin conjugate.

PURPOSE: To investigate a novel mechanism for oral controlled release of drugs involving a continuous degradation of a phospholipid prodrug along the intestine. An indomethacin-lecithin conjugate with the drug attached to the sn-2 position of the phospholipid through a 5-carbon linker (DP-155) was used as a model molecule. METHODS: The pharmacokinetics of DP-155 and free indomethacin liberated from the prodrug following intravenous, oral or intra-colon administration was investigated in rats, and evaluated in comparison to free indomethacin administration. Degradation by phospholipase A(2) (PLA(2)) enzymes was assessed in-vitro. The impact of the linker length was evaluated in comparison to an indomethacin-phospholipid conjugate with a shorter linker (2-carbons). RESULTS: Following oral or intra-colon DP-155 administration, free indomethacin was liberated along the intestine and absorbed into the systemic circulation, resulting in a controlled release profile of indomethacin in the plasma. The shorter linker caused a 20-fold decrease in the subsequent indomethacin absorption. DP-155 in-vitro degradation by PLA(2) was over 60%, while shorter linkers were profoundly less degradable. CONCLUSIONS: DP-155 caused a continuous input of free indomethacin into the plasma following degradation by PLA(2) in the gut lumen. Since the rate of drug release is not formulation dependent, the prodrug can be compounded even in a liquid dosage form. The phospholipid-drug conjugate is thus a potential novel mechanism for oral controlled release of drugs.

A peptide based on an anti-DNA autoantibody downregulates matrix metalloproteinases in murine models of lupus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the increased production of autoantibodies and by T cell dysfunction associated with general clinical manifestations. A model of induced experimental SLE by the immunization with the human monoclonal anti-DNA 16/6Id(+) autoantibody and a model of the SLE-prone mice (NZB x NZW)F1 were used in the present study. Two peptides based on the complementarity determining regions (CDR) 1 and 3 of a murine monoclonal anti-DNA 16/6Id(+) autoantibody were shown to ameliorate spontaneous and induced SLE in mice. We demonstrate here that levels of matrix metalloproteinase (MMP)-3 and MMP-9 were elevated in plasma and kidneys of SLE-afflicted mice. Levels of both MMP-3 and MMP-9 were elevated in kidneys of mice with the 16/6Id induced experimental SLE already in the early phases of disease development. However, increased levels of only MMP-3 were detected in the plasma at the early stages of disease, while MMP-9 activity was elevated later, when clinical manifestations were already observed. Treatment of SLE-afflicted mice, with the CDR1-based peptide that ameliorates disease manifestations in mice, led to a reduction in MMP-9 activity and in MMP-3 protein levels both in plasma and in kidneys. We thus suggest that these enzymes may play a pathogenic role in the disease and may serve as markers for the determination of disease progression or amelioration.