Triple-Hybrid BioScaffold Based on Silk Fibroin, Chitosan, and nano-Biphasic Calcium Phosphates: Preparation, Characterization of Physiochemical and Biopharmaceutical Properties.

Bioscaffolds, which promote cell regeneration and restore tissues' functions, have emerged as significant need in clinic. The hybrid of several biomaterials in a bioscaffold renders clinically advanced and relevant properties for applications yet add challenges in cost efficiency, production, and clinical investigation. This study proposes a facile and sustainable method to formulate a triple-hybrid bioscaffold based on Vietnamese cocoon origin Silk Fibroin, Chitosan, and nano-Biphasic Calcium Phosphates (nano-BCP) that can be easily molded, has high porosity (55-80%), and swelling capacity that facilitates cell proliferation and nutrient diffusion. Notably, their mechanical properties, in particular compressive strength, can easily be tuned in a range from 50 - 200 kPa by changing the amount of nano-BCP addition, which is comparable to the successful precedents for productive cell regeneration. The latter parts investigate the biopharmaceutical properties of a representative bioscaffold, including drug loading and release studies with two kinds of active compounds, salmon calcitonin and methylprednisolone. Furthermore, the bioscaffold is highly biocompatible as the results of hemocompatibility and hemostasis tests, as well as ovo chick chorioallantoic membrane investigation. The findings of the study suggest the triple-hybrid scaffold as a promising platform for multi-functional drug delivery and bone defect repair.

Clathrin light chain-conjugated drug delivery for cancer.

Targeted drug delivery systems hold the remarkable potential to improve the therapeutic index of anticancer medications markedly. Here, we report a targeted delivery platform for cancer treatment using clathrin light chain (CLC)-conjugated drugs. We conjugated CLC to paclitaxel (PTX) through a glutaric anhydride at high efficiency. Labeled CLCs localized to 4T1 tumors implanted in mice, and conjugation of PTX to CLC enhanced its delivery to these tumors. Treatment of three different mouse models of cancer-melanoma, breast cancer, and lung cancer-with CLC-PTX resulted in significant growth inhibition of both the primary tumor and metastatic lesions, as compared to treatment with free PTX. CLC-PTX treatment caused a marked increase in apoptosis of tumor cells and reduction of tumor angiogenesis. Our data suggested HSP70 as a binding partner for CLC. Our study demonstrates that CLC-based drug-conjugates constitute a novel drug delivery platform that can augment the effects of chemotherapeutics in treating a variety of cancers. Moreover, conjugation of therapeutics with CLC may be used as means by which drugs are delivered specifically to primary tumors and metastatic lesions, thereby prolonging the survival of cancer patients.

The Derivatives of Cromolyn Ameliorate the Abnormal Misfolding of Amyloid Proteins and Neuroinflammation in the Neural Cells.

BACKGROUND: The representative symptom of Alzheimer's Disease (AD) has mainly been mentioned to be misfolding of amyloid proteins, such as amyloid-beta (Aß) and tau protein. In addition, the neurological pathology related to neuroinflammatory signaling has recently been raised as an important feature in AD. Currently, numerous drug candidates continue to be investigated to reduce symptoms of AD, including amyloid proteins misfolding and neuroinflammation. OBJECTIVE: Our research aimed to identify the anti-AD effects of two chemical derivatives modified from cromoglicic acid, CNU 010 and CNU 011. METHODS: CNU 010 and CNU 011 derived from cromoglicic acid were synthesized. The inhibitory effects of Aß and tau were identified by thioflavin T assay. Moreover, western blots were conducted with derivates CNU 010 and CNU 011 to confirm the effects on inflammation. RESULTS: CNU 010 and CNU 011 significantly inhibited the aggregation of Aß and tau proteins. Moreover, they reduced the expression levels of mitogen-activated protein (MAP) kinase and nuclear factor kappa-light-chain-enhancer of activated B cells (NF- κB) signaling proteins, which are representative early inflammatory signaling markers. Also, the inhibitory effects on the lipopolysaccharide (LPS)-induced cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) expression referring to late inflammation were confirmed. CONCLUSION: Our results showing multiple beneficial effects of cromolyn derivatives against abnormal aggregation of amyloid proteins and neuroinflammatory signaling provide evidence that CNU 010 and CNU 011 could be further developed as potential drug candidates for AD treatment.

Cromolyn platform suppresses fibrosis and inflammation, promotes microglial phagocytosis and neurite outgrowth.

Neurodegenerative diseases are characterized by chronic neuroinflammation and may perpetuate ongoing fibrotic reactions within the central nervous system. Unfortunately, there is no therapeutic available that treats neurodegenerative inflammation and its sequelae. Here we utilize cromolyn, a mast cell inhibitor with anti-inflammatory capabilities, and its fluorinated analogue F-cromolyn to study fibrosis-related protein regulation and secretion downstream of neuroinflammation and their ability to promote microglial phagocytosis and neurite outgrowth. In this report, RNA-seq analysis shows that administration of the pro-inflammatory cytokine TNF-α to HMC3 human microglia results in a robust upregulation of fibrosis-associated genes. Subsequent treatment with cromolyn and F-cromolyn resulted in reduced secretion of collagen XVIII, fibronectin, and tenascin-c. Additionally, we show that cromolyn and F-cromolyn reduce pro-inflammatory proteins PLP1, PELP1, HSP90, IL-2, GRO-α, Eotaxin, and VEGF-Α, while promoting secretion of anti-inflammatory IL-4 in HMC3 microglia. Furthermore, cromolyn and F-cromolyn augment neurite outgrowth in PC12 neuronal cells in concert with nerve growth factor. Treatment also differentially altered secretion of neurogenesis-related proteins TTL, PROX1, Rab35, and CSDE1 in HMC3 microglia. Finally, iPSC-derived human microglia more readily phagocytose Aß42 with cromolyn and F-cromolyn relative to controls. We propose the cromolyn platform targets multiple proteins upstream of PI3K/Akt/mTOR, NF-κB, and GSK-3ß signaling pathways to affect cytokine, chemokine, and fibrosis-related protein expression.

New Approaches to Profile the Microbiome for Treatment of Neurodegenerative Disease.

Progressive neurodegenerative diseases represent some of the largest growing treatment challenges for public health in modern society. These diseases mainly progress due to aging and are driven by microglial surveillance and activation in response to changes occurring in the aging brain. The lack of efficacious treatment options for Alzheimer's disease (AD), as the focus of this review, and other neurodegenerative disorders has encouraged new approaches to address neuroinflammation for potential treatments. Here we will focus on the increasing evidence that dysbiosis of the gut microbiome is characterized by inflammation that may carry over to the central nervous system and into the brain. Neuroinflammation is the common thread associated with neurodegenerative diseases, but it is yet unknown at what point and how innate immune function turns pathogenic for an individual. This review will address extensive efforts to identify constituents of the gut microbiome and their neuroactive metabolites as a peripheral path to treatment. This approach is still in its infancy in substantive clinical trials and requires thorough human studies to elucidate the metabolic microbiome profile to design appropriate treatment strategies for early stages of neurodegenerative disease. We view that in order to address neurodegenerative mechanisms of the gut, microbiome and metabolite profiles must be determined to pre-screen AD subjects prior to the design of specific, chronic titrations of gut microbiota with low-dose antibiotics. This represents an exciting treatment strategy designed to balance inflammatory microglial involvement in disease progression with an individual's manifestation of AD as influenced by a coercive inflammatory gut.

Cromolyn inhibits the secretion of inflammatory cytokines by human microglia (HMC3).

Cromolyn is a known mast cell stabilizer and is approved for treatment of asthma and for other allergic indications. Cromolyn, in a new redesigned dry powder formulation, is being tested in a pivotal clinical trial in combination with low dose ibuprofen to treat early Alzheimer's Disease (AD) subjects. To better understand the mechanistic effect cromolyn has in slowing down or halting the neuroinflammatory response associated with AD progression, we tested the effect of cromolyn to dampen the inflammatory response in the human HMC3 microglia cell line. The direct effect of cromolyn on HMC3 microglia is on cytokines and chemokines production following their activation by the inflammatory cytokine TNF-α. Cromolyn and a new fluorinated analog dramatically reduced the secretion of a wide spectrum of inflammatory mediators, which included cytokines such as IL-1ß, IL-6, IL-8 and IFN-γ, and chemokines such as CXCL10, CCL2, CCL3 and CCL4. These results bolster our understanding of how our cromolyn platform modulates toxic microglia behavior as a dynamic future treatment option for neurodegenerative disorders.

Evaluation of Fluorinated Cromolyn Derivatives as Potential Therapeutics for Alzheimer's Disease.

BACKGROUND: Cromolyn is an anti-neuroinflammatory modulator with a multifactorial mechanism of action that has been shown to inhibit amyloid-ß (Aß) aggregation and enhance microglial uptake and clearance of Aß. OBJECTIVE: We report the effects of fluoro-cromolyn derivatives on microglial cell toxicity and microglial clearance of Aß42. METHODS: Microglial cell toxicity for cromolyn derivatives were determined in naive BV2 microglial cells. Microglial clearance assays were performed with Aß42 in naive BV2 microglial cell line and single cell clone BV2 line expressing CD33WT. PET imaging was performed for three F-18 analogs in a rhesus macaque. RESULTS: All compounds but derivative 8 exhibited low microglial cell toxicity. Cromolyn 1 and derivatives 2, 4, and 7 displayed an increased uptake on Aß42 in naïve BV2 microglial cells. Derivative 4 increased Aß42 uptake in a dose-dependent manner and at 75µM resulted in a one-fold increase in Aß42 uptake in BV2-CD33WT. PET imaging for three [18F]cromolyn analogs revealed the order of brain tracer penetration to be 4a > 10 > 2a. Tracer 4a exhibited enhanced uptake in areas of high perfusion (putamen, grey matter, and cerebellum) and lower signal in areas of lower perfusion (caudate, thalamus, and white matter). CONCLUSION: Substantial uptake of Aß42 in both naïve BV2 and BV2-CD33WT cells observed with 4 indicate conversion of microglial cells from a pro-inflammatory to an activation state favoring Aß phagocytosis/clearance. These findings suggest that a fluoro-cromolyn analog could reduce fibril-prone Aß42in vivo and thereby serve as a therapeutic for the treatment and prevention of AD.

Developing Effective Alzheimer's Disease Therapies: Clinical Experience and Future Directions.

Alzheimer's disease (AD) clinical trials, focused on disease modifying drugs and conducted in patients with mild to moderate AD, as well as prodromal (early) AD, have failed to reach efficacy endpoints in improving cognitive function in most cases to date or have been terminated due to adverse events. Drugs that have reached clinical stage were reviewed using web resources (such as clinicaltrials.gov, alzforum.org, company press releases, and peer reviewed literature) to identify late stage (Phase II and Phase III) efficacy clinical trials and summarize reasons for their failure. For each drug, only the latest clinical trials and ongoing trials that aimed at improving cognitive function were included in the analysis. Here we highlight the potential reasons that have hindered clinical success, including clinical trial design and choice of outcome measures, heterogeneity of patient populations, difficulties in diagnosing and staging the disease, drug design, mechanism of action, and toxicity related to the long-term use. We review and suggest approaches for AD clinical trial design aimed at improving our ability to identify novel therapies for this devastating disease.

Cromolyn Reduces Levels of the Alzheimer's Disease-Associated Amyloid ß-Protein by Promoting Microglial Phagocytosis.

Amyloid-beta protein (Aß) deposition is a pathological hallmark of Alzheimer's disease (AD). Aß deposition triggers both pro-neuroinflammatory microglial activation and neurofibrillary tangle formation. Cromolyn sodium is an asthma therapeutic agent previously shown to reduce Aß levels in transgenic AD mouse brains after one-week of treatment. Here, we further explored these effects as well as the mechanism of action of cromolyn, alone, and in combination with ibuprofen in APPSwedish-expressing Tg2576 mice. Mice were treated for 3 months starting at 5 months of age, when the earliest stages of ß-amyloid deposition begin. Cromolyn, alone, or in combination with ibuprofen, almost completely abolished longer insoluble Aß species, i.e. Aß40 and Aß42, but increased insoluble Aß38 levels. In addition to its anti-aggregation effects on Aß, cromolyn, alone, or plus ibuprofen, but not ibuprofen alone, increased microglial recruitment to, and phagocytosis of ß-amyloid deposits in AD mice. Cromolyn also promoted Aß42 uptake in microglial cell-based assays. Collectively, our data reveal robust effects of cromolyn, alone, or in combination with ibuprofen, in reducing aggregation-prone Aß levels and inducing a neuroprotective microglial activation state favoring Aß phagocytosis versus a pro-neuroinflammatory state. These findings support the use of cromolyn, alone, or with ibuprofen, as a potential AD therapeutic.

Pharmacokinetics of Cromolyn and Ibuprofen in Healthy Elderly Volunteers.

BACKGROUND AND OBJECTIVES: The combination of cromolyn and ibuprofen is being investigated as a treatment for early Alzheimer's disease (AD). This study investigated the pharmacokinetics, safety, and tolerability of cromolyn and ibuprofen co-administration in healthy elderly adult volunteers. METHODS: In this open-labeled study, 26 subjects, aged 55-75 years, received co-administration of inhaled cromolyn (single dose 17.1 mg; double dose 34.2 mg total) and oral ibuprofen (single dose 10 mg; double dose 20 mg total). Blood sampling was performed for 6 h after co-administration in all subjects; cerebrospinal fluid (CSF) was collected in three to four subjects per cohort for 4 h following co-administration. Safety parameters, including adverse events (AEs), were monitored throughout the study. RESULTS: For cromolyn, the mean (±SD) maximum observed concentration (C max) in plasma was 46.69 ± 32.97 and 96.75 ± 46.22 ng/ml after single- and double-dose inhalation, respectively [time to C max (t max) ~22 min for each; terminal elimination half-life (t ½) ~1.8 h for each]. For ibuprofen, the plasma C max was 1090.98 ± 474.64 ng/ml and 2062.96 ± 655.13 ng/ml after single- and double-dose oral administration, respectively (t max ~1.6-1.8 h; t ½ ~1.9 h for each). For cromolyn, the CSF C max was 0.24 ± 0.08 ng/ml at 3.72 ± 0.70 h after single-dose administration and 0.34 ± 0.17 ng/ml at 3.45 ± 0.95 h after double-dose administration, and for ibuprofen, the CSF C max was 3.94 ± 1.29 ng/ml at 2.55 ± 0.96 h after single-dose administration and 8.93 ± 3.29 ng/ml at 3.15 ± 1.05 h after double-dose administration. Three (12%) subjects reported mild or moderate AEs which were unlikely to be related to study drug. CONCLUSIONS: The combination of cromolyn and ibuprofen was safe and well tolerated. The concentrations of cromolyn and ibuprofen observed in the CSF are considered sufficient to titrate the estimated daily amyloid production and the associated inflammatory response in patients with AD.

A Food and Drug Administration-approved asthma therapeutic agent impacts amyloid ß in the brain in a transgenic model of Alzheimer disease.

Interfering with the assembly of Amyloid ß (Aß) peptides from monomer to oligomeric species and fibrils or promoting their clearance from the brain are targets of anti-Aß-directed therapies in Alzheimer disease. Here we demonstrate that cromolyn sodium (disodium cromoglycate), a Food and Drug Administration-approved drug already in use for the treatment of asthma, efficiently inhibits the aggregation of Aß monomers into higher-order oligomers and fibrils in vitro without affecting Aß production. In vivo, the levels of soluble Aß are decreased by over 50% after only 1 week of daily intraperitoneally administered cromolyn sodium. Additional in vivo microdialysis studies also show that this compound decreases the half-life of soluble Aß in the brain. These data suggest a clear effect of a peripherally administered, Food and Drug Administration-approved medication on Aß economy, supporting further investigation of the potential long-term efficacy of cromolyn sodium in Alzheimer disease.

Crown ethers attenuate aggregation of amyloid beta of Alzheimer's disease.

In this report, we reasoned that non-covalent modification of amyloid beta (Aß) by crown ethers could inhibit its aggregation. We demonstrated that PiB-C, a conjugate PiB and crown ether, could significantly reduce the aggregation in vitro. Additionally, two-photon imaging showed that PiB-C could efficiently label Aß plaques and CAAs in AD mice.

Membrane potential-dependent uptake of 18F-triphenylphosphonium--a new voltage sensor as an imaging agent for detecting burn-induced apoptosis.

BACKGROUND: Mitochondrial dysfunction has been closely related to many pathologic processes, such as cellular apoptosis. Alterations in organelle membrane potential are associated with mitochondrial dysfunction. A fluorine-18 labeled phosphonium compound: (18)F-triphenylphosphonium ((18)F-TPP) was prepared to determine its potential use as a mitochondria-targeting radiopharmaceutical to evaluate cellular apoptosis. METHODS: Studies were conducted in both ex vivo cell lines and in vivo using a burned animal model. Uptake of (18)F-TPP was assessed in PC-3 cells by gamma counting under the following conditions: graded levels of extracellular potassium concentrations, incubation with carbonyl cyanide m-chlorophenylhydrazone and staurosporine. Apoptosis was studied in a burn animal model using terminal deoxynucleotidyl transferase dUTP nick end labeling staining and simultaneous assessment of (18)F-TPP uptake by biodistribution. RESULTS: We found that stepwise membrane depolarization by potassium (K) resulted in a linear decrease in (18)F-TPP uptake, with a slope of 0.62 ± 0.08 and a correlation coefficient of 0.936 ± 0.11. Gradually increased concentrations of m-chlorophenylhydrazone lead to decreased uptake of (18)F-TPP. Staurosporine significantly decreased the uptake of (18)F-TPP in PC-3 cells from 14.2 ± 3.8% to 5.6 ± 1.3% (P < 0.001). Burn-induced significant apoptosis (sham: 4.4 ± 1.8% versus burn: 24.6 ± 6.7 %; P < 0.005) and a reduced uptake of tracer in the spleens of burn-injured animals as compared with sham burn controls (burn: 1.13 ± 0.24% versus sham: 3.28 ± 0.67%; P < 0.005). Biodistribution studies demonstrated that burn-induced significant reduction in (18)F-TPP uptake in spleen, heart, lung, and liver, which were associated with significantly increased apoptosis. CONCLUSIONS: (18)F-TPP is a promising new voltage sensor for detecting mitochondrial dysfunction and apoptosis in various tissues.

Mannose-binding lectin binds to amyloid ß protein and modulates inflammation.

Mannose-binding lectin (MBL), a soluble factor of the innate immune system, is a pattern recognition molecule with a number of known ligands, including viruses, bacteria, and molecules from abnormal self tissues. In addition to its role in immunity, MBL also functions in the maintenance of tissue homeostasis. We present evidence here that MBL binds to amyloid ß peptides. MBL binding to other known carbohydrate ligands is calcium-dependent and has been attributed to the carbohydrate-recognition domain, a common feature of other C-type lectins. In contrast, we find that the features of MBL binding to Aß are more similar to the reported binding characteristics of the cysteine-rich domain of the unrelated mannose receptor and therefore may involve the MBL cysteine-rich domain. Differences in MBL ligand binding may contribute to modulation of inflammatory response and may correlate with the function of MBL in processes such as coagulation and tissue homeostasis.

4-[18F]-tetraphenylphosphonium as a PET tracer for myocardial mitochondrial membrane potential.

OBJECTIVES: This study tested the hypothesis that 4-[(18)F]fluorophenyltriphenylphosphonium ((18)F-TPP) is useful for in vivo positron emission tomography (PET) measurement of mitochondrial membrane potential (ΔΨm). Its utility as a blood flow tracer also was evaluated. BACKGROUND: Tetraphenylphosphonium is useful for in vitro measurement of ΔΨm. In vivo measurement of ΔΨm has potential value in the assessment of heart failure pathophysiology and therapy as well as assessment of myocardial viability and so may be a very useful clinical tool. METHODS: Anesthetized swine (N = 6) with a balloon catheter in the left anterior descending coronary artery were studied. Microsphere measurements of myocardial blood flow (MBF) were made after balloon inflation (baseline) and ∼10 min after intravenous administration of adenosine and phenylephrine after which ∼10 mCi (18)F-TPP was injected intravenously and dynamic PET data acquisition obtained for 30 min. After the swine were killed, the hearts were sectioned for microsphere measurement of MBF and (18)F-TPP measured by well counter in these same samples. PET images provided whole blood and myocardial (18)F-TPP concentration for determination of ΔΨm by the Nernst equation, corrected for nonspecific (18)F-TPP binding. Microsphere MBF, absolute (ml/min/g) and relative, was compared with PET data (standard uptake value and K1). RESULTS: Nonspecific binding of (18)F-TPP overestimated ΔΨm measured by -37 ± 4 mV (mean ± SD). Normal zone ΔΨm of ex vivo samples (-91 ± 11 mV; N = 52; sample weight, 1.07 ± 0.18 g) correlated strongly (R(2)= 0.93) with normal zone by PET (-81 ± 13 mV). Both ex vivo and PET normal zone ΔΨm, although somewhat lower, compared well with that reported for tritium labeled triphenylphosphonium in normal working Langendorff rat heart (-100 mV). Although the relative MBF by (18)F-TPP correlated strongly with relative microsphere MBF (R(2)= 0.83), there was no correlation between absolute MBF by (18)F-TPP and microsphere MBF. CONCLUSIONS: (18)F-TPP is a promising tracer for noninvasive PET measurement of ΔΨm in living subjects. It is useful as well for assessment of relative but not absolute MBF.

Evaluation of (4-[18F]Fluorophenyl)triphenylphosphonium ion. A potential myocardial blood flow agent for PET.

PURPOSE: The lipophilic cationic compound, (4-[¹8F]fluorophenyl)triphenylphosphonium ion (¹8F-FTPP) was synthesized and evaluated as a potential positron emission tomography (PET) myocardial perfusion agent. PROCEDURE: ¹8F-FTPP was prepared from (4-nitrophenyl)triphenylphosphonium nitrate and ammonium [¹8F]fluoride by nucleophilic aromatic substitution and was purified by high performance liquid chromatography before use. Biodistribution studies were performed in rats at 5, 30, 60 min (five rats per time point). Three rats were evaluated by microPET imaging after injection of ¹8F-FTPP. In addition, microPET imaging in rabbits (three) was performed before and after occlusion of the left anterior descending (LAD) artery with ¹³NH3 (111 MBq) and ¹8F-FTPP (74 MBq). RESULTS: Biodistribution data in rats showed rapid blood clearance and high levels of accumulation in the heart; 75:1 heart-to-blood ratio at 30 min. Uptake of radioactivity in the heart was 1.64% ID/G, 1.51% ID/g, and 1.57% ID/g at 5, 30, and 60 min. At 5, 30, and 60 min, lung activity was 0.69% ID/g, 0.03% ID/g, and 0.38% ID/g, and liver uptake was 0.34% ID/g, 0.18% ID/g, and 0.17% ID/g. Heart-to-lung ratios at 5, 30, and 60 min were 2, 5, and 4. Bone accumulation was minimal. MicroPET imaging in both rats and rabbits after injection of ¹8F-FTPP demonstrated an initial spike of activity in the myocardium corresponding to blood flow followed by a plateau after 1 min. Region of interest analysis of microPET images of normal and LAD-occluded rabbits with ¹³NH3 and ¹8F-FTPP indicated similar distributions of the two tracers in both normal and altered blood flow regions. CONCLUSION: The excellent heart-to-blood ratio of ¹8F-FTPP and its correlation with ¹³NH3 distribution in normal and LAD-occluded rabbits suggest that this radiopharmaceutical may have potential as a PET agent for characterizing mitochondrial damage and/or myocardial blood flow.

Dopamine transporter (DAT) inhibitors alleviate specific parkinsonian deficits in monkeys: association with DAT occupancy in vivo.

Viable dopamine neurons in Parkinson's disease express the dopamine transporter (DAT) and release dopamine (DA). We postulated that potent DAT inhibitors, with low affinity for the serotonin transporter (SERT), may elevate endogenously released extracellular dopamine levels to provide therapeutic benefit. The therapeutic potential of eight DAT inhibitors was investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated cynomolgus monkeys (Macaca fascicularis), with efficacy correlated with DAT occupancy as determined by positron emission tomography imaging in striatum. Four potent DAT inhibitors, with relatively high norepinephrine transporter, but low SERT affinities, that occupied the DAT improved activity in parkinsonian monkeys, whereas three high-affinity DAT inhibitors with low DAT occupancy did not. 2beta-Carbomethoxy-3alpha-(3,4-dichlorophenyl)-7beta-hydroxy-8-methyl-8-azabicyclo[3.2.1.]octane (O-1163) occupied the DAT but had short-lived pharmacological effects. The benztropine analog difluoropine increased general activity, improved posture, reduced body freeze, and produced sleep disturbances at high doses. (1R)-2beta-(1-Propanoyl)-3alpha-(4-fluorophenyl)tropane (O-1369) alleviated parkinsonian signs in advanced parkinsonian monkeys, by increasing general activity, improving posture, reducing body freeze, and sedation, but not significantly reducing bradykinesia or increasing locomotor activity. In comparison with the D(2)-D(3) DA receptor agonist quinelorane, O-1369 elicited oral/facial dyskinesias, whereas quinelorane did not improve posture or reduce balance and promoted stereotypy. In conclusion, DAT inhibitors with therapeutic potential combine high DAT affinity in vitro and high DAT occupancy of brain striatum in vivo with enduring day-time effects that do not extend into the nighttime. Advanced parkinsonian monkeys (80% DAT loss) respond more effectively to DAT inhibitors than mild parkinsonian monkeys (46% DAT loss). The therapeutic potential of dopamine transport inhibitors for Parkinson's disease warrants preclinical investigation.

Anti-viral activity of human antithrombin III.

Soluble inhibitory factors produced by CD8+ T-cells have been shown to inhibit HIV-1 replication and may play a critical role in vivo in anti-viral host defense. CD8+ T-cell-modified antithrombin III (ATIII) accounts for some of the described CD8+ T-cell anti-viral activity. We demonstrate that CD4+ T-cells, CD8+ T-cells, and natural killer cells react to an ATIII gradient by cell migration. Furthermore, exogenously added ATIII induced a G-protein-coupled signal transduction process in CD4+ T-cells and inhibited TNF-alpha-induced NF-kappaB activation. Heat and/or heparin treatment prior to the anti-viral inhibition test increased the anti-HIV activity up to 1000-fold. Our data indicate that anti-viral inactive ATIII can be activated having promising anti-viral properties as complementary candidate for the treatment of HIV infection.

Evaluation of trans-9-18F-fluoro-3,4-Methyleneheptadecanoic acid as a PET tracer for myocardial fatty acid imaging.

UNLABELLED: This study describes the radiosynthesis and preliminary biologic evaluation of trans-9(RS)-(18)F-fluoro-3,4(RS,RS)-methyleneheptadecanoic acid ((18)F-FCPHA) as a new potential probe for assessing myocardial fatty acid metabolism by PET. This fatty acid, containing a cyclopropyl moiety in the beta,gamma-position, was designed to enter the myocardium by the same mechanism as natural fatty acids and to undergo partial metabolism before being trapped in the cell. METHODS: (18)F-FCPHA and the beta-methyl analog 8(RS)-(18)F-fluoro-3(RS)-methylheptadecanoic acid ((18)F-FBMHA) were prepared from their corresponding mesylate precursors by nucleophilic substitution. The precursors used for labeling were fully characterized, and the data were consistent with the proposed structures. Biodistribution studies of each tracer were performed with Sprague-Dawley rats at 5 and 60 min after injection. Sequential imaging of a rhesus monkey injected with 222 MBq of (18)F-FCPHA was performed by use of a microPET camera. RESULTS: At 5 and 60 min, heart uptake values measured as mean +/- SD percentage injected dose per gram (%ID/g) in rats for (18)F-FCPHA were 1.55 +/- 0.72 and 1.43 +/- 0.14, respectively. The heart-to-blood ratios at 5 and 60 min, an indication of target definition, were 25.8 and 20.4, respectively. The heart-to-lung ratios at 5 and 60 min were 3.3 and 4.6, respectively. Bone accumulation (%ID/g), an indication of defluorination, was 0.16 +/- 0.03 at 5 min and increased to 0.70 +/- 0.39 at 60 min. The heart-to-blood ratio obtained with (18)F-FBMHA was 2.6 at 5 min and did not change significantly at 60 min. Imaging of the monkey heart after injection of (18)F-FCPHA showed an initial spike of activity corresponding to blood flow followed by a plateau at 10 min. CONCLUSION: The cyclopropyl moiety in (18)F-FCPHA does have a significant influence on heart accumulation, as suggested by the high heart-to-blood ratio and the fast blood clearance in rats. These results, along with the remarkable quality of the PET images, indicate the potential of this new class of labeled fatty acids for use in studying heart disease by PET.

Novel (bisarylmethoxy)butylpiperidine analogues as neurotransmitter transporter inhibitors with activity at dopamine receptor sites.

A series of (bisarylmethoxy)butylpiperidine derivatives was prepared and evaluated in vitro and in vivo to determine the structural requirements necessary for dual activity at the DAT and DA/5-HT receptor sites. These hybrid ligands, constructed by combining pharmacophores specific for the DAT and DA/5-HT receptors, could be useful drugs for treating cocaine addiction by assisting cocaine addicts in maintaining abstinence. The series was evaluated in vitro for DAT and DA/5-HT receptor activity and then selected compounds were tested in vivo for their effects on cocaine-induced hyperlocomotor activity (LMA). The majority of the new compounds demonstrated high to moderate affinity (4-191 nM) for the DAT with 4-hydroxy-4-phenylpiperidine analogues 14 and 15 possessing the greatest affinity. Compounds 15 and 22 exhibited the highest ratio of reuptake inhibition-to-binding (discrimination ratio, DR), 111 and 323, respectively. These derivatives had modest affinity and antagonistic activity for dopamine D(2)/D(3) receptors. Compounds 9 and 15 (DR=0.9 and 111, respectively) stimulated locomotor activity, whereas the other compounds suppressed this response. All compounds tested except for 17 and 21 attenuated cocaine-induced hyperlocomotion.