Target-specific projections of amygdala somatostatin-expressing neurons to the hypothalamus and brainstem.

Somatostatin neurons in the central nucleus of the amygdala (CeA/Sst) can be parsed into subpopulations that project either to the nucleus of the solitary tract (NST) or parabrachial nucleus (PBN). We have shown recently that inhibition of CeA/Sst-to-NST neurons increased the ingestion of a normally aversive taste stimulus, quinine HCl (QHCl). Because the CeA innervates other forebrain areas such as the lateral hypothalamus (LH) that also sends axonal projections to the NST, the effects on QHCl intake could be, in part, the result of CeA modulation of LH-to-NST neurons. To address these issues, the present study investigated whether CeA/Sst-to-NST neurons are distinct from CeA/Sst-to-LH neurons. For comparison purposes, additional experiments assessed divergent innervation of the LH by CeA/Sst-to-PBN neurons. In Sst-cre mice, two different retrograde transported flox viruses were injected into the NST and the ipsilateral LH or PBN and ipsilateral LH. The results showed that 90% or more of retrograde-labeled CeA/Sst neurons project either to the LH, NST, or PBN. Separate populations of CeA/Sst neurons projecting to these different regions suggest a highly heterogeneous population in terms of synaptic target and likely function.

PQQ Dietary Supplementation Prevents Alkylating Agent-Induced Ovarian Dysfunction in Mice.

Alkylating agents (AAs) that are commonly used for cancer therapy cause great damage to the ovary. Pyrroloquinoline-quinine (PQQ), which was initially identified as a redox cofactor for bacterial dehydrogenases, has been demonstrated to benefit the fertility of females. The aim of this study was to investigate whether PQQ dietary supplementation plays a protective role against alkylating agent-induced ovarian dysfunction. A single dose of busulphan (20 mg/kg) and cyclophosphamide (CTX, 120 mg/kg) were used to establish a mouse model of ovarian dysfunction. Feed containing PQQNa2 (5 mg/kg) was provided starting 1 week before the establishment of the mouse model until the date of sacrifice. One month later, estrous cycle period of mice were examined and recorded for consecutive 30 days. Three months later, some mice were mated with fertile male mice for fertility test. The remaining mice were sacrificed to collect serum samples and ovaries. One day before sacrifice, some mice received a single injection of BrdU to label proliferating cells. Serum samples were used for test hormonal levels. Ovaries were weighted and used to detect follicle counts, cell proliferation, cell apoptosis and cell senescence. In addition, the levels of inflammation, oxidative damage and Pgc1α expression were detected in ovaries. Results showed that PQQ treatment increased the ovarian weight and size, partially normalized the disrupted estrous cycle period and prevented the loss of follicles of mice treated with AAs. More importantly, we found that PQQ treatment significantly increased the pregnancy rate and litter size per delivery of mice treated with AAs. The protective effects of PQQ appeared to be directly mediated by promoting cell proliferation of granulosa, and inhibiting cell apoptosis of granulosa and cell senescence of ovarian stromal cells. The underlying mechanisms may attribute to the anti-oxidative stress, anti-inflammation and pro-mitochondria biogenesis effects of PQQ.Our study highlights the therapeutic potential of PQQ against ovarian dysfunction caused by alkylating agents.

Quinine-Based Semisynthetic Ion Transporters with Potential Antiproliferative Activities.

Synthetic ion transporters have attracted tremendous attention for their therapeutic potential against various ion-transport-related diseases, including cancer. Inspired by the structure and biological activities of natural products, we synthesized a small series of squaramide and thiourea derivatives of quinine and investigated their ion transport activities. The involvement of a quinuclidine moiety for the cooperative interactions of Cl- and H+ ions with the thiourea or squaramide moiety resulted in an effectual transport of these ions across membranes. The interference of ionic equilibrium by the potent Cl- ion carrier selectively induced cancer cell death by endorsing caspase-arbitrated apoptosis. In vivo assessment of the potent ionophore showed an efficient reduction in tumor growth with negligible immunotoxicity to other organs.

Quinine Enhances Photo-Inactivation of Gram-Negative Bacteria.

BACKGROUND: Antimicrobial resistance is a significant concern to public health, and there is a pressing need to develop novel antimicrobial therapeutic modalities. METHODS: In this study, we investigated the capacity for quinine hydrochloride (Q-HCL) to enhance the antimicrobial effects of antimicrobial blue light ([aBL] 405 nm wavelength) against multidrug-resistant (MDR) Gram-negative bacteria in vitro and in vivo. RESULTS: Our findings demonstrated the significant improvement in the inactivation of MDR Pseudomonas aeruginosa and Acinetobacter baumannii (planktonic cells and biofilms) when aBL was illuminated during Q-HCL exposure. Furthermore, the addition of Q-HCL significantly potentiated the antimicrobial effects of aBL in a mouse skin abrasion infection model. In addition, combined exposure of aBL and Q-HCL did not result in any significant apoptosis when exposed to uninfected mouse skin. CONCLUSIONS: In conclusion, aBL in combination with Q-HCL may offer a novel approach for the treatment of infections caused by MDR bacteria.

Exploring Structure-Activity Relationship in Tacrine-Squaramide Derivatives as Potent Cholinesterase Inhibitors.

Tacrine was the first drug to be approved for Alzheimer's disease (AD) treatment, acting as a cholinesterase inhibitor. The neuropathological hallmarks of AD are amyloid-rich senile plaques, neurofibrillary tangles, and neuronal degeneration. The portfolio of currently approved drugs for AD includes acetylcholinesterase inhibitors (AChEIs) and N-methyl-d-aspartate (NMDA) receptor antagonist. Squaric acid is a versatile structural scaffold capable to be easily transformed into amide-bearing compounds that feature both hydrogen bond donor and acceptor groups with the possibility to create multiple interactions with complementary sites. Considering the relatively simple synthesis approach and other interesting properties (rigidity, aromatic character, H-bond formation) of squaramide motif, we combined this scaffold with different tacrine-based derivatives. In this study, we developed 21 novel dimers amalgamating squaric acid with either tacrine, 6-chlorotacrine or 7-methoxytacrine representing various AChEIs. All new derivatives were evaluated for their anti-cholinesterase activities, cytotoxicity using HepG2 cell line and screened to predict their ability to cross the blood-brain barrier. In this contribution, we also report in silico studies of the most potent AChE and BChE inhibitors in the active site of these enzymes.

Mouse Parabrachial Neurons Signal a Relationship between Bitter Taste and Nociceptive Stimuli.

Taste and somatosensation both mediate protective behaviors. Bitter taste guides avoidance of ingestion of toxins while pain sensations, such as noxious heat, signal adverse conditions to ward off harm. Although brain pathways for taste and somatosensation are typically studied independently, prior data suggest that they intersect, potentially reflecting their common protective role. To investigate this, we applied electrophysiologic and optogenetic techniques in anesthetized mice of both sexes to evaluate relationships between oral somatosensory and taste activity in the parabrachial nucleus (PbN), implicated for roles in gustation and pain. Spikes were recorded from taste-active PbN neurons tested with oral delivery of thermal and chemesthetic stimuli, including agonists of nocisensitive transient receptor potential (TRP) ion channels on somatosensory fibers. Gustatory neurons were also tested to follow electrical pulse stimulation of an oral somatosensory region of the spinal trigeminal subnucleus caudalis (Vc), which projects to the PbN. Neurons composed classic taste groups, including sodium, electrolyte, appetitive, or bitter cells. Across groups, most neurons spiked to Vc pulse stimulation, implying that trigeminal projections reach PbN gustatory neurons. Among such cells, a subpopulation responsive to the bitter taste stimuli quinine and cycloheximide, and aversive concentrations of sodium, cofired to agonists of nocisensitive TRP channels, including capsaicin, mustard oil, and noxious heat. Such neurons populated the lateral PbN. Further, nociceptive activity in PbN bitter taste neurons was suppressed during optogenetic-assisted inhibition of the Vc, implying convergent trigeminal input contributed to such activity. Our results reveal a novel role for PbN gustatory cells in cross-system signaling related to protection.SIGNIFICANCE STATEMENT Prior data suggest that gustatory and trigeminal neural pathways intersect and overlap in the parabrachial area. However, no study has directly examined such overlap and why it may exist. Here we found that parabrachial gustatory neurons can receive afferent projections from trigeminal nuclei and fire to oral nociceptive stimuli that excite somatosensory receptors and fibers. Activation to aversive nociceptive stimuli in gustatory cells was associated with responding to behaviorally avoided bitter tastants. We were further able to show that silencing trigeminal projections inhibited nociceptive activity in parabrachial bitter taste neurons. Our results imply that in the parabrachial area, there is predictable overlap between taste and somatosensory processing related to protective coding and that classically defined taste neurons contribute to this process.

Functional Components from Nature-Derived Drugs for the Treatment of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic systemic disorder characterized by persistent synovitis and systemic inflammation. Currently, the widely used drugs for the treatment of RA are disease-modifying antirheumatic drugs, biological agents and glucocorticoids. But their clinical use has been limited because of their adverse effects with a high frequency and high cost of treatment. It is essential to find novel candidate agents. Traditional Chinese medicine (TCM) has been used for RA treatment for a long period of time. In recent years, significant amounts of studies have shown that some TCMs and their active ingredients have obvious therapeutic effects on RA. In this review, the compounds in TCMs that have an effect in clinic or animal experiments of RA are critically reviewed and summarized. Moreover, the relationship between chemical structures of the compound and their activities is analyzed. The relevant researches are described from the aspects of source, methods, result, and related mechanism analysis. The existing studies show that most effective compounds in TCM for RA treatment belong to alkaloids, flavonoids, terpenoids, phenols and quinines. It is hoped that the data summarized in this review will be beneficial to the screening of new nature-derived antirheumatic drugs.

Identification of Compounds with Efficacy against Malaria Parasites from Common North American Plants.

Some of the most valuable antimalarial compounds, including quinine and artemisinin, originated from plants. While these drugs have served important roles over many years for the treatment of malaria, drug resistance has become a widespread problem. Therefore, a critical need exists to identify new compounds that have efficacy against drug-resistant malaria strains. In the current study, extracts prepared from plants readily obtained from local sources were screened for activity against Plasmodium falciparum. Bioassay-guided fractionation was used to identify 18 compounds from five plant species. These compounds included eight lupane triterpenes (1-8), four kaempferol 3-O-rhamnosides (10-13), four kaempferol 3-O-glucosides (14-17), and the known compounds amentoflavone and knipholone. These compounds were tested for their efficacy against multi-drug-resistant malaria parasites and counterscreened against HeLa cells to measure their antimalarial selectivity. Most notably, one of the new lupane triterpenes (3) isolated from the supercritical extract of Buxus sempervirens, the common boxwood, showed activity against both drug-sensitive and -resistant malaria strains at a concentration that was 75-fold more selective for the drug-resistant malaria parasites as compared to HeLa cells. This study demonstrates that new antimalarial compounds with efficacy against drug-resistant strains can be identified from native and introduced plant species in the United States, which traditionally have received scant investigation compared to more heavily explored tropical and semitropical botanical resources from around the world.

Mouth rinsing and ingestion of a bitter-tasting solution increases corticomotor excitability in male competitive cyclists.

PURPOSE: Recently, we have shown that the combination of mouth rinsing and ingesting a bitter-tasting quinine solution immediately prior to the performance of a maximal 30-s cycling sprint significantly improves mean and peak power output. This ergogenic effect was proposed to be related to the activation of the corticomotor pathway by afferent taste signals originating from bitter taste receptors in the oral cavity. The aim of the present study was to use single-pulse transcranial magnetic stimulation to investigate whether mouth rinsing and ingestion of a bitter quinine solution increases corticomotor excitability. METHODS: A series of 10 motor-evoked potentials (MEPs) were recorded from the relaxed first dorsal interosseus muscle in 16 male competitive cyclists immediately before and after they rinsed their mouth for 10 s and then ingested either a 2 mM bitter quinine solution or plain water. RESULTS: Mean MEP amplitude was significantly increased in response to quinine administration by 16% (p < 0.05), with no evidence of a time-dependent effect over the 10 pulses. Mean MEP amplitude also increased by 10% in response to water administration (p < 0.05), though this increase was significantly smaller than the response to quinine (p < 0.05). CONCLUSIONS: We conclude that the activation of bitter taste receptors in the oral cavity and upper gastrointestinal tract has the capacity to increase corticomotor excitability in male competitive cyclists.

Cuantificación de quinina en extractos de Cinchona pubescens y evaluación de la actividad antiplasmodial y citotóxica / Quantification of quinine in extracts from stems Cinchona pubescens and evaluation of antiplasmodial and cytotoxic activity

Plants belonging to the genus Cinchona L. (Rubiaceae), whose active ingredient is quinine, was used for centuries to treat malaria. Plants of this genus are a potential source of new structural templates in the search for new antimalarial candidates. This study aimed to the identification, quantification of quinine and other metabolites present in extracts of different polarity of the stems of Cinchona pubescens Vahl. , oxoquinovic acid isolation, antiplasmodial activity, and measuring its cytotoxic effect. The results show a high activity to antiplasmodial alkaloids extract (IC50 = 2.20 +/- 0.0325 ug/mL), cytotoxicity (CC50 = 80.2 +/- 12.2 ug/mL), and a quinine content of 21.3+/-0.0247 ppm. The compound known as acid antiplasmodial activity oxoquinovic presented in IC50 = 11.3 +/- 0.741 ug/mL, and cytotoxicity CC50 = 72.4 +/- 3.85 ug/mL. These results motivate phytochemical studies in the search for active structural analogues quinine and quinolinic core as a source of new antimalarial agents.

Las plantas pertenecientes al género Cinchona L. (familia Rubiaceae), cuyo principio activo es la quinina, fueron utilizadas durante siglos para tratar la malaria. Este género es una fuente potencial de nuevas plantillas estructurales en la búsqueda de nuevos candidatos antimaláricos. El presente trabajo tuvo como objetivo la identificación y, cuantificación de la quinina y de otros metabolitos presentes en los extractos de diferente polaridad, de los tallos de Cinchona pubescens Vahl. , el aislamiento del ácido oxoquinóvico, la actividad antiplasmodial y, además, la medición de su efecto citotóxico. Los resultados muestran una alta actividad antiplasmodial para el extracto de los alcaloides (IC50 = 2,20 +/- 0,0325 ug/mL), una baja citotoxicidad (CC50 = 80,2 +/- 12,2 ug/mL), y un alto contenido de quinina el cual fue 21,3+/-0,0247 ppm. El compuesto ácido oxoquinóvico presentó una actividad antiplasmodial de IC50 = 11,3 +/- 0.741 ug/mL, y una citotoxicidad de CC50 = 72,4 +/- 3,85 ug/mL. Estos resultados motivan los estudios fitoquímicos en la búsqueda de principios activos y análogos estructurales en diferentes especies de Cinchonas como una fuente de nuevos agentes antimaláricos.

Functional mechanotransduction is required for cisplatin-induced hair cell death in the zebrafish lateral line.

Cisplatin, one of the most commonly used anticancer drugs, is known to cause inner ear hair cell damage and hearing loss. Despite much investigation into mechanisms of cisplatin-induced hair cell death, little is known about the mechanism whereby cisplatin is selectively toxic to hair cells. Using hair cells of the zebrafish lateral line, we found that chemical inhibition of mechanotransduction with quinine and EGTA protected against cisplatin-induced hair cell death. Furthermore, we found that the zebrafish mutants mariner (myo7aa) and sputnik (cad23) that lack functional mechanotransduction were resistant to cisplatin-induced hair cell death. Using a fluorescent analog of cisplatin, we found that chemical or genetic inhibition of mechanotransduction prevented its uptake. These findings demonstrate that cisplatin-induced hair cell death is dependent on functional mechanotransduction in the zebrafish lateral line.

Quinine controls body weight gain without affecting food intake in male C57BL6 mice.

BACKGROUND: Quinine is a natural molecule commonly used as a flavouring agent in tonic water. Diet supplementation with quinine leads to decreased body weight and food intake in rats. Quinine is an in vitro inhibitor of Trpm5, a cation channel expressed in taste bud cells, the gastrointestinal tract and pancreas. The objective of this work is to determine the effect of diet supplementation with quinine on body weight and body composition in male mice, to investigate its mechanism of action, and whether the effect is mediated through Trpm5. RESULTS: Compared with mice consuming AIN, a regular balanced diet, mice consuming AIN diet supplemented with 0.1% quinine gained less weight (2.89 ± 0.30 g vs 5.39 ± 0.50 g) and less fat mass (2.22 ± 0.26 g vs 4.33 ± 0.43 g) after 13 weeks of diet, and had lower blood glucose and plasma triglycerides. There was no difference in food intake between the mice consuming quinine supplemented diet and those consuming control diet. Trpm5 knockout mice gained less fat mass than wild-type mice. There was a trend for a diet-genotype interaction for body weight and body weight gain, with the effect of quinine less pronounced in the Trpm5 KO than in the WT background. Faecal weight, energy and lipid contents were higher in quinine fed mice compared to regular AIN fed mice and in Trpm5 KO mice compared to wild type mice. CONCLUSION: Quinine contributes to weight control in male C57BL6 mice without affecting food intake. A partial contribution of Trpm5 to quinine dependent body weight control is suggested.

In vitro susceptibility of Plasmodium falciparum Welch field isolates to infusions prepared from Artemisia annua L. cultivated in the Brazilian Amazon.

Artemisinin is the active antimalarial compound obtained from the leaves of Artemisia annua L. Artemisinin, and its semi-synthetic derivatives, are the main drugs used to treat multi-drug-resistant Plasmodium falciparum (one of the human malaria parasite species). The in vitro susceptibility of P. falciparum K1 and 3d7 strains and field isolates from the state of Amazonas, Brazil, to A. annua infusions (5 g dry leaves in 1 L of boiling water) and the drug standards chloroquine, quinine and artemisinin were evaluated. The A. annua used was cultivated in three Amazon ecosystems (várzea, terra preta de índio and terra firme) and in the city of Paulínia, state of São Paulo, Brazil. Artemisinin levels in the A. annua leaves used were 0.90-1.13% (m/m). The concentration of artemisinin in the infusions was 40-46 mg/L. Field P. falciparum isolates were resistant to chloroquine and sensitive to quinine and artemisinin. The average 50% inhibition concentration values for A. annua infusions against field isolates were 0.11-0.14 µL/mL (these infusions exhibited artemisinin concentrations of 4.7-5.6 ng/mL) and were active in vitro against P. falciparum due to their artemisinin concentration. No synergistic effect was observed for artemisinin in the infusions.

In vitro susceptibility of Plasmodium falciparum Welch field isolates to infusions prepared from Artemisia annua L. cultivated in the Brazilian Amazon

Artemisinin is the active antimalarial compound obtained from the leaves of Artemisia annua L. Artemisinin, and its semi-synthetic derivatives, are the main drugs used to treat multi-drug-resistant Plasmodium falciparum (one of the human malaria parasite species). The in vitro susceptibility of P. falciparum K1 and 3d7 strains and field isolates from the state of Amazonas, Brazil, to A. annua infusions (5 g dry leaves in 1 L of boiling water) and the drug standards chloroquine, quinine and artemisinin were evaluated. The A. annua used was cultivated in three Amazon ecosystems (várzea, terra preta de índio and terra firme) and in the city of Paulínia, state of São Paulo, Brazil. Artemisinin levels in the A. annua leaves used were 0.90-1.13% (m/m). The concentration of artemisinin in the infusions was 40-46 mg/L. Field P. falciparum isolates were resistant to chloroquine and sensitive to quinine and artemisinin. The average 50% inhibition concentration values for A. annua infusions against field isolates were 0.11-0.14 μL/mL (these infusions exhibited artemisinin concentrations of 4.7-5.6 ng/mL) and were active in vitro against P. falciparum due to their artemisinin concentration. No synergistic effect was observed for artemisinin in the infusions.

Extracellular heme enhances the antimalarial activity of artemisinin.

Artemisinin exerts the antimalarial activity through activation by heme. The hemolysis in malaria results in the elevated levels of plasma heme which may affect the activity of artemisinin. We hypothesized that the extracellular heme would potentiate the antimalarial activity of artemisinin. Hemin (ferric heme) at the pathologic concentrations enhanced the activity of artemisinin against Plasmodium falciparum in vitro and increased the levels of the lipid peroxidation products in the presence of artemisinin. The antimalarial activity of artemisinin and potentiation by hemin was decreased by vitamin E. Hemin had no effect on the activity of quinoline drugs (chloroquine, quinine and mefloquine). Furthermore, the oxidative effect of hemin in the presence of artemisinin or quinoline drugs was studied using low-density lipoprotein (LDL) oxidation as a model. Artemisinin enhanced the effects of hemin on lipid peroxidation and a decrease of tryptophan fluorescence in LDL whereas the quinoline drugs inhibited the oxidation by hemin. In conclusion, the extracellular hemin enhances the antimalarial activity of artemisinin as a result of the increasing oxidative effect of hemin.

Genetic epidemiology of induced CYP3A4 activity.

AIM: The cytochrome P450 3A4 (CYP3A4) enzyme is implicated in the metabolism of more than 50% of all prescribed medications and its activity - including induced or inhibited activity - is deemed to be a crucial determinant of interindividual variability in drug disposition, poor therapeutic efficacy, and adverse response to medication. METHODS: We used the classical twin model in conjunction with an induction experiment to uncover the relative contribution of genetic and environmental factors to interindividual variation in induced CYP3A4 activity. A total of 367 healthy twins participated in the study. Each volunteer was administered a potent inducer of CYP3A4 (St John's Wort) for 14 days and the activity of CYP3A4 was quantified through the metabolism of the exogenously administered probe drug quinine sulfate. RESULTS: Baseline and induced CYP3A4 activity were highly variable with a seven-fold and 11-fold difference among our population, respectively. Alcohol consumption, BMI, and smoking were significantly associated with induced CYP3A4 activity, collectively explaining 20% of the variation (P<1×10(-4)). The narrow-sense heritability of induced CYP3A4 activity was estimated at 66%, whereas the remainder of the variation was attributed to unique environmental factors. CONCLUSION: To our knowledge, this is the first genetic epidemiological study of induced CYP3A4 activity. Our results motivate further research to identify common and rarer genetic variants that underpin the heritable component of variation in induced CYP3A4 activity.

Plasmodium falciparum: Solanum nudum SN-1 steroid antiplasmodial activity when combined with antimalarial drugs.

The effect of 16 alpha-acetoxy-26-hydroxycholest-4-ene-3,22-dione (SN-1) isolated from Solanum nudum Dunal (a Solanaceae traditionally used for treating fever in Colombia) on Plasmodium falciparum erythrocyte stages and its in vitro antiplasmodial activity when combined with the following conventional drugs was studied: chloroquine (CQ), amodiaquine (AQ), desethylamodiaquine (desethyl-AQ), quinine (QN), artemisinin (AR), atovaquone (ATV) and quinine (QN). It was found that SN-1 targeted trophozoites and had a synergistic effect when combined with CQ and QN; however, it had an antagonist effect when used with the other combinations.

Evidence for an integrated oral sensory module in the human anterior ventral insula.

Taste, which is almost always accompanied by other oral sensations, serves to identify potential nutrients and toxins. The present study was designed to determine the influence of sensory modality (chemesthetic vs. gustatory) and physiological significance (potentially nutritive vs. potentially harmful) on insular response to oral stimulation. Sixteen subjects underwent functional magnetic resonance imaging scanning while receiving 2 potentially nutritive solutions (sucrose and NaCl), 2 potentially harmful solutions (quinine and capsaicin, a chemesthetic stimulus), and a tasteless control solution. We identified a region of anterior ventral insula that responded to oral stimulation irrespective of modality or physiological significance. However, when subjects tasted a potentially nutritive stimulus, the connectivity between the insula and a feeding network including the hypothalamus, ventral pallidum, and striatum was greater than when tasting a potentially harmful stimulus. No differential connectivity was observed as a function of modality (gustatory vs. chemesthetic). These results support the existence of an integrated supramodal flavor system in the anterior ventral insula that preferentially communicates with the circuits guiding feeding when the flavor is potentially nutritive.

In vitro activity of pyronaridine against Plasmodium falciparum and comparative evaluation of anti-malarial drug susceptibility assays.

BACKGROUND: Pyronaridine, a Mannich base anti-malarial with high efficacy against drug resistant Plasmodium falciparum, is currently evaluated as a fixed dose combination with artesunate for the treatment of uncomplicated malaria. In this study, the in vitro activity of pyronaridine against clinical isolates of P. falciparum from Lambaréné, Gabon, was assessed in order to obtain baseline data on its activity prior to its future use in routine therapy. Moreover, follow-up assessment on the in vitro activity of chloroquine, artesunate and quinine was performed. METHODS: In vitro response of field isolates of P. falciparum to pyronaridine, chloroquine, artesunate and quinine was assessed using the traditional WHO microtest. In addition, the histidine-rich protein 2 (HRP-2) assay was performed and evaluated for its future implementation for follow-up of drug susceptibility testing. RESULTS: Pyronaridine exhibited a high in vitro activity against P. falciparum, with a geometric mean cut-off concentration of 9.3 nmol/l. Fifty percent effective concentrations were 1.9 nmol/l and 2.0 nmol/l in the WHO microtest and HRP-2 assay, respectively. Results matched closely in vivo findings from a recent clinical trial on pyronaridine-artesunate treatment. One isolate showed diminished sensitivity to artesunate. For chloroquine and quinine resistance levels were comparable to prior studies from Lambaréné. Results from the novel HRP-2 assay corresponded well to those obtained by the WHO microtest. CONCLUSION: Pyronaridine is highly active in chloroquine-resistant parasites and seems a promising partner drug for artemisinin-based combination therapy in Africa.