Parkinson’s disease (PD) is a debilitating movement disorder resulted from a progressive degeneration of the nigrostriatal dopaminergic pathway and depletion of neurotransmitter dopamine in the striatum. Molecular cloning studies have identified nearly a dozen genes or loci that are associated with small clusters of mostly early onset and genetic forms of PD. The etiology of the vast majority of PD cases remains unknown and the precise molecular and biochemical processes governing the selective and progressive degeneration of the nigrostriatal dopaminergic pathway is poorly understood. Current drug therapies for PD are symptomatic and appear to bear little impact on the progressive neurodegenerative process. Studies of post mortem PD brains and various cellular and animal models of PD in the last two decades strongly suggest that the generation of pro-inflammatory and neurotoxic factors by the resident brain immune cells, microglia, plays a prominent role in mediating the progressive neurodegenerative process. This review will discuss literature supporting the possibility of modulating the activity of microglia as a neuroprotective strategy for the treatment of PD.
Dopamine neuron; Parkinson’s disease; Movement disorder; Microglia; Neuroprotection; Free radical
New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at μ and δ opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in G-protein coupled receptors (GPCRs) as illustrated by the δ opioid receptor.
drug design; neuropathic pain; bifunctional ligands; plasmon waveguide resonance spectroscopy; GPCRs; opioid receptors; cholecystokinin receptors
Parkinson’s disease (PD) is a debilitating movement disorder resulting from a progressive degeneration of the nigrostriatal dopaminergic pathway and depletion of neurotransmitter dopamine in the striatum. Molecular cloning studies have identified nearly a dozen genes or loci that are associated with small clusters of mostly early onset and genetic forms of PD. The etiology of the vast majority of PD cases remains unknown, and the precise molecular and biochemical processes governing the selective and progressive degeneration of the nigrostriatal dopaminergic pathway are poorly understood. Current drug therapies for PD are symptomatic and appear to bear little effect on the progressive neurodegenerative process. Studies of postmortem PD brains and various cellular and animal models of PD in the last 2 decades strongly suggest that the generation of proinflammatory and neurotoxic factors by the resident brain immune cells, microglia, plays a prominent role in mediating the progressive neurodegenerative process. This review discusses literature supporting the possibility of modulating the activity of microglia as a neuroprotective strategy for the treatment of PD.
Dopamine neuron; Parkinson’s disease; movement disorder; microglia; neuroprotection; free radical
New modalities providing safe and effective treatment of pain, especially prolonged pathological pain, have not appeared despite much effort. In this mini-review/overview we suggest that new paradigms of drug design are required to counter the underlying changes that occur in the nervous system that may elicit chronic pain states. We illustrate this approach with the example of designing, in a single ligand, molecules that have agonist activity at μ and σ opioid receptors and antagonist activities at cholecystokinin (CCK) receptors. Our findings thus far provide evidence in support of this new approach to drug design. We also report on a new biophysical method, plasmon waveguide resonance (PWR) spectroscopy, which can provide new insights into information transduction in g-protein coupled receptors (GPCRs) as illustrated by the δ opioid receptor.
drug design; neuropathic pain; bifunctional ligands; plasmon waveguide resonance spectroscopy; GPCRs; opioid receptors; cholecystokinin receptors
Replacement therapy with exogenous recombinant factor VIII (rFVIII) to control bleeding episodes results in the development of inhibitory antibodies in 15% to 30% of hemophilia A patients. The inhibitory antibodies are mainly directed against specific and universal immunodominant epitopes located in the C2 domain. Previously we have shown that complexation of O-phospho-L-serine (phosphatidylserine head group) with the phospholipid binding region of the C2 domain can lead to an overall reduction in the immunogenicity of rFVIII. Here, we have investigated the hypothesis that dicaproyl phosphatidylserine, a short-chain water-soluble phospholipid, can reduce the immunogenicity of rFVIII. Circular dichroism and fluorescence spectroscopy studies suggest that dicaproyl phosphatidyl-serine interacts with rFVIII, causing subtle changes in the tertiary and secondary structure of the protein. Sandwich enzyme-linked immunosorbent assay studies indicate that dicaproyl phosphatidylserine probably interacts with the phospholipid binding region of the C2 domain. The immunogenicity of FVIII-dicaproyl phosphatidylserine complexes prepared at concentrations above and below the critical micellar concentrations of the lipid were evaluated in hemophilia A mice. Our results suggest that micellar dicaproyl phosphatidylserine may be useful to reduce the immunogenicity of rFVIII preparations.
Hemophilia A; inhibitor development; aggregation; recombinant human factor VIII; protein folding; factor VIII-DCPS complex
It was the purpose of this study to evaluate the potential of different molecular-weight chitosan-EDTA conjugates as a carrier matrix for nanoparticulate gene delivery systems. Covalent binding of EDTA to more than one chitosan chain provides a cross-linked polymer that is anticipated to produce stabilized particles. pDNA/chitosan-EDTA particles, generated via coazervation, were characterized in size and zeta potential by electrophoretic light scattering and electron microscopy. Stability was investigated at different pH values by enzymatic degradation and subsequent gel retardation assay. Lactate dehydrogenase assay was performed to determine toxicity. Furthermore, transfection efficiency into Caco-2 cells was assessed using a beta-galactosidase reporter gene. Chitosan-EDTA produced from low-viscous chitosan with 68% amino groups being modified by the covalent attachment of EDTA showed the highest complexing efficacy resulting in nanoparticles of 43 nm mean size and exhibiting a zeta potential of +6.3 mV. These particles were more stable at pH 8 than chitosan control particles. The cytotoxicity of chitosan-EDTA particles was below 1% over a time period of 4 hours. These new nanoplexes showed 35% improved in vitro transfection efficiency compared with unmodified chitosan nanoparticles. According to these results, the chitosan-EDTA conjugate may be a promising polymer for gene transfer.
nanoparticles; stability; cross-linking; gene transfer; cytotoxicity; Caco-2
GTI-2040 is a 20-mer phosphorothioate oligonucleotide, which is complementary to the messenger ribonucleic acid (mRNA) of the R2 subunit of ribonucleotide reductase. This study characterized both the in vivo and in vitro metabolism of GTI-2040. A highly specific ion-pair reversed-phase electrospray ionization (IP-RP-ESI) liquid chromatographymass spectrometry (LC-MS) method was used for the identification of GTI-2040 and metabolites from a variety of biological samples including exonuclease enzyme solutions, plasma, urine, mouse liver/kidney homogenates, and human liver microsomes. Progressively chain-shortened metabolites trucated from the 3′ terminal of GTI-2040 were detected in all of the evaluated biological samples. GTI-2040 was found to be a good substrate for 3′ but not 5′ exonuclease. While the pattern of n-1 chain-shortened 3′-exonucleolytic degradation was similar in the mouse liver and kidney homogenates, the latter was found to contain a larger number of shortenmers, the kidneys appeared to possess higher enzymatic reactivity toward GTI-2040. Thus, metabolism of GTI-2040 was found to occur in a variety of biological samples, mainly mediated by the 3′ exonuclease.
Metabolism; phosphorothioate oligonucleotides; GTI-2040; liquid chromatography/mass spectrometry
In comparison to classical medicines, gene therapy has the potential to mediate the highest possible level of therapeutic specificity. Every normal or diseased cell can switch on or off a gene expression cassette in a tissue-, disease-, and time-dependent fashion, by use of specific transcription factors that are active only in a given unique situation. In practice, we face the problem in realizing the concept: the delivery of nucleic acids into target cells is very ineffective and presents a formidable challenge. Key issues for future developments include improved targeting, enhanced intracellular uptake, and reduced toxicity of gene vectors. The currently used classes of vectors have complementary characteristics, such as high intracellular efficiency of viral vectors on the one hand and low immunogenicity and greater flexibility of nonviral vectors on the other hand. The merge of viral and nonviral vector technologies is highlighted as an encouraging strategy for the future; concepts include chemically modified viral vectors (“chemo-viruses”) and synthesis of virus-like systems (“synthetic viruses”). Examples for the development of vectors toward artificial synthetic viruses are presented.
Gene transfer; nonviral vector; receptor targeting; synthetic virus; viral vector
Steroid hormone receptors (SHRs), such as glucocorticoid receptors (GR) and progesterone receptors (PR), are shuttling proteins that undergo continuous nuclear import and export. Various mechanisms have been proposed to explain the localization of SHRs. It has been suggested that the ligand-binding domain (LBD) of SHRs is important in determining the subcellular localization. We have studied the localization of GR-LBD and PR-LBD alone, as well as of full-length GR and PR in the presence of geldanamycin (GA), a benzoquinoid ansamycin that specifically inhibits heat shock protection (Hsp90), using transient transfections and fluorescent microscopy. Our studies have indicated that GR-LBD and PR-LBD are retained in the cytoplasm via interaction with Hsp90. It was observed that in the unliganded state, treatment with GA translocates these LBDs to the nucleus. Similar results were obtained for full-length PR and GR. Additionally, it was found that after ligand induction, GA accelerated reexport of SHRs after ligand washout, implicating Hsp90 in nuclear retention of SHRs in the washout state. We also propose that a recently found “export” signal present in the LBD of SHRs is involved in interactions with Hsp90 and hence cytoplasmic retention of these receptors. After ligand induction, Hsp90 also may play a role in nuclear retention of SHRs following hormone washout.
Hsp90; ligand binding domain; progesterone receptor; glucocorticoidreceptor; geldanamycin; cytoplasmic retention
High-throughput liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) methodology for the determination of methamphetamine (METH), amphetamine (AMP), 4-hydroxymethamphetamine (4-OH-METH), and 4-hydroxyamphetamine (4-OH-AMP) was developed and validated using simple trichloroacetic acid sample treatment. The method was validated in rat serum, brain, and testis. Lower limits-of-quantitation (LOQ) for METH and AMP were 1 ng·mL−1 using positive ion electrospray tandem mass spectrometry (MS/MS). The accuracy of the method was within 25% of the actual values over a wide range of analyte concentrations. The within-assay precision was better than 12% (coefficient of variation). The method was linear over a wide dynamic range (0.3–1000 ng·mL−1). Quantitation was possible in all 3 matrices using only serum standards because of minimal matrix-associated ion effects or the use of an internal standard. Finally, the LC-MS/MS method was used to determine serum, brain, and testis METH and AMP concentrations during a subcutaneous infusion (5.6 mg kg−1 day−1) of METH in rats. Concentrations of 4-OH-AMP and 4-OH-METH were below the LOQ in experimental samples. The bias introduced by using serum calibrators for the determination of METH and AMP concentrations in testis and brain was less than 8% and insignificant relative to the interanimal variability.
methamphetamine; rats; LC-MS/MS; matrix ion effects
Cannabinoids are antinociceptive in animal models of acute, tissue injury-, and nerve injury-induced nociception. This review examines the biology of endogenous cannabinoids (endocannabinoids) and behavioral, neurophysiological, and neuroanatomical evidence supporting the notion that cannabioids play a role in pain modulation. Behavioral pharmacological approaches, in conjunction with the identification and quantification of endocannabinoids through the use of liquid and gas chromatography mass spectrometry, have providedinsight into the functional roles of endocannabinoids in pain modulation. Here we examine the distribution of cannabinoid receptors and endocannabinoid-hydrolyzing enzymes within pain modulatory circuits together with behavioral, neurochemical, and neurophysiological studies that suggest a role for endocannabinoid signaling in pain modulation. This review will provide a comprehensive evaluation of the roles of the endocannabinoids 2-arachidonoyl-glycerol and anandamide in stress-induced analgesia. These findings provide a functional framework with which to understand the roles of endocannabinoids in nociceptive processing at the supraspinal level.
2-arachidonoylglycerol; anandamide; CB1; fatty acid amide hydrolase; monoacylglycerol lipase; periaqueductal gray; rostral ventromedial medulla
In the central nervous, system, vesicular monoamine transporter 2 (VMAT2) is the only transporter that moves cytoplasmic dopamine (DA) into synaptic vesicles for storage and subsequent exocytotic release. Pharmacologically enhancing DA sequenstration by VMAT2, and thus preventing the oxidation of DA in the cytoplasm, may be a strategy for treating diseases such as Parkinson's disease. VMAT2 may also be a novel target for the development of treatments for psychostimulant abuse. This review summarizes the possible role of VMAT2 as a therapeutic target, VMAT2 ligands reported in the literature, and the structure-activity relationship of these ligands, including tetrabenazine analogs, ketanserin analogs, lobeline analogs, and 3-amine-2-phenylpropene analogs. The molecular structure of VMAT2 and its relevance to ligand binding are briefly discussed.
vesicular monoamine transporter 2; Parkinson's disease; psychostimulant abuse; tetrabenazine; ketanserin; lobeline
The purpose of this study was to assess the effect of hyperlipoproteinemia on the biodistribution of cyclosporine A (CyA), an extensively lipoprotein bound immunosuppressant, in a rat model and to determine the potential toxicological significance of this effect. Normolipidemic and hyperlipoproteinemic rats were given a single 5 mg/kg dose of CyA as intravenous bolus and at selected times postdose, tissues, blood, and plasma were harvested and assayed for CyA content. Hyperlipoproteinemia was induced by intraperitoneal injection of 1 g/kg poloxamer 407. Compared with normolipidemic animals, hyperlipoproteinemic rats had higher plasma, blood, kidney, and liver CyA concentrations. In contrast, in heart and spleen the concentrations were decreased in hyperlipoproteinemia. The nephrotoxic effect of CyA was also evaluated in normolipidemic and hyperlipoproteinemic rats after 7 days of dosing with 20 mg/kg/day. In both groups of animals, repeated doses of CyA were associated with equivalent decreases in creatinine and urea clearances compared with matching control and predose baseline measures. The concentrations of drug in kidney were equivalent at the conclusion of the study. However, despite these similarities there was microscopic evidence of more severe changes in the kidneys in the hyperlipoproteinemic rats, which also experienced a significant decrease in body weight compared with the normolipedemic animals. In conclusion, the distribution of CyA to kidneys was enhanced in poloxamer 407-induced hyperlipoproteinemic rats after single doses, and with repeated doses there was an apparent greater adverse effect on these animals compared with normolipidemic animals.
Biodistribution; hyperlipoproteinemia; protein binding; nephrotoxicity
Interest in cannabinoid pharmacology increased dramatically upon the identification of the first cannabinoid receptor (CB1) in 1998 and continues to expand as additional endocannabinoids and cannabinoid receptors are discovered. Using CB1 receptor (CB1R) systems, medicinal chemistry programs began screening libraries searching for cannabinoid ligands, ultimately leading to the discovery of the first potent cannabinoid receptor antagonist, SR141716A (Rimonabant). Its demonstrated efficacy in treating obesity and facilitating smoking cessation, among other impressive pharmacological activities, has furthered the interest in cannabinoid receptor antagonists as therapeutics, such that the number of patents and publications covering this class of compounds continues to grow at an impressive rate. At this time, medicinal chemistry approaches including combinatorial chemistry, conformational constraint, and scaffold hopping are continuing to generate a large number of cannabinoid antagonists. These molecules provide an opportunity to gain insight into the 3-dimensional structure-activity relationships that appear crucial for CB1R-ligand interaction. In particular, studies in which conformational constraints have been imposed on the various pyrazole ring substituents of SR141716A provide a direct opportunity to characterize changes in conformation/conformational freedom within a single class of compounds. While relatively few conformationally constrained molecules have been synthesized to date, the structure-activity information is often more readily interpreted than in studies where entire substituents are replaced. Thus, it is the focus of this mini-review to examine the structural properties of SR141716A, and to use conformationally constrained molecules to illustrate the importance of conformation and conformational freedom to CB1R affinity, selectivity, and efficacy.
cannabinoid receptor; CB1; antagonists; SR141716A; structure-activity relationships; conformational analysis; molecular modeling
The United States Pharmacopeial Convention (the USP Convention), which meets at 5-year intervals, last convened in 2005. At that meeting, the convention membership elected a new Council of Experts for the 2005–2010 cycle. In turn, the Council elected members of Expert Committees charged with updating and revising the United States Pharmacopeia-National Formulary (USP-NF) and developing other authoritative standards and information. As one of their final activities, Expert Committees from the 2000–2005 cycle and USP staff carefully reviewed their work from the 2000–2005 cycle and reexamined the contents of USP-NF. From this comprehensive inventory emerged an updated and more focused new work plan directed toward acquiring missing monographs, updating monographs (typically because of advances in analytical technologies), and attending to General Chapter work (eg, dividing the General Chapter Chromatography <621> into smaller chapters) during the 2005–2010 cycle. Several elements of the work plan also speak to Resolutions adopted at the March 2005 Convention (available at www.usp.org/aboutUSP/resolutions. html) and prior ones as well. Because the work plan involves new approaches that affect both General Chapters (and thus the performance of tests and procedures) and monograph specifications—as well as the function of Pharmacopeial Forum and the introduction of new products—USP expects the plan to have a broad impact. This article briefly reviews some of these anticipated changes, informs constituents about how they can remain updated about progress and upcoming modifications to official texts, and invites participation in the standards-setting process.
US Pharmacopeia; United States Pharmacopeia-National Formulary; USP-NF; Pharmacopeial Forum; monograph
We have synthesized 4,4-dimethoxyoxazoline derivatives of several fatty acids associated with the endocannabinoid metabolome using tris(hydroxymethyl)aminomethane in a 1-step reaction by microwave irradiation. The derivatization incorporates a nitrogen into the final product, which allows for improved detection by liquid chromatographymass spectrometry in positive atmospheric pressure chemical ionization (APCI) mode. Palmitic and oleic acid derivatives show a 200-fold increase in sensitivity compared with the free acids when analyzed in negative-mode APCI. In addition to improving sensitivity, the oxazoline derivatization creates a similar ionization response for the fatty acids tested, which simplifies their quantitation. Fatty acid oxazoline derivatives can be detected using the same conditions optimized for the endocannabinoids, which allows for a simultaneous quantitation of the entire endocannabinoid metabolome.
fatty acids; LC-MS; metabolome; oxazoline derivatives; microwave synthesis
Sirtuins are recently discovered NAD+-dependent deacetylases that remove acetyl groups from acetyllysine-modified proteins, thereby regulating the biological function of their targets. Sirtuins have been shown to increase organism and tissue survival in diverse organisms, ranging from yeast to mammals. Evidence indicates that NAD+ metabolism and sirtuins contribute to mechanisms that influence cell survival under conditions of stress and toxicity. For example, recent work has shown that sirtuins and increased NAD+ biosynthesis provide protection against neuron axonal degeneration initiated by genotoxicity or trauma. In light of their protective effects, sirtuins and NAD+ metabolism could represent therapeutic targets for treatment of acute and chronic neurodegenerative conditions. Our work has focused on elucidating the enzymatic functions of sirtuins and quantifying perturbations of cellular NAD+ metabolism. We have developed mass spectrometry methods to quantitate cellular NAD+ and nicotinamide. These methods allow the quantitation of changes in the amounts of these metabolites in cells caused by chemical and genetic interventions. Characterization of the biochemical properties of sirtuins and investigations of NAD+ metabolism are likely to provide new insights into mechanisms by which NAD+ metabolism regulates sirtuin activities in cells. To develop new strategies to improve cell stress resistance, we have initiated proof of concept studies on pharmacological approaches that target sirtuins and NAD+ metabolism, with the goal of enhancing cell protection against genotoxicity.
Sirtuins; Sir2; gene silencing; toxicity; genotoxins; longevity; NAD; metabolism; SIRT1
The physical structure and polymorphism of nimodipine were studied by means of micro-Raman, WAXD, DSC, and SEM for cases of the pure drug and its solid dispersions in PEG 4000, prepared by both the hot-melt and solvent evaporation methods. The dissolution rates of nimodipine/PEG 4000 solid dispersions were also measured and discussed in terms of their physicochemical characteristics. Micro-Raman and WAXD revealed a significant amorphous portion of the drug in the samples prepared by the hot-melt method, and that saturation resulted in local crystallization of nimodipine forming, almost exclusively, modification I crystals (racemic compound). On the other hand, mainly modification II crystals (conglomerate) were observed in the solid dispersions prepared by the solvent evaporation method. However, in general, both drug forms may appear in the solid dispersions. SEM and HSM microscopy studies indicated that the drug particle size increased with drug content. The dissolution rates were substantially improved for nimodipine from its solid dispersions compared with the pure drug or physical mixtures. Among solid dispersions, those resulting from solvent coevaporation exhibited a little faster drug release at drug concentrations lower than 20 wt%. Drug amorphization is the main reason for this behavior. At higher drug content the dissolution rates became lower compared with the samples from melt, due to the drug crystallization in modification II, which results in higher crystallinity and increased particle size. Overall, the best results were found for low drug content, for which lower drug crystallinity and smaller particle size were observed.
nimodipine; solid dipersion; raman; polymorphism
In developing and manufacturing protein biopharmaceuticals, aggregation is a parameter that needs careful monitoring to ensure the quality and consistency of the final biopharmaceutical drug product. The analytical method of choice used to perform this task is size-exclusion chromatography (SEC). However, it is becoming more and more apparent that considerable care is required in assessing the accuracy of SEC data. One old analytical tool that is now reappearing to help in this assessment is analytical ultracentrifugation (AUC). Developments in AUC hardware and, more importantly, recent developments in AUC data analysis computer programs have converged to provide this old biophysical tool with the ability to extract very high resolution size information about the molecules in a given sample from a simple sedimentation velocity experiment. In addition, AUC allows sample testing to be conducted in the exact or nearly exact liquid formulation or reconstituted liquid formulation of the biopharmaceutical in the vial, with minimal surface area contact with extraneous materials. As a result, AUC analysis can provide detailed information on the aggregation of a biopharmaceutical, while avoiding many of the major problems that can plague SEC, thus allowing AUC to be used as an orthogonal method to verity SEC aggregation information and the associating properties of biopharmaceuticals.
Protein aggregation; analytical ultracentrifugation; size-exclusion chromatography; SEDFIT
Analytical ultracentrifugation (AUC) and field flow fractionation (FFF) are 2 important biophysical methods for measuring protein aggregates. Both methods can separate protein monomer from its aggregate forms under a broad range of solution conditions. Recent advances in instrumentation and data analysis, particularly in the field of analytical ultracentrifugation technology, have significantly improved the capability and sensitivity of these biophysical methods for detecting protein aggregates. These advances have resulted in an increased use of these methods in the biopharmaceutical industry for characterization of therapeutic proteins. However, despite their many advantages over conventional methods, the difficulty in the use of the instrumentation and the complexity of data analysis process, have often hampered the widespread use and proper interpretation of data. This article reviews the recent progress in both technologies, and a few case studies are also presented to discuss their advantages and limitations.
Analytical ultracentrifuge; sedimentation velocity; field flow fractionation; protein aggregates
Protein aggregation is a common issue encountered during manufacture of biotherapeutics. It is possible to influence the amount of aggregate produced during the cell culture and purification process by carefully controlling the environment (eg, media components) and implementing appro-priate strategies to minimize the extent of aggregation. Steps to remove aggregates have been successfully used at a manufacturing scale. Care should be taken when developing a process to monitor the compatibility of the equipment and process with the protein to ensure that potential aggregation is minimized.
Aggregation; self-association; cell culture; purification; filling; manufacture