Dried Whole Plant Artemisia annua as an Antimalarial Therapy

Drugs are primary weapons for reducing malaria in human populations. However emergence of resistant parasites has repeatedly curtailed the lifespan of each drug that is developed and deployed. Currently the most effective anti-malarial is artemisinin, which is extracted from the leaves of Artemisia annua. Due to poor pharmacokinetic properties and prudent efforts to curtail resistance to monotherapies, artemisinin is prescribed only in combination with other anti-malarials composing an Artemisinin Combination Therapy (ACT). Low yield in the plant, and the added cost of secondary anti-malarials in the ACT, make artemisinin costly for the developing world. As an alternative, we compared the efficacy of oral delivery of the dried leaves of whole plant (WP) A. annua to a comparable dose of pure artemisinin in a rodent malaria model (Plasmodium chabaudi). We found that a single dose of WP (containing 24 mg/kg artemisinin) reduces parasitemia more effectively than a comparable dose of purified drug. This increased efficacy may result from a documented 40-fold increase in the bioavailability of artemisinin in the blood of mice fed the whole plant, in comparison to those administered synthetic drug. Synergistic benefits may derive from the presence of other anti-malarial compounds in A. annua. If shown to be clinically efficacious, well-tolerated, and compatible with the public health imperative of forestalling evolution of drug resistance, inexpensive, locally grown and processed A. annua might prove to be an effective addition to the global effort to reduce malaria morbidity and mortality.     

Development of a Specific Monoclonal Antibody-Based ELISA to Measure the Artemether Content of Antimalarial Drugs

Artemether is one of the artemisinin derivatives that are active ingredients in antimalarial drugs. Counterfeit and substandard antimalarial drugs have become a serious problem, which demands reliable analytical tools and implementation of strict regulation of drug quality. Structural similarity among artemisinin analogs is a challenge to develop immunoassays that are specific to artemisinin derivatives. To produce specific antibodies to artemether, we used microbial fermentation of artemether to obtain 9-hydroxyartemether, which was subsequently used to prepare a 9-O-succinylartemether hapten for conjugation with ovalbumin as the immunogen. A monoclonal antibody (mAb), designated as 2G12E1, was produced with high specificity to artemether. 2G12E1 showed low cross reactivities to dihydroartemisinin, artemisinin, artesunate and other major antimalarial drugs. An indirect competitive enzyme linked immunosorbent assay (icELISA) developed showed a concentration causing 50% of inhibition for artemether as 3.7 ng mL⁻¹ and a working range of 0.7–19 ng mL⁻¹. The icELISA was applied for determination of artemether content in different commercial drugs and the results were comparable to those determined by high-performance liquid chromatography analysis. In comparison with reported broad cross activity of anti-artemisinin mAbs, the most notable advantage of the 2G12E1-based ELISA is its high specificity to artemether only.     

Combined antimalarial therapy using artemisinin

The existing armamentarium of drugs for the treatment and prevention of malaria is limited primarily by resistance (and cross-resistance between closely related drugs). However, most of these drugs still have a place and their life-span could be prolonged if better deployed and used, and also by rationally combining them based on pharmacodynamic and pharmacokinetic properties. Newer compounds are also being developed. The nature of malaria disease and its prevalence in the developing world call for innovative approaches to develop new affordable drugs and to safeguard the available ones. According to WHO, the concept of combination therapy is based on the synergistic or additive potential of two or more drugs, to improve therapeutic efficacy and also delay the development of resistance to the individual components of the combination. Combination therapy (CT) with antimalarial drugs is the simultaneous use of two or more blood schizontocidal drugs with independent modes of action and different biochemical targets in the parasite. In the context of this definition, multiple-drug therapies that include a nonantimalarial drug to enhance the antimalarial effect of a blood schizontocidal drug are not considered combination therapy. Similarly, certain antimalarial drugs that fit the criteria of synergistic fixed-dose combinations are operationally considered as single products in that neither of the individual components would be given alone for anti-malarial therapy. An example is sulfadoxine-pyrimethamine. Artemisinin-based combination therapies have been shown to improve treatment efficacy and also contain drug resistance in South-East Asia. However, major challenges exist in the deployment and use of antimalarial drug combination therapies, particularly in Africa. These include: 1) the choice of drug combinations best suited for the different epidemiological situations; 2) the cost of combination therapy; 3) the timing of the introduction of combination therapy; 4) the operational obstacles to implementation, especially compliance. As a response to increasing levels of antimalarial resistance, the World Health Organization (WHO) recommends that all countries experiencing resistance to conventional monotherapies, such as chloroquine, amodiaquine or sulfadoxine/pyrimethamine, should use combination therapies, preferably those containing artemisinin derivatives (ACTs--artemisinin-based combination therapies) for malaria caused by Plasmodium falciparum. There is a promising role of such compounds in replacing or complementing current options. Since 1979, several different formulations of artemisinin and its derivatives have been produced and studied in China in several thousand patients for either P. falciparum or P. vivax malaria. To date, there is no evidence of drug resistance to these compounds. The use of artemisinin, artemether, arteether and artesunate for either uncomplicated or severe malaria is now spreading through almost all malarious areas of the world, although some of they have no patent protection, their development (with few exceptions) has not followed yet full international standards. Both artesunate, artemether and arteether are rapidly and extensively converted to their common bioactive metabolite, dihydroarte-misinin. WHO currently recommends the following therapeutic options: 1) artemether/lumefantrine; 2) artesunate plus amodiaquine; 3) artesunate plus sulfadoxine/pyrimethamine (in areas where SP efficacy remains high); 4) artesunate plus mefloquine (in areas with low to moderate transmission); and 5) amodiaquine plus sulfadoxine/pyrimethamine, in areas where efficacy of both amodiaquine and sulfadoxine/pyrimethamine remains high (mainly limited to countries in West Africa). This non artemisinin-based combination therapy is reserved as an interim option for countries, which, for whatever reason, are unable immediately to move to ACTs.     

Artemisinin, an endoperoxide antimalarial, disrupts the hemoglobin catabolism and heme detoxification systems in malarial parasite.

Endoperoxide antimalarials based on the ancient Chinese drug Qinghaosu (artemisinin) are currently our major hope in the fight against drug-resistant malaria. Rational drug design based on artemisinin and its analogues is slow as the mechanism of action of these antimalarials is not clear. Here we report that these drugs, at least in part, exert their effect by interfering with the plasmodial hemoglobin catabolic pathway and inhibition of heme polymerization. In an in vitro experiment we observed inhibition of digestive vacuole proteolytic activity of malarial parasite by artemisinin. These observations were further confirmed by ex vivo experiments showing accumulation of hemoglobin in the parasites treated with artemisinin, suggesting inhibition of hemoglobin degradation. We found artemisinin to be a potent inhibitor of heme polymerization activity mediated by Plasmodium yoelii lysates as well as Plasmodium falciparum histidine-rich protein II. Interaction of artemisinin with the purified malarial hemozoin in vitro resulted in the concentration-dependent breakdown of the malaria pigment. Our results presented here may explain the selective and rapid toxicity of these drugs on mature, hemozoin-containing, stages of malarial parasite. Since artemisinin and its analogues appear to have similar molecular targets as chloroquine despite having different structures, they can potentially bypass the quinoline resistance machinery of the malarial parasite, which causes sublethal accumulation of these drugs in resistant strains.     

Potent in vivo antimalarial activity of 3,15-di-O-acetylbruceolide against Plasmodium berghei infection in mice

The antimalarial activity of the O-acylated bruceolide derivative, 3,15-di-O-acetylbruceolide, was evaluated against Plasmodium berghei in vivo. The concentration of 3,15-di-O-acetylbruceolide required for 50% suppression (ED50) of P. berghei in mice was 0.46 +/- 0.06 mg/kg/day, whereas bruceolide was only half as effective as 3,15-di-O-acetylbruceolide. Two antimalarial drugs used clinically, chloroquine and artemisinin, demonstrated only low activity corresponding to 1/4 and 1/12 of the ED50 value of 3,15-di-O-acetylbruceolide, respectively. These results may be helpful in the design of better chemotherapeutic bruceolides against falciparum malaria.     

Artemisinin Directly Targets Malarial Mitochondria through Its Specific Mitochondrial Activation

The biological mode of action of artemisinin, a potent antimalarial, has long been controversial. Previously we established a yeast model addressing its mechanism of action and found mitochondria the key in executing artemisinin''s action. Here we present data showing that artemisinin directly acts on mitochondria and it inhibits malaria in a similar way as yeast. Specifically, artemisinin and its homologues exhibit correlated activities against malaria and yeast, with the peroxide bridge playing a key role for their inhibitory action in both organisms. In addition, we showed that artemisinins are distributed to malarial mitochondria and directly impair their functions when isolated mitochondria were tested. In efforts to explore how the action specificity of artemisinin is achieved, we found strikingly rapid and dramatic reactive oxygen species (ROS) production is induced with artemisinin in isolated yeast and malarial but not mammalian mitochondria, and ROS scavengers can ameliorate the effects of artemisinin. Deoxyartemisinin, which lacks an endoperoxide bridge, has no effect on membrane potential or ROS production in malarial mitochondria. OZ209, a distantly related antimalarial endoperoxide, also causes ROS production and depolarization in isolated malarial mitochondria. Finally, interference of mitochondrial electron transport chain (ETC) can alter the sensitivity of the parasite towards artemisinin. Addition of iron chelator desferrioxamine drastically reduces ETC activity as well as mitigates artemisinin-induced ROS production. Taken together, our results indicate that mitochondrion is an important direct target, if not the sole one, in the antimalarial action of artemisinins. We suggest that fundamental differences among mitochondria from different species delineate the action specificity of this class of drugs, and differing from many other drugs, the action specificity of artemisinins originates from their activation mechanism.     

Improving Pharmacokinetic-Pharmacodynamic Modeling to Investigate Anti-Infective Chemotherapy with Application to the Current Generation of Antimalarial Drugs

Mechanism-based pharmacokinetic-pharmacodynamic (PK/PD) modelling is the standard computational technique for simulating drug treatment of infectious diseases with the potential to enhance our understanding of drug treatment outcomes, drug deployment strategies, and dosing regimens. Standard methodologies assume only a single drug is used, it acts only in its unconverted form, and that oral drugs are instantaneously absorbed across the gut wall to their site of action. For drugs with short half-lives, this absorption period accounts for a significant period of their time in the body. Treatment of infectious diseases often uses combination therapies, so we refined and substantially extended the PK/PD methodologies to incorporate (i) time lags and drug concentration profiles resulting from absorption across the gut wall and, if required, conversion to another active form; (ii) multiple drugs within a treatment combination; (iii) differing modes of action of drugs in the combination: additive, synergistic, antagonistic; (iv) drugs converted to an active metabolite with a similar mode of action. This methodology was applied to a case study of two first-line malaria treatments based on artemisinin combination therapies (ACTs, artemether-lumefantrine and artesunate-mefloquine) where the likelihood of increased artemisinin tolerance/resistance has led to speculation on their continued long-term effectiveness. We note previous estimates of artemisinin kill rate were underestimated by a factor of seven, both the unconverted and converted form of the artemisinins kill parasites and the extended PK/PD methodology produced results consistent with field observations. The simulations predict that a potentially rapid decline in ACT effectiveness is likely to occur as artemisinin resistance spreads, emphasising the importance of containing the spread of artemisinin resistance before it results in widespread drug failure. We found that PK/PD data is generally very poorly reported in the malaria literature, severely reducing its value for subsequent re-application, and we make specific recommendations to improve this situation.     

Plasmodium falciparum malaria: rosettes are disrupted by quinine, artemisinin, mefloquine, primaquine, pyrimethamine, chloroquine and proguanil.

An assay was developed measuring the disruption of rosettes between Plasmodium falciparuminfected (trophozoites) and uninfected erythrocytes by the antimalarial drugs quinine, artemisinin mefloquine, primaquine, pyrimethamine, chloroquine and proguanil. At 4 hr incubation rosettes were disrupted by all the drugs in a dose dependent manner. Artemisinin and quinine were the most effective anti-malarials at disrupting rosettes at their therapeutic concentrations with South African RSA 14, 15, 17 and The Gambian FCR-3 P. falciparum strains. The least effective drugs were proguanil and chloroquine. A combination of artemisinin and mefloquine was more effective than each drug alone. The combinations of pyrimethamine or primaquine, with quinine disrupted more rosettes than quinine alone. Quinine may be an effective drug in the treatment of severe malaria because the drug efficiently reduces the number of rosettes.     

无载体固定化黄花蒿细胞生产青蒿素

疟疾是一种严重危害人类健康的流行病,主要由疟原虫经蚊虫叮咬引起。目前,在临床上疟原虫对治疗疟疾的药物(如氯奎等)有较强的耐药性,并表现出明显的交叉耐药性。来自黄花蒿的青蒿素具有极其明显的抗疟活性,成为临床首选的药物,因此青蒿素的获取成为关键。本研究采用无载体固定化法培养黄花蒿生产青蒿素,初步研究了无载体固定化细胞的生长特性。检测发现,利用该方法生产的青蒿素是常规细胞培养法的9倍,因此该方法有望成为青蒿素生产的首选方法。     

Re-evaluation of how artemisinins work in light of emerging evidence of in vitro resistance

There are more than half a billion cases of malaria every year. Combinations of an artemisinin with other antimalarial drugs are now recommended treatments for Plasmodium falciparum malaria in most endemic areas. These treatment regimens act rapidly to relieve symptoms and effect cure. There is considerable controversy on how artemisinins work and over emerging indications of resistance to this class of antimalarial drugs. Several individual molecules have been proposed as targets for artemisinins, in addition to the idea that artemisinins might have many targets at the same time. Our suggestion that artemisinins inhibit the parasite-encoded sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) PfATP6 has gained support from recent observations that a polymorphism in the gene encoding PfATP6 is associated with in vitro resistance to artemether in field isolates of P. falciparum.     

Rapid determination of artemisinin and related analogues using high-performance liquid chromatography and an evaporative light scattering detector

Artemisinin and its analogues are a class of compounds of current interest in the treatment of drug-resistant malaria. These antimalarials are preferentially taken up into malaria infected erythrocytes as compared to uninfected erythrocytes, a fact that may represent an important parameter in drug potency. Numerous methods for the analysis of specific artemisinin analogues have been developed, but most are not widely adaptable to a large range of analogues. In this paper we describe a high-performance liquid chromatographic method developed and validated for artemisinin and several analogues of artemisinin using a readily available evaporative light scattering detector. This quantitation method was found to be straight forward, rapid, inexpensive and reproducible. Standard calibration curves constructed for six artemisinin compounds were linear with the detection limit determined between 6 and 60 ng. The intra- and inter-day accuracy were found to be 2.75% and 4.15%, respectively with less than 3% variation in precision. The validated assay was applied to a mixture of artemisinin derivatives, where they were easily separated and quantitated.     

Pilot Study of Essential Drug Quality in Two Major Cities in India

Background

India is an increasingly influential player in the global pharmaceutical market. Key parts of the drug regulatory system are controlled by the states, each of which applies its own standards for enforcement, not always consistent with others. A pilot study was conducted in two major cities in India, Delhi and Chennai, to explore the question/hypothesis/extent of substandard and counterfeit drugs available in the market and to discuss how the Indian state and federal governments could improve drug regulation and more importantly regulatory enforcement to combat these drugs.

Methodology/Principal Findings

Random samples of antimalarial, antibiotic, and antimycobacterial drugs were collected from pharmacies in urban and peri-urban areas of Delhi and Chennai, India. Semi-quantitative thin-layer chromatography and disintegration testing were used to measure the concentration of active ingredients against internationally acceptable standards. 12% of all samples tested from Delhi failed either one or both tests, and were substandard. 5% of all samples tested from Chennai failed either one or both tests, and were substandard. Spatial heterogeneity between pharmacies was observed, with some having more or less substandard drugs (30% and 0% respectively), as was product heterogeneity, with some drugs being more or less frequently substandard (12% and 7% respectively).

Conclusions/Significance

In a study using basic field-deployable techniques of lesser sensitivity rather than the most advanced laboratory-based techniques, the prevalence of substandard drugs in Delhi and Chennai is confirmed to be roughly in accordance with the Indian government''s current estimates. However, important spatial and product heterogeneity exists, which suggests that India''s substandard drug problem is not ubiquitous, but driven by a subset of manufacturers and pharmacies which thrive in an inadequately regulated environment. It is likely that the drug regulatory system in India needs to be improved for domestic consumption, and because India is an increasingly important exporter of drugs for both developed and developing countries. Some poor countries with high burdens of disease have weak drug regulatory systems and import many HIV/AIDS, tuberculosis and malaria drugs from India.     

Novel polymorphisms in Plasmodium falciparum ABC transporter genes are associated with major ACT antimalarial drug resistance

Chemotherapy is a critical component of malaria control. However, the most deadly malaria pathogen, Plasmodium falciparum, has repeatedly mounted resistance against a series of antimalarial drugs used in the last decades. Southeast Asia is an epicenter of emerging antimalarial drug resistance, including recent resistance to the artemisinins, the core component of all recommended antimalarial combination therapies. Alterations in the parasitic membrane proteins Pgh-1, PfCRT and PfMRP1 are believed to be major contributors to resistance through decreasing intracellular drug accumulation. The pfcrt, pfmdr1 and pfmrp1 genes were sequenced from a set of P.falciparum field isolates from the Thai-Myanmar border. In vitro drug susceptibility to artemisinin, dihydroartemisinin, mefloquine and lumefantrine were assessed. Positive correlations were seen between the in vitro susceptibility responses to artemisinin and dihydroartemisinin and the responses to the arylamino-alcohol quinolines lumefantrine and mefloquine. The previously unstudied pfmdr1 F1226Y and pfmrp1 F1390I SNPs were associated significantly with artemisinin, mefloquine and lumefantrine in vitro susceptibility. A variation in pfmdr1 gene copy number was also associated with parasite drug susceptibility of artemisinin, mefloquine and lumefantrine. Our work unveils new candidate markers of P. falciparum multidrug resistance in vitro, while contributing to the understanding of subjacent genetic complexity, essential for future evidence-based drug policy decisions.     

Bangladesh in the era of malaria elimination

Bangladesh has dramatically reduced malaria by 93% from 2008 to 2020. The strategy has been district-wise, phased elimination; however, the last districts targeted for elimination include remote, forested regions which present several challenges for prevention, detection, and treatment of malaria. These districts border Myanmar which harbors Plasmodium falciparum malaria parasites resistant to artemisinins, key drugs used in artemisinin-based combination therapies (ACTs) that have been vital for control programs. Challenges in monitoring emergence of artemisinin resistance (AR), tracking parasite reservoirs, changes in vector behavior and responses to insecticides, as well as other environmental and host factors (including the migration of Forcibly Displaced Myanmar Nationals; FDMNs) may pose added hazards in the final phase of eliminating malaria in Bangladesh.     

The Plasmodium falciparum Ca(2+)-ATPase PfATP6: insensitive to artemisinin, but a potential drug target

The disease malaria, caused by the parasite Plasmodium falciparum, remains one of the most important causes of morbidity and mortality in sub-Saharan Africa. In the absence of an efficient vaccine, the medical treatment of malaria is dependent on the use of drugs. Since artemisinin is a powerful anti-malarial drug which has been proposed to target a particular Ca2+-ATPase (PfATP6) in the parasite, it has been important to characterize the molecular properties of this enzyme. PfATP6 is a 139?kDa protein composed of 1228 amino acids with a 39% overall identity with rabbit SERCA1a (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1a). PfATP6 conserves all sequences and motifs that are important for the function and/or structure of a SERCA, such as two high-affinity Ca2+-binding sites, a nucleotide-binding site and a phosphorylation site. We have been successful in isolating PfATP6 after heterologous expression in yeast and affinity chromatography in a pure, active and stable detergent-solubilized form. With this preparation, we have characterized and compared with the eukaryotic SERCA1a isoform the substrate (Ca2+ and ATP) -dependency for PfATP6 activity as well as the specific inhibition/interaction of the protein with drugs. Our data fully confirm that PfATP6 is a SERCA, but with a distinct pharmacological profile: compared with SERCA1a, it has a lower affinity for thapsigargin and much higher affinity for cyclopiazonic acid. On the other hand, we were not able to demonstrate any inhibition by artemisinin and were also not able to monitor any binding of the drug to the isolated enzyme. Thus it is unlikely that PfATP6 plays an important role as a target for artemisinin in the parasite P. falciparum.     

Beyond malaria: The inhibition of viruses by artemisinin-type compounds

Natural products represent valuable chemical scaffolds for drug development. A recent success story in this context was artemisinin, which is not only active against malaria but also to other diseases. This raised the interest of artemisinin's potential for drug repurposing. On the present review, we give an overview on artemisinin's antiviral activity. There is good in vitro and in vivo evidence for the activity of artemisinin and its derivatives against DNA viruses of the Herpesviridae and Hepadnaviridae families such as cytomegaloviruses, human herpesvirus 6, herpes simplex viruses 1 and 2, Epstein-Barr virus and Hepatitis B virus. The evidence is weaker for Polyomaviruses and papilloma viruses. Weaker or no inhibitory activity in vitro has been reported for RNA viruses such as human immunodeficiency viruses 1 and 2, hepatitis C virus, influenza virus and others. Interestingly, the artemisinin derivative artesunate did not exert cross-resistance to ganciclovir-resistant HCMV and exerted synergistic inhibition in combination with several clinically established antiviral standard drugs. The antiviral activity of first generation artemisinin derivatives (e.g. artesunate, artemether, etc.) was enhanced by novel derivatives, including dimer and trimer molecules. First results on patients indicating activity in a subset of HCMV patients. Novel developments in the field of nanotechnology and synthetic biology to bioengineer microorganisms for artemisinin production may pave the way for novel drugs to fight viral infections with artemisinin-based drugs.     

青蒿素的发现与研究进展

1967年北京《5.23抗疟计划》付诸实施,在全国多个研究单位协作下,组织植物化学与药理学等专业200多人,搜寻对抗耐氯喹恶性疟疾的新药.他们追索我国历代抗疟方剂,用约200种草药制成380多种抽提物,再筛查其对小鼠疟疾模型的疗效.最后确定,在60℃用乙醚萃取中药青蒿的黄花蒿所得第191号中性抽提物,对感染鼠疟和猴疟的小鼠与猴以及21例恶性疟、间日疟患者均能奏效.1972年11月,从该抽提物分离出相对分子质量0.282 kD的无色结晶,命名为青蒿素.它属于一种新型的倍半萜内酯.继后青蒿素纯品的胶囊被用于临床数千例疟疾患者.2006年以来,为克服耐药性,世界卫生组织(WHO)宣布改用青蒿素的联合疗法(ACT),挽救了80个国家100多万人的生命.因在发现青蒿素过程中的关键作用,屠呦呦被授予2011年度拉斯克临床医学研究奖.本文对青蒿素发现的争议,如何恰当评价其他科学家的贡献,以及开发植物界以外青蒿素新药源的近况,均作了简要报道.     

Interaction between artemisinin and heme. A Density Functional Theory study of structures and interaction energies

Malaria is an infectious disease caused by the unicellular parasite Plasmodium sp. Currently, the malaria parasite is becoming resistant to the traditional pharmacological alternatives, which are ineffective. Artemisinin is the most recent advance in the chemotherapy of malaria. Since it has been proven that artemisinin may act on intracellular heme, we have undertaken a systematic study of several interactions and arrangements between artemisinin and heme. Density Functional Theory calculations were employed to calculate interaction energies, electronic states, and geometrical arrangements for the complex between the heme group and artemisinin. The results show that the interaction between the heme group and artemisinin at long distances occurs through a complex where the iron atom of the heme group retains its electronic features, leading to a quintet state as the most stable one. However, for interaction at short distances, due to artemisinin reduction by the heme group, the most stable complex has a septet spin state. These results suggest that a thermodynamically favorable interaction between artemisinin and heme may happen.     

In vitro schizontocidal activity of standard antimalarial drugs on chloroquine-sensitive and chloroquine-resistant isolates of Plasmodium falciparum

The expanding foci of multiple drug resistant malaria and emergence of different strains requires the reassessment of antimalarial activity with various drugs. In vitro response of a chloroquine sensitive and a chloroquine resistant isolate of P. falciparum to a group of 6 quinine derived and 3 artemisinin derived standard drugs has been screened, to evaluate schizontocidal activity of the drugs. In a conventional test system the IC50s were derived from the log dose response curves and evaluated by a rigorous statistical interpretation. Analysis by Tukey's test was significant for the quinine related drugs (Q < or = 0.01) and excludes the statistical significance of artemisinin related drugs in these isolates. The dose-responses of these two isolates vary with quinine derivatives, with some overlap at lower doses for the sensitive isolate than for the resistant one which manifests at higher doses.