Noise-induced trauma produces a temporal pattern of change in blood levels of the outer hair cell biomarker prestin.

Biomarkers in easy-to-access body fluid compartments, such as blood, are commonly used to assess health of various organ systems in clinical medicine. At present, no such biomarkers are available to inform on the health of the inner ear. Previously, we proposed the outer-hair-cell-specific protein prestin, as a possible biomarker and provided proof of concept in noise- and cisplatin-induced hearing loss. Our ototoxicity data suggest that circulatory prestin changes after inner ear injury are not static and that there is a temporal pattern of change that needs to be further characterized before practical information can be extracted. To achieve this goal, we set out to 1) describe the time course of change in prestin after intense noise exposure, and 2) determine if the temporal patterns and prestin levels are sensitive to severity of injury. After assessing auditory brainstem thresholds and distortion product otoacoustic emission levels, rats were exposed to intense octave band noise for 2 h at either 110 or 120 dB SPL. Auditory function was re-assessed 1 and 14 days later. Blood samples were collected at baseline, 4, 24, 48, 72 h and 7 and 14 days post exposure and prestin concentrations were measured using enzyme-linked immunosorbent assay (ELISA). Functional measures showed temporary hearing loss 1 day after exposure in the 110 dB SPL group, but permanent loss through Day 14 in the 120 dB SPL group. Prestin levels temporarily increased 5% at 4 h after 120 dB SPL exposure, but not in the 110 dB SPL group. There was a gradual decline in prestin levels in both groups thereafter, with prestin being below baseline on Day 14 by 5% in the 110 dB group (NS) and more than 10% in the 120 dB SPL group (p = 0.043). These results suggest that there is a temporal pattern of change in serum prestin level after noise-induced hearing loss that is related to severity of hearing loss. Circulatory levels of prestin may be able to act as surrogate biomarker for hearing loss involving OHC loss.

Oral Administration of Clinical Stage Drug Candidate SENS-401 Effectively Reduces Cisplatin-induced Hearing Loss in Rats.

HYPOTHESIS: SENS-401, an oral clinical-stage drug, may reduce cisplatin-induced hearing loss and cochlear damage in an in vivo model. BACKGROUND: Cisplatin is commonly associated with hearing loss, causing significant learning and behavioral difficulties in the pediatric cancer population, and for which there are currently no clinical solutions. SENS-401 has previously been shown to improve acoustic trauma-induced hearing loss in vivo. METHODS: The effect of SENS-401 (R-azasetron besylate) on cisplatin IC50 values was evaluated in a panel of cisplatin-sensitive cell lines (NIH:OVCAR-3, SK-N-AS, NCI-H460, FaDu). Auditory brainstem response and distortion product otoacoustic emission tests were performed in a rat model of cisplatin-induced hearing-loss (8 mg/kg, day 1) at baseline, and after 14 days of SENS-401 (6.6, 13.2, 26.4 mg/kg/d). Cochlear outer hair cells were counted after immunolabeling for myosin-VIIa. RESULTS: Cisplatin cytotoxicity was not impacted by the addition of SENS-401 (up to 10 µM) in any of the cell types evaluated. In vivo, all SENS-401 doses significantly improved auditory brainstem response threshold shift (up to 30 dB) and distortion product otoacoustic emission amplitude loss (up to 19 dB) over placebo. Body weight and survival were not significantly different between rats receiving placebo and those receiving 26.4 mg/kg SENS-401. Significantly more surviving outer hair cells were present after SENS-401 treatment compared with placebo (p < 0.001), with up to 11-fold more in the basal turn of the cochlea. CONCLUSION: In vivo and in vitro data support the otoprotective potential and tolerability of SENS-401 without impacting chemotherapeutic potential. Oral SENS-401 is a promising candidate for treating cisplatin-induced ototoxicity.

High Accumulation and In Vivo Recycling of the New Antimalarial Albitiazolium Lead to Rapid Parasite Death.

Albitiazolium is the lead compound of bisthiazolium choline analogues and exerts powerful in vitro and in vivo antimalarial activities. Here we provide new insight into the fate of albitiazolium in vivo in mice and how it exerts its pharmacological activity. We show that the drug exhibits rapid and potent activity and has very favorable pharmacokinetic and pharmacodynamic properties. Pharmacokinetic studies in Plasmodium vinckei-infected mice indicated that albitiazolium rapidly and specifically accumulates to a great extent (cellular accumulation ratio, >150) in infected erythrocytes. Unexpectedly, plasma concentrations and the area under concentration-time curves increased by 15% and 69% when mice were infected at 0.9% and 8.9% parasitemia, respectively. Albitiazolium that had accumulated in infected erythrocytes and in the spleen was released into the plasma, where it was then available for another round of pharmacological activity. This recycling of the accumulated drug, after the rupture of the infected erythrocytes, likely extends its pharmacological effect. We also established a new viability assay in the P. vinckei-infected mouse model to discriminate between fast- and slow-acting antimalarials. We found that albitiazolium impaired parasite viability in less than 6 and 3 h at the ring and late stages, respectively, while parasite morphology was affected more belatedly. This highlights that viability and morphology are two parameters that can be differentially affected by a drug treatment, an element that should be taken into account when screening new antimalarial drugs.

New insight into the mechanism of accumulation and intraerythrocytic compartmentation of albitiazolium, a new type of antimalarial.

Bis-thiazolium salts constitute a new class of antihematozoan drugs that inhibit parasite phosphatidylcholine biosynthesis. They specifically accumulate in Plasmodium- and Babesia-infected red blood cells (IRBC). Here, we provide new insight into the choline analogue albitiazolium, which is currently being clinically tested against severe malaria. Concentration-dependent accumulation in P. falciparum-infected erythrocytes reached steady state after 90 to 120 min and was massive throughout the blood cycle, with cellular accumulation ratios of up to 1,000. This could not occur through a lysosomotropic effect, and the extent did not depend on the food vacuole pH, which was the case for the weak base chloroquine. Analysis of albitiazolium accumulation in P. falciparum IRBC revealed a high-affinity component that was restricted to mature stages and suppressed by pepstatin A treatment, and thus likely related to drug accumulation in the parasite food vacuole. Albitiazolium also accumulated in a second high-capacity component present throughout the blood cycle that was likely not related to the food vacuole and also observed with Babesia divergens-infected erythrocytes. Accumulation was strictly glucose dependent, drastically inhibited by H+/K+ and Na+ ionophores upon collapse of ionic gradients, and appeared to be energized by the proton-motive force across the erythrocyte plasma membrane, indicating the importance of transport steps for this permanently charged new type of antimalarial agent. This specific, massive, and irreversible accumulation allows albitiazolium to restrict its toxicity to hematozoa-infected erythrocytes. The intraparasitic compartmentation of albitiazolium corroborates a dual mechanism of action, which could make this new type of antimalarial agent resistant to parasite resistance.

Potent in vivo anti-malarial activity and representative snapshot pharmacokinetic evaluation of artemisinin-quinoline hybrids.

BACKGROUND: Because Plasmodium falciparum displays increase tolerance against the recommended artemisinin combination therapies (ACT), new classes of anti-malarial drugs are urgently required. Previously synthesized artemisinin-aminoquinoline hybrids were evaluated to ascertain whether the potent low nanomolar in vitro anti-plasmodial activity would carry over in vivo against Plasmodium vinckei. A snapshot pharmacokinetic analysis was carried out on one of the hybrids to obtain an indication of the pharmacokinetic properties of this class of anti-malarial drugs. METHODS: In vitro activity of hybrids 2 and 3 were determined against the 3D7 strain of P. falciparum. Plasmodium vinckei-infected mice were treated with hybrids 1 - 3 for four days at a dosage of 0.8 mg/kg, 2.5 mg/kg, 7.5 mg/kg or 15 mg/kg intraperitoneally (ip), or orally (per os) with 2.7 mg/kg, 8.3 mg/kg, 25 mg/kg or 50 mg/kg. Artesunate was used as reference drug. A snapshot oral and IV pharmacokinetic study was performed on hybrid 2. RESULTS: Hybrids 1 - 3 displayed potent in vivo anti-malarial activity with ED50 of 1.1, 1.4 and <0.8 mg/kg by the ip route and 12, 16 and 13 mg/kg per os, respectively. Long-term monitoring of parasitaemia showed a complete cure of mice (without recrudescence) at 15 mg/kg via ip route and at 50 mg/kg by oral route for hybrid 1 and 2, whereas artesunate was only able to provide a complete cure at 30 mg/kg ip and 80 mg/kg per os. CONCLUSIONS: These compounds provide a new class of desperately needed anti-malarial drug. Despite a short half-life and moderate oral bioavailability, this class of compounds was able to cure malaria in mice at very low dosages. The optimum linker length for anti-malarial activity was found to be a diaminoalkyl chain consisting of two carbon atoms either methylated or unmethylated.

New bis-thiazolium analogues as potential antimalarial agents: design, synthesis, and biological evaluation.

Bis-thiazolium salts are able to inhibit phosphatidylcholine biosynthesis in Plasmodium and to block parasite proliferation in the low nanomolar range. However, due to their physicochemical properties (i.e., permanent cationic charges, the flexibility, and lipophilic character of the alkyl chain), the oral bioavailability of these compounds is low. New series of bis-thiazolium-based drugs have been designed to overcome this drawback. They feature linker rigidification via the introduction of aromatic rings and/or a decrease in the overall lipophilicity through the introduction of heteroatoms. On the basis of the structure-activity relationships, a few of the promising compounds (9, 10, and 11) were found to exhibit potent antimalarial in vitro and in vivo activities (EC(50) < 10 nM and ED(50) ip < 0.7 mg/kg).

Antimalarial and anticancer activities of artemisinin-quinoline hybrid-dimers and pharmacokinetic properties in mice.

Malaria, one of the three most important life-threatening infectious diseases, is recommended to be treated with ACT (artemisinin combination therapy) against which Plasmodium falciparum already displayed resistance. Two artemisinin-4-amino-quinoline hybrid-dimers (1 and 2), previously synthesized, possessed low nanomolar in vitro antiplasmodial activity, while poorly toxic against mammalian cells. They are here investigated to ascertain whether this antimalarial activity would be carried on in vivo against Plasmodium vinckei. During the four day treatment, parasitemia of less than 1% were observed on day 5 after doses from 2.5 mg/kg ip and 50 mg/kg po for hybrid-dimer 1, and from 7.5 mg/kg ip and 25 mg/kg po for hybrid-dimer 2. Snapshot pharmacokinetic analysis demonstrated that the antiplasmodial activity of these C-10-acetal artemisinin dimers may be due to active metabolites, which were confirmed by in silico findings. Hybrid-dimer 1 also displayed potent in vitro activity against tumor cells and was found to be more active than etoposide against TK10, UACC62 and MCF7 cell lines (TGI values 3.45 vs. 43.33 µM, 2.21 vs. 45.52 µM and 2.99 vs. >100 µM, respectively). The 1,3-diaminopropane linker, present in hybrid-dimer 1, was therefore identified as the optimum linker.

Evaluation of bis-alkylamidoxime O-alkylsulfonates as orally available antimalarials.

The main threat to controlling malaria is the emerging multidrug resistance of Plasmodium sp. parasites. Bis-alkylamidines were developed as a potential new chemotherapy that targets plasmodial phospholipid metabolism. Unfortunately, these compounds are not orally available. To solve this absorption issue, we investigated a prodrug strategy based on sulfonate derivatives of alkylamidoximes. A total of 25 sulfonates were synthesized as prodrug candidates of one bis-N-alkylamidine and of six N-substituted bis-C-alkylamidines. Their antimalarial activities were evaluated in vitro against P. falciparum and in vivo against P. vinckei in mice to define structure-activity relationships. Small alkyl substituents on the sulfonate group of both C-alkyl- and N-alkylamidines led to the best oral antimalarial activities; alkylsulfonate derivatives are chemically transformed into the corresponding alkylamidines.

Pharmacokinetic properties and metabolism of a new potent antimalarial N-alkylamidine compound, M64, and its corresponding bioprecursors.

Antimalarial activities and pharmacokinetics of the bis-alkylamidine, M64, and its amidoxime, M64-AH, and O-methylsulfonate, M64-S-Me, derivatives were investigated. M64 and M64-S-Me had the most potent activity against the Plasmodium falciparum growth (IC(50)<12nM). The three compounds can clear the Plasmodium vinckei infection in mice (ED(50)<10mg/kg). A liquid chromatography-mass spectrometry method was validated to simultaneously quantify M64 and M64-AH in human and rat plasma. M64 is partially metabolized to M64-monoamidoxime and M64-monoacetamide by rat and mouse liver microsomes. The amidoxime M64-AH undergoes extensive metabolism forming M64, M64-monoacetamide, M64-diacetamide and M64-monoamidoxime. Strong interspecies differences were observed. The pharmacokinetic profiles of M64, M64-AH and M64-S-Me were studied in rat after intravenous and oral administrations. M64 is partially metabolized to M64-AH; while M64-S-Me is rapidly and totally converted to M64 and M64-AH. M64-AH is mostly oxidized to the inactive M64-diacetamine while its N-reduction to the efficient M64 is a minor metabolic pathway. Oral dose of M64-AH was well absorbed (38%) and converted to M64 and M64-diacetamide. This study generated substantial information about the properties of this class of antimalarial drugs. Other routes of synthesis will be explored to prevent oxidative transformation of the amidoxime and to favour the N-reduction.

Reliability of antimalarial sensitivity tests depends on drug mechanisms of action.

In vitro antimalarial activity tests play a pivotal role in malaria drug research or for monitoring drug resistance in field isolates. We applied two isotopic tests, two enzyme-linked immunosorbent assays (ELISA) and the SYBR green I fluorescence-based assay, to test artesunate and chloroquine, the metabolic inhibitors atovaquone and pyrimethamine, our fast-acting choline analog T3/SAR97276, and doxycycline, which has a delayed death profile. Isotopic tests based on hypoxanthine and ethanolamine incorporation are the most reliable tests provided when they are applied after one full 48-h parasite cycle. The SYBR green assay, which measures the DNA content, usually requires 72 h of incubation to obtain reliable results. When delayed death is suspected, specific protocols are required with increasing incubation times up to 96 h. In contrast, both ELISA tests used (pLDH and HRP2) appear to be problematic, leading to disappointing and even erroneous results for molecules that do not share an artesunatelike profile. The reliability of these tests is linked to the mode of action of the drug, and the conditions required to get informative results are hard to predict. Our results suggest some minimal conditions to apply these tests that should give rise to a standard 50% inhibitory concentration, regardless of the mechanism of action of the compounds, and highlight that the most commonly used in vitro antimalarial activity tests do not have the same potential. Some of them might not detect the antimalarial potential of new classes of compounds with innovative modes of action, which subsequently could become promising new antimalarial drugs.

Comparison of the cellular and biochemical properties of Plasmodium falciparum choline and ethanolamine kinases.

The proliferation of the malaria-causing parasite Plasmodium falciparum within the erythrocyte is concomitant with massive phosphatidylcholine and phosphatidylethanolamine biosynthesis. Based on pharmacological and genetic data, de novo biosynthesis pathways of both phospholipids appear to be essential for parasite survival. The present study characterizes PfCK (P. falciparum choline kinase) and PfEK (P. falciparum ethanolamine kinase), which catalyse the first enzymatic steps of these essential metabolic pathways. Recombinant PfCK and PfEK were expressed as His6-tagged fusion proteins from overexpressing Escherichia coli strains, then purified to homogeneity and characterized. Using murine polyclonal antibodies against recombinant kinases, PfCK and PfEK were shown to be localized within the parasite cytoplasm. Protein expression levels increased during erythrocytic development. PfCK and PfEK appeared to be specific to their respective substrates and followed Michaelis-Menten kinetics. The Km value of PfCK for choline was 135.3+/-15.5 microM. PfCK was also able to phosphorylate ethanolamine with a very low affinity. PfEK was found to be an ethanolamine-specific kinase (Km=475.7+/-80.2 microM for ethanolamine). The quaternary ammonium compound hemicholinium-3 and an ethanolamine analogue, 2-amino-1-butanol, selectively inhibited PfCK or PfEK. In contrast, the bis-thiazolium compound T3, which was designed as a choline analogue and is currently in clinical trials for antimalarial treatment, affected PfCK and PfEK activities similarly. Inhibition exerted by T3 was competitive for both PfCK and PfEK and correlated with the impairment of cellular phosphatidylcholine biosynthesis. Comparative analyses of sequences and structures for both kinase types gave insights into their specific inhibition profiles and into the dual capacity of T3 to inhibit both PfCK and PfEK.

NG2 and Olig2 expression provides evidence for phenotypic deregulation of cultured central nervous system and peripheral nervous system neural precursor cells.

Neural stem cells cultured with fibroblast growth factor 2 (FGF2)/epidermal growth factor (EGF) generate clonal expansions called neurospheres (NS), which are widely used for therapy in animal models. However, their cellular composition is still poorly defined. Here, we report that NS derived from several embryonic and adult central nervous system (CNS) regions are composed mainly of remarkable cells coexpressing radial glia markers (BLBP, RC2, GLAST), oligodendrogenic/neurogenic factors (Mash1, Olig2, Nkx2.2), and markers that in vivo are typical of the oligodendrocyte lineage (NG2, A2B5, PDGFR-alpha). On NS differentiation, the latter remain mostly expressed in neurons, together with Olig2 and Mash1. Using cytometry, we show that in growing NS the small population of multipotential self-renewing NS-forming cells are A2B5(+) and NG2(+). Additionally, we demonstrate that these NS-forming cells in the embryonic spinal cord were initially NG2(-) and rapidly acquired NG2 in vitro. NG2 and Olig2 were found to be rapidly induced by cell culture conditions in spinal cord neural precursor cells. Olig2 expression was also induced in astrocytes and embryonic peripheral nervous system (PNS) cells in culture after EGF/FGF treatment. These data provide new evidence for profound phenotypic modifications in CNS and PNS neural precursor cells induced by culture conditions.

Exogenous and fibroblast growth factor 2/epidermal growth factor-regulated endogenous cytokines regulate neural precursor cell growth and differentiation.

Neurospheres (NSs) are clonal cellular aggregates composed of neural stem cells and progenitors. A comprehensive description of their proliferation and differentiation regulation is an essential prerequisite for their use in biotherapies. Cytokines are essential molecules regulating cell precursor fate. Using a gene-array strategy, we conducted a descriptive and functional analysis of endogenous cytokines and receptors expressed by spinal cord-derived NSs during their growth or their differentiation into neuronal and glial cells. NSs were found to express approximately 100 receptor subunits and cytokine/secreted developmental factors. Several angiogenic factors and receptors that could mediate neural precursor cell-endothelial cell relationships were detected. Among them, receptor B for endothelins was highly expressed, and endothelins were found to increase NS growth. In contrast, NSs express receptors for ciliary neurotrophic factor (CNTF), bone morphogenetic protein (BMP), interferon (IFN)-gamma, or tumor necrosis factor (TNF)-alpha, which, when added in the growth phase, led to a dramatic growth reduction followed by a reduction or a loss of oligodendrocyte formation on differentiation. In addition, NSs synthesize fibroblast growth factor 2/epidermal growth factor (FGF2/EGF)-regulated endogenous cytokines that participate in their growth and differentiation. Notably, BMP-7 and CNTF were expressed during expansion, but upon differentiation there was a remarkable switch from BMP-7 to BMP-4 and -6 and a sharp increase of CNTF. Reintroduction of growth factors reverses the BMP expression profile, indicating growth factor-BMP cross-regulations. The role of endogenous CNTF was investigated by deriving NSs from CNTF knockout mice. These NSs have an increased growth rate associated with reduction of apoptosis and generate astrocytes with a reduced glial fibulary acidic protein (GFAP) content. These results demonstrate the combined role of endogenous and exogenous cytokines in neural precursor cell growth and differentiation.