Mode of Action of RZL-012, a New Fat-Reducing Molecule.

BACKGROUND: RZL-012 (5-[3,6-dibromo-9H-carbazol-9-yl]-N,N,N-trimethylpentan-1-aminium chloride) is a novel investigational drug injected subcutaneously into fat tissues in patients with fat-related disorders (Dercum disease) or subjects seeking aesthetic changes. OBJECTIVE: Preclinical studies were undertaken to understand RZL-012's mechanism of action. MATERIALS AND METHODS: The effects of RZL-012 were tested in vitro by measuring adipocyte cell killing, membrane integrity, cytosolic calcium, and mitochondrial membrane potential (MMP). In vivo studies in pigs evaluated RZL-012's adipocyte killing effect and measured pig fat thickness in the injected areas. RESULTS: RZL-012 triggered adipocyte cell killing with IC50 values ranging from 25 to 106 µM. RZL-012 demonstrated initial effects on membrane integrity and calcium levels with delayed alterations in MMP. Incubation of RZL-012 with nanoghosts increased membrane permeability, culminating in full membrane destruction. Analysis of injected areas in pigs revealed liponecrosis 24 hours after dosing followed by an inflammatory response and formation of fibrotic tissue. Three months after dosing, an 18% reduction in mean fat thickness was observed in RZL-012 treated pigs. CONCLUSION: RZL-012 destroys adipocytes by directly disrupting cell membrane integrity. Replacement of dead fat tissue by fibrotic tissue enables healing and causes contraction of the injected area. These effects are translated into significant reduction in fat tissue volume.

Comparing the biological impact of glatiramer acetate with the biological impact of a generic.

For decades, policies regarding generic medicines have sought to provide patients with economical access to safe and effective drugs, while encouraging the development of new therapies. This balance is becoming more challenging for physicians and regulators as biologics and non-biological complex drugs (NBCDs) such as glatiramer acetate demonstrate remarkable efficacy, because generics for these medicines are more difficult to assess. We sought to develop computational methods that use transcriptional profiles to compare branded medicines to generics, robustly characterizing differences in biological impact. We combined multiple computational methods to determine whether differentially expressed genes result from random variation, or point to consistent differences in biological impact of the generic compared to the branded medicine. We applied these methods to analyze gene expression data from mouse splenocytes exposed to either branded glatiramer acetate or a generic. The computational methods identified extensive evidence that branded glatiramer acetate has a more consistent biological impact across batches than the generic, and has a distinct impact on regulatory T cells and myeloid lineage cells. In summary, we developed a computational pipeline that integrates multiple methods to compare two medicines in an innovative way. This pipeline, and the specific findings distinguishing branded glatiramer acetate from a generic, can help physicians and regulators take appropriate steps to ensure safety and efficacy.

Metabolism, mechanism of action and sensitivity profile of fluorocyclopentenylcytosine (RX-3117; TV-1360).

A novel cytidine analog fluorocyclopentenylcytosine (RX-3117; TV-1360) was characterized for its cytotoxicity in a 59-cell line panel and further characterized for cytotoxicity, metabolism and mechanism of action in 15 additional cancer cell lines, including gemcitabine-resistant variants. In both panels sensitivity varied 75-fold (IC50: 0.4- > 30 µM RX-3117). RX-3117 showed a different sensitivity profile compared to cyclopentenyl-cytosine (CPEC) and azacytidine, substrates for uridine-cytidine-kinase (UCK). Dipyridamole, an inhibitor of the equilibrative-nucleoside-transporter protected against RX-3117. Uridine and cytidine protected against RX-3117, but deoxycytidine (substrate for deoxycytidine-kinase [dCK]) not, although it protected against gemcitabine, demonstrating that RX-3117 is a substrate for UCK and not for dCK. UCK activity was abundant in all cell lines, including the gemcitabine-resistant variants. RX-3117 was a very poor substrate for cytidine deaminase (66,000-fold less than gemcitabine). RX-3117 was rapidly metabolised to its nucleotides predominantly the triphosphate, which was highest in the most sensitive cells (U937, A2780) and lowest in the least sensitive (CCRF-CEM). RX-3117 did not significantly affect cytidine and uridine nucleotide pools. Incorporation of RX-3117 into RNA and DNA was higher in sensitive A2780 and low in insensitive SW1573 cells. In sensitive U937 cells 1 µM RX-3117 resulted in 90% inhibition of RNA synthesis but 100 µM RX-3117 was required in A2780 and CCRF-CEM cells. RX-3117 at IC50 values did not affect the integrity of RNA. DNA synthesis was completely inhibited in sensitive U937 cells at 1 µM, but in other cells even higher concentrations only resulted in a partial inhibition. At IC50 values RX-3117 downregulated the expression of DNA methyltransferase. In conclusion, RX-3117 showed a completely different sensitivity profile compared to gemcitabine and CPEC, its uptake is transporter dependent and is activated by UCK. RX-3117 is incorporated into RNA and DNA, did not affect RNA integrity, depleted DNA methyltransferase and inhibited RNA and DNA synthesis. Nucleotide formation is related with sensitivity.

Guidelines for preclinical animal research in ALS/MND: A consensus meeting.

The development of therapeutics for ALS/MND is largely based on work in experimental animals carrying human SOD mutations. However, translation of apparent therapeutic successes from in vivo to the human disease has proven difficult and a considerable amount of financial resources has been apparently wasted. Standard operating procedures (SOPs) for preclinical animal research in ALS/MND are urgently required. Such SOPs will help to establish SOPs for translational research for other neurological diseases within the next few years. To identify the challenges and to improve the research methodology, the European ALS/MND group held a meeting in 2006 and published guidelines in 2007 (1). A second international conference to improve the guidelines was held in 2009. These second and improved guidelines are dedicated to the memory of Sean F. Scott.

Guidelines for the preclinical in vivo evaluation of pharmacological active drugs for ALS/MND: report on the 142nd ENMC international workshop.

A transgenic animal model for anterior horn cell loss was established in 1994. This model is based on the insertion of a high copy number of disease-causing human Cu/Zn SOD mutations into the intact mouse genome. It serves to establish hypotheses for the pathogenesis of anterior horn cell death, but also to test potential pharmacological approaches to therapy in human ALS. Today, more than 100 -- published and unpublished -- compounds have been tested in this animal model, a large part of them being reported as successful. However, it proved to be difficult to translate these therapeutic successes in the animal model into human trials. Also, a number of disease-modifying strategies were difficult to reproduce, even by the same group. On the other hand, the step from mice to men means a huge investment for the sponsors of clinical trials and the scientific community. Therefore, establishment of standard methods for drug testing in ALS models is mandatory. In this workshop, clinical and preclinical researchers established in the field of ALS/MND met in Holland in March 2006 in order to establish guidelines for the community for drug testing in mouse models.

A new method of wet scanning electron microscopy for the analysis of myelination in EAE mouse model of multiple sclerosis.

Development of effective therapies for multiple sclerosis (MS) is dependent on the advancement of improved tools for evaluation of progression of this disease in animal models. We present a novel technique utilizing scanning electron microscopy (SEM) for imaging wet biological specimens thus enabling rapid and high-resolution imaging of myelin in mouse spinal cord (SC). We demonstrate the advantages of using the wet SEM technique to image myelin in a murine model of MS, experimental autoimmune encephalomyelitis (EAE) induced in the Biozzi (antibody-high) mouse, by sensitization with spinal cord homogenate (SCH) in adjuvant. Our studies show that the methodology allows easy identification of normal and pathological components with great clarity, which is then correlated with light microscopy (LM) and validated thereby. Furthermore, we demonstrate gold immunolabeling of specific epitopes. We conclude that the new technique provides a quick, accurate, and detailed structural evaluation of the SC that can be applied to advance the research of MS.

In vivo measurement of brain monoamine oxidase B occupancy by rasagiline, using (11)C-l-deprenyl and PET.

UNLABELLED: In recent years, monoamine oxidase B (MAO-B) inhibitors have become widely used in the treatment of early-stage Parkinson's disease. (11)C-l-deprenyl PET has been used by others to characterize MAO-B ligands in terms of their in vivo potency toward MAO-B and duration of action. In this study, we used (11)C-l-deprenyl PET to demonstrate the specific binding characteristics of the new irreversible selective MAO-B inhibitor rasagiline in 3 healthy volunteers. METHODS: The healthy volunteers received 1 mg of rasagiline daily for 10 d. Dynamic (11)C-l-deprenyl PET brain scans were acquired before the first treatment (scan 1) and immediately (scan 2), 2-3 wk (scan 3), and 4-6 wk (scan 4) after the final treatment. RESULTS: On scan 1, all subjects showed the highest l-deprenyl uptake in the thalamus and basal ganglia, with fairly high activity also in the cortex and cerebellum and much lower activity in the white matter. The areas of high uptake were absent from scan 2, on which activity throughout the brain was comparable to that in white matter, presumably because of blocking of MAO-B binding sites by rasagiline. Gradual recovery toward the baseline state was observed in the weeks after termination of treatment (scans 3 and 4). CONCLUSION: (11)C-l-deprenyl PET showed binding of rasagiline to MAO-B, confirming blocking of MAO-B sites after 10 d of treatment with 1 mg of rasagiline per day, with immediate post-rasagiline treatment tracer uptake and metabolism in the basal ganglia compatible only with nonspecific binding. Subsequent gradual recovery was also seen, with return to near-baseline uptake. This finding is compatible with the known rate of de novo synthesis of MAO-B, confirming the irreversible binding of rasagiline.

Rasagiline alone and in combination with riluzole prolongs survival in an ALS mouse model.

Rasagiline is an antiapoptotic compound with neuroprotective potential. We examined its neuroprotective effect alone and in combination with the putative glutamate release blocker riluzole in the G93A model of familial amyotrophic lateral sclerosis (fALS). Endpoints of experimental treatment were survival and motor activity. The drug had a significant dose-dependent therapeutic effect on both preclinical and clinical motor function and survival of the animals. We also found that the combination of rasagiline with riluzole is safe and increases survival by about 20 % in a dose-dependent manner. Therefore, we conclude that the combination of rasagiline and riluzole is a promising clinical combination for the improvement of current neuroprotective treatment strategies of ALS.

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.