Programma di ricerca del Ministero della Salute "Trasferimento di interventi di documentata efficacia nella pratica assistenziale" (TRIPSS III) Progetto di ricerca sanitaria finalizzata della Regione Piemonte Direzione Sanità Pubblica "Costruzione e applicazione di un percorso integrato ospedale e territorio nella gestione dello scompenso cardiaco LINEE GUIDA AZIENDALI
Most often, when a man first has problems with lowered potency or with sexual dysfunction viagra australia shipping to pharmacies, paying purveyors, training personnel, or having a huge advertising budget.
Antiplasmodial activity, in vivo pharmacokinetics and anti-malarial efficacy evaluation of hydroxypyridinone hybrids in a mouse model
Dambuza et al. Malar J (2015) 14:505 Open Access
Antiplasmodial activity, in vivo pharmacokinetics and anti-malarial efficacy evaluation of hydroxypyridinone hybrids in a mouse modelNtokozo S. Dambuza1*, Peter Smith1, Alicia Evans1, Jennifer Norman1, Dale Taylor1, Andrew Andayi2, Timothy Egan2, Kelly Chibale2 and Lubbe Wiesner1 Abstract
Background: During the erythrocytic stage in humans, malaria parasites digest haemoglobin of the host cell, and
the toxic haem moiety crystallizes into haemozoin. Chloroquine acts by forming toxic complexes with haem mol-
ecules and interfering with their crystallization. In chloroquine-resistant strains, the drug is excluded from the site of
action, which causes the parasites to accumulate less chloroquine in their acid food vacuoles than chloroquine-sen-
sitive parasites. 3-Hydroxylpyridin-4-ones are known to chelate iron; hydroxypyridone-chloroquine (HPO-CQ) hybrids
were synthesized in order to determine whether they can inhibit parasites proliferation in the parasitic digestive
vacuole by withholding iron from plasmodial parasite metabolic pathway.
Methods: Two HPO-CQ hybrids were tested against Plasmodium falciparum chloroquine-sensitive (D10 and 3D7) and
-resistant strains (Dd2 and K1). The pharmacokinetic properties of active compounds were determined using a mouse
model and blood samples were collected at different time intervals and analysed using LC–MS/MS. For in vivo efficacy
the mice were infected with Plasmodium berghei in a 4-day Peters' test. The parasitaemia was determined from day 3
and the course of the infection was followed by microscopic examination of stained blood films every 2–3 days until a
rise in parasitaemia was observed in all test subjects.
Results: IC50 values of the two compounds for sensitive and resistant strains were 0.064 and 0.047 µM (compound
1), 0.041 and 0.122 µM (compound 2) and 0.505 and 0.463 µM (compound 1), 0.089 and 0.076 µM (compound 2),
respectively. Pharmacokinetic evaluation of these compounds showed low oral bioavailability and this affected in vivo
efficacy when compounds were dosed orally. However, when dosed intravenously compound 1 showed a clearance
rate of 28 ml/min/kg, an apparent volume of distribution of 20 l/kg and a half-life of 4.3 h. A reduction in parasitaemia
was observed when compound 1 was dosed intravenously for four consecutive days in P. berghei-infected mice. How-
ever, a rise in parasitaemia levels was observed on day 6 and on day 9 for chloroquine-treated mice.
Conclusion: The hybrid compounds that were tested were able to reduce parasitaemia levels in P. berghei-infected
mice when dosed intravenously, but parasites recrudesced 24 h after the administration of the least dose. Despite low
oral bioavailability, the IV data obtained suggests that further structural modifications may lead to the identification of
more HPO-CQ hybrids with improved pharmacokinetic properties and in vivo efficacy.
Keywords: Malaria, Pharmacokinetics, Hydroxypyridone-chloroquine, In vivo efficacy
*Correspondence: [email protected] 1 Division of Clinical Pharmacology, Department of Medicine, University of Cape Town, Observatory, Cape Town 7925, South AfricaFull list of author information is available at the end of the article 2015 Dambuza et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (mits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (o the data made available in this article, unless otherwise stated.
Dambuza et al. Malar J (2015) 14:505 obtained from the malaria reagent depository, Malaria Malaria is a disease caused by an eukaryote parasite of Research and Reference Reagent Resource Center (MR4) the genus Plasmodium and remains a major global public (ATCC, Manassas, VA, USA). The parasites were con- health problem, that was responsible for 584,000 deaths tinuously cultured in vitro according to the method in 2013, with most occurring in Africa and most deaths described by Trager and Jensen but with modifications in children under 5 years of age ]. Plasmodium fal- [ultures were maintained at 37 °C in O-positive ciparum and Plasmodium vivax, are responsible for (O+) human erythrocytes in complete culture medium, most cases of malaria and the control of these parasites which contained RPMI 1640 (Gibco-BRL Laboratories) by chloroquine and other known anti-malarial drugs has growth medium supplemented with 25 mM 4-(2-hydrox- been compromised by the emergence and spread of drug yethyl)piperazine-1-ethanesulfonic acid (HEPES) (Sigma- resistance in many parts of the world, primarily in P. fal- Aldrich Chemical Company) buffer, 22 mM glucose ciparum strains [, ]. This has severely limited the use (Sigma), 5 g/l albumax (Gibco-BRL Laboratories), 25 mM of many effective anti-malarials, and has become a major sodium carbonate (Sigma) and 0.3 mM hypoxanthine threat to malaria elimination efforts, causing increased (Sigma). In order to control microbial contamination morbidity and mortality and a financial burden due to 50 µg/l gentamicin (Sigma) was added to the culture sustenance of replacement thera medium, and to obtain a loosely synchronous ring stage, When the parasite infects the erythrocyte, it uses the 5 % sterile aqueous d-sorbitol (Sigma) was used. The endolysosomal system to digest the haemoglobin in an antiplasmodial assay was initiated with the parasites in acidic food vacuole, producing an oxidized form of haem, the trophozoite stage and with a parasitaemia and haem- ferriprotoporphyrin IX (FP-IX), which is the iron con- atocrit of 2 %.
taining non-protein component of haemoglobin, as a by- The test compounds were tested at a concentration producree FP-IX is toxic and can lyse the cell range of 0.15–150 mM and chloroquine (Sigma) was and affect the function of lysozomal enzymes. The para- tested at a concentration range of 0.003–3 and 0.0003– site disposes the toxic FP-IX by a polymerization process 0.3 mM for resistant strain and sensitive strains, respec- that crystallizes at least 95 % of FP-IX as haemozoin, and tively. Non-parasitized erythrocytes were used as a this allows an uninterrupted growth and proliferation negative control and parasitized erythrocytes, without of the parasite [on chelators have been studied any test compound, were used as positive control. A full as alternative malaria drugs for many years because of day dose–response was performed for all compounds to their ability to interact with available iron in the nucleus determine the concentration inhibiting 50 % of parasite and parasite cytosol, thereby interfering with the iron- growth (IC50 value). The plates were incubated for 48 h dependent metabolism of malaria parasites and inhibit- at 37 °C in a gassing chamber containing a mixture of 5 % ing their developmen CO2, 3 % O2 and 92 % N3. In order to quantify parasite Hydroxypyridones are iron-chelating agents known viability and to determine the effect of the compounds to suppress malaria growth in vivo and in vitro [, on the parasite, a parasite lactate dehydrogenase (pLDH) oth hydroxypyridones and chloroquine target assay was used as described by Makler et ], but the erythrocytic stage of the malaria life cycle, which is with modifications.
highly dependent on iron. For the purpose of this study, N-alkyl-3-hydroxypyridin-4-ones were combined with Pharmacokinetic studies
chloroquine in an attempt to enhance antiplasmodial A comprehensive pharmacokinetic (PK) study of com- effect against chloroquine-resistant strains of P. falcipa- pound 1 and 2 was performed on 10-week old male and rum when compared to inhibition by chloroquine alone female C57BL/6 mice (20–30 g) obtained from the Uni- [pound 1 and 2 were selected among a series of versity of Cape Town Medical School Animal Unit. The hydroxypyridone-chloroquine (HPO-CQ) hybrid com- mice were housed in ventilated cages at room tempera- pounds in order to assess their antiplasmodial activity ture (approximately 22 °C) with constant supply of food in vitro and efficacy in vivo and to evaluate their pharma- and water and were monitored twice daily. The study was authorized by the Faculty of Health Science Animal Research Ethics Committee before commencement: Ref- erence No. 012/020. All the work was performed accord- In vitro antiplasmodial activity
ing to the guidelines established by Austin e].
The compounds were prepared in oil-in-water (o/w) The human parasite P. falciparum strains used in this microemulsions, which consist of 5 % ethyl linoleate study were chloroquine-sensitive (CQS) (3D7 and D10) (Sigma), 11 % Tween 80 (Sigma), 4 % ethanol (Merck), and chloroquine-resistant (CQR) (Dd2 and K1) and were and 80 % water. In each experiment a group of five Dambuza et al. Malar J (2015) 14:505 animals were dosed orally (10 ml/kg) at 20 mg/kg and Table 1 Mass spectrometer settings and MS parameters
intravenously (5 ml/kg) via the dorsal penile vein at 4 mg/ used for the detection of the test compounds on an API
kg under anaesthesia. For oral dosage, a gavage nee- 3200 Q-Trap
dle was used for the administration of test compounds Parameter
directly into the lower oesophagus or stomach. Blood samples (approximately 30 µl) were collected serially by Q1 mass (Da) needle prick on the tail vein, near to the tip of the tail, Q3 mass (Da) at 0, 0.17, 0.5, 1, 2, 3, 5, 7, and 9 h post-dosing. Lithium Dwell time (ms) heparin-coated MiniCollect® Plasma Tubes (Lasec, Declustering potential (V) South Africa) were used to collect the blood samples. The Collision energy (V) collected blood samples were placed on ice immediately Entrance potential (V) after sampling, and were frozen at −80 °C until analysis.
Collision cell exit potential (V) Source temperature (°C) Curtain gas (psi) The blood samples stored at −80 °C were thawed at room Gas 1 (psi) temperature and then mixed by vortex to ensure homo- geneity. Twenty µl of blood was mixed with 50 µl Milli-Q CAD gas water (Millipore, USA) and 150 µl acetonitrile (Merck). Ion spray voltage (kV) The mixture was vortexed for 15 s, sonicated for 10 min Ionization mode and centrifuged at 13,000g for 5 min. The supernatant was transferred to a flat-bottomed glass insert and placed in a glass vial and placed in the autosampler for analysis.
exposure [AUC0–α (µM min)], volume of distribution (l/ kg) and plasma clearance [CL (l/min/kg)].
Liquid chromatography–mass spectrometry summary
A liquid chromatography–mass spectrometry (LC/MS/
In vivo anti‑malarial efficacy of compound 1 against a
MS) system was employed for the quantification of the chloroquine‑sensitive Plasmodium berghei strain
compounds in mouse blood. The LC system employed The chloroquine-sensitive Plasmodium berghei (ANKA was an ultra-fast liquid chromatography (UFLC) system strain) was used to assess in vivo anti-malarial efficacy of (Shimadzu) and the separation of the compounds was the test compounds. The parasites were maintained in a performed on a Phenomenex, Luna 5 μm PFP (2), 100 Å, C57BL/6 mouse by inoculation with 250 µl of a 1:1 (v/v) 50 mm × 2 mm analytical column. The mobile phase A suspension of erythrocytes infected with P. berghei in consisted of 0.1 % formic acid in water (v/v) and mobile phosphate buffered saline (PBS). On the day of the exper- phase B consisted of acetonitrile. The flow rate was set at iment the host mouse was anaesthetized intraperitoneally 500 µl/min and the temperature of the column was main- with a mixture of ketamine (120 mg/kg) and xylazine tained at 40 °C. For the separation of the compounds, the (16 mg/kg). Whole blood from the host mouse was mobile phase was increased from 5 to 95 % B over 4 min, drawn by cardiac puncture into a Vacuette® heparin tube after that, phase B was returned to 5 % within 0.1 min, and a suspension of P. berghei parasitized erythrocytes then equilibrated for 3 min.
(1 × 107) in PBS was prepared and the test mice were The detection of the compounds was performed on infected with 200 µl of this suspension intraperitoneally.
an AB Sciex 3200 Q-Trap mass spectrometer which Evaluation of the curative potential of the test com- was operated at unit resolution in the multiple reaction pounds was performed using Peters' 4-day test as monitoring (MRM) mode. The calibration range for all describe]. The mice were dosed orally at 20 and the compounds was between 7.8 and 1000 ng/ml and the 40 mg/kg and intravenously at 4 and 8 mg/kg 2 h post- accuracy (% Nom) for the calibration curves was between infection and for three consecutive days (D0 to D3). On 90.3 and 104.0 %. Table gives an overview of the MS the first day (D0), blood samples were collected serially parameters and the instrument settings.
from each mouse at 0.5, 1, 3, and 7 h post-dosing in order to provide quantitative measurements of drug exposure which is needed for the sound interpretation of the effi- Non-compartmental analysis was performed on each cacy of the anti-malarialhloroquine was used as individual set of data using PK Solutions 2.0 Pharma- a reference drug and was dosed orally at 10 mg/kg. The cokinetic Analysis Software (Summit Research Services, mice were also dosed orally with PBS as a control. The Montrose, USA). The following PK parameters were parasitaemia was determined from the third day (D4) by calculated: apparent terminal half-life [t½ (min)], total preparing thin blood films from the tail of each mouse Dambuza et al. Malar J (2015) 14:505 and the smears were fixed with methanol and stained The IC50 values calculated from the dose response with Giemsa. This was repeated every 2–3 days to moni- curves were in the range of 0.041–0.064 µM against 3D7 tor the efficacy of the test compounds. The formula used and 0.047–0.122 µM against D10. These values were to calculate % parasitaemia and % reduction is described higher than that of chloroquine, with IC50 values of 0.019 below, respectively. The data for % parasitaemia and % and 0.023 µM against 3D7 and D10, respectively. The reduction of parasitaemia is presented in Table .
IC50 values of the compounds against the resistant strain K1 and Dd2 were compared to those of chloroquine (IC values of 0.279 and 0.180 µM for K1 and Dd2, respec- No. of parasitized RBC out of 500 erythrocytes tively). Compound 2 was more active than chloroquine Total no. of RBC counted with IC50 values of 0.089 and 0.076 µM for K1 and Dd2, respectively. Compound 1 on the other hand was less active than chloroquine with IC50 values of 0.505 and 0.463 µM for K1 and Dd2, respectively. The data pre- sented in Table how that the difference in the activity Parasitamia of placebo − Parasitamia of test compound of the HPO-CQ hybrids when compared to chloroquine Parasitamia of placebo is insignificant at P > 0.05 in all chloroquine-sensitive strains. However, activity of the two compounds against the resistant strains differed significantly. The compounds Antiplasmodial action of the compounds in vitro
had lower RI values than chloroquine. Compound 2 had Compounds 1 and 2 were synthesized in order to an RI value of 2.2, which is over five times less than that enhance the activity of chloroquine by combining it with of chloroquine and the RI value of compound 1 was 10.7 an iron chelator. These compounds were tested for anti- plasmodial activity against sensitive strains 3D7 and D10 and resistant strains K1 and Dd2 and the IC50 values (in Pharmacokinetics
µM) of compounds 1 and 2 are summarized in Table . The compounds were administered to mice orally and The P values were calculated using the non-parametric intravenously at 20 and 4 mg/kg, respectively. The blood Mann–Whitney U test in order to compare the activity levels for both compounds in the mice dosed orally could of chloroquine with that of the HPO-CQ hybrids. The not be detected because the values were below the limit resistance index (RI), the ratio of the IC50 of the resist- of quantitation (LOQ). The concentration versus time ant strain to that of the sensitive strain, was calculated intravenously (IV) profiles of both compounds is pre- for these compounds using the lowest IC50 value of the sented in Fig. . The mean concentration–time data were sensitive and the highest IC50 of the resistant strain for used to calculate the PK parameters by non-compart- each compound, which should give the highest RI value mental analysis using PK Solutions 2.0 Pharmacokinetic for that comp]. This value provides a quantita- Analysis Software. The PK parameters are presented in tive measurement of the antiplasmodial activity against Table .
CQR strains relative to that against CQ Compound 1 showed a clearance rate of 28 ml/min/kg,
higher the RI value, the higher the level of resistance. The a high apparent volume of distribution of 20 l/kg and a RI value was calculated according to this formula [ half-life of 4.3 h. The area under the curve (AUC) value was also at 196 µM min. Even with the half-life of 4.3 h, − resistant strain IC50 the compound remained at detectable levels of 0.16 µM Chloroquine − sensitive strain IC50 Table 2 In vitro IC50 values (µM) of compounds 1 and 2
The values obtained from antiplasmodial tests represent the mean of three independent experiments each performed in triplicateRI value, values calculated using the highest CQR IC50 value and lowest CQS IC50 value; n/a, not applicable Dambuza et al. Malar J (2015) 14:505 Fig. 1 The structures of synthesized hydroxypyridone-chloroquine hybrids (compound 1 and 2)
Fig. 2 Blood concentrations of compounds 1 (a) and 2 (b) in C57BL/6 mice blood after intravenous administration of 4 mg/kg. Data represent
mean ± standard deviation of data points obtained from five single mice
Table 3 Pharmacokinetic parameters of hydroxypyridone-
Compound 2, on the other hand, had a very short half chloroquine hybrids after intravenous administration
life of 0.7 h and after 2 h the blood levels dropped to Compound 1
0.03 µM because of a high clearance rate of 366 ml/min/ kg. The volume of distribution was also high at 154.1 l/kg.
Anti‑malarial effect of compound 1 on Plasmodium
AUC0– (µM min) The PK evaluation shows that compound 1 and 2 lev- Data represent mean ± standard deviation of data points obtained from five els were below the LOQ. They both presented poor PK properties following oral administration. However, the ND indicate that the value was not determined LC–MS/MS assay only detected the parent compound and no assays were conducted to measure or identify any after 9 h at the end of the dosing interval, indicating a possible metabolites. The decision to perform efficacy slow elimination rate from circulation. When consider- studies was based on the possibility that compounds 1 ing these properties and IC and 2 may have high first pass effect which may cause low 50 values of 0.064–0.505 µM against sensitive and resistant strains, respectively, com- bioavailability. After the infected mice were dosed orally pound 1 was selected for in vivo efficacy studies. Even and intravenously with compound 1 and 2, the blood though the compound could not be detected in blood fol- concentrations were measured in P. berghei-infected mice lowing an oral dosage, investigating the efficacy of com- at different time intervals to measure the exposure of pound 1 after an intravenous dosage was considered.
the infected mice to the drug. The blood concentration levels of the mice that were dosed orally were below the Dambuza et al. Malar J (2015) 14:505 after intraperitoneal inoculation . This differ- ence could possibly be due to the variation in age and To evaluate the anti-malarial efficacy of compound 1 in infected mice, the parasitaemia level was calculated at days 4, 6 and 9 post infection, and the % reduction of par- asitaemia was calculated for day 9 only because parasite levels were observed in all treated mice on day 9.
In the mice that were dosed orally, the parasites were visible at a parasitaemia of ±5 % on day 4. These data correlate well with the PK data because the blood levels of compound 1 following oral dosage were too low to Time (hours)
have any effect on reducing parasitaemia, and thus allow- Fig. 3 Blood levels of compound 1 in C57BL/6 mice blood infected
ing the disease to progress as indicated by a rise in the with P. berghei after intravenous administration of 4 and 8 mg/kg of parasitaemia levels, as shown in Tat was also noted compound. Data represent mean ± standard deviation of data points that the parasitaemia in mice that were dosed orally was obtained from five mice higher than the parasitaemia in the placebo group. This may mean that high oral doses may cause compound 1 to be immunosuppressive, thus allowing the infection to be detection level in both 20 and 40 mg/kg groups. Figure severe in the oral group when compared to the placebo shows concentration versus time profiles of compound group. High parasitemia levels in the 40 mg/kg/day oral 1 following IV administration in mice infected with P. group resulted in a negative value of % reduction of para- berghei. For compound 2 the IV data showed a very high sitaemia (Ta clearance rate compared to compound 1 and blood con- As expected, blood levels remained high for a longer centration levels were very low. High toxicity was also period for mice dosed intravenously with 4 and 8 mg/ detected when dosed orally and intravenously. The PK kg of compound 1 as shown in the concentration ver- studies were only conducted for 9 h, and therefore the sus time profiles in Fig. se data are consistent with symptoms of toxicity were not severe. However, with effi- the comprehensive PK study presented in Table , which cacy studies the mice are dosed every day for 4 days, and shows that compound 1 had a half-life of 4.3 h following this might cause distress to the mice because the toxic intravenous dosage and a clearance rate of 28 ml/min/kg. effect of the drug will be more severe. The efficacy study Figure shows that after 7 h following intravenous dos- for compound 2 is not reported in this paper.
age the blood concentration levels were relatively high for When considering compound 1 blood concentration 4 and 8 mg/kg. This resulted in a reduced parasite mul- of healthy animals in Fig. a and P. berghei-infected tiplication rate as indicated by undetected to low parasi- animals in Fig. , it was noted that higher drug lev- taemia until day 4 and 6. However, the data suggest that els were observed in infected mice. It is unlikely that compound 1 suppressed the parasitaemia without clear- the presence of parasites could be the reason for such ing all parasites, therefore, surviving parasites in the mice a significant difference in blood levels because the blood began to multiply after the 4-day course of treat- mouse exposure study was performed 2 h post infec- ment, causing a parasite reduction to decrease to 62 tion and parasitized erythrocytes are rapidly absorbed and 83 % in the mice dosed at 4 and 8 mg/kg on day 9, Table 4 In vivo antiplasmodial efficacy of compounds 1 in C57BL/6 mice blood infected with P. berghei
Average % parasitemia
Oral (20 mg/kg/day) Oral (40 mg/kg/day) Oral CQ (10 mg/kg/day) Data represent mean ± standard data points obtained from five mice Dambuza et al. Malar J (2015) 14:505 respectively, as shown in Table . This also shows that the Competing interests
efficacy of compound 1 is dose dependent.
The authors declare that they have no competing interests.
Received: 15 July 2015 Accepted: 2 December 2015 Conclusion
The aim of synthesizing HPO-CQ hybrids was to develop
anti-malarial candidates that were potent against sensi- tive and resistant P. falciparum strains. This study showed that combining chloroquine with a HPO can cause a References
1. Prakash A, Sharma SK, Mohapatra PK, Bhattacharjee K, Gogoi K, Gogoi change in the physicochemical properties of the hybrid, P, et al. In vitro and in vivo antiplasmodial activity of the root extracts of thus affecting its efficacy and PK properties. Chloroquine Brucea mollis Wall. ex Kurz. Parasitol Res. 2013;112:637–42.
alone has an oral bioavailability of 79–90 % and a half-life 2. WHO. World malaria report 2014. Geneva: World Health of 7 h in mice, as reported by Salakven though compound 2 was more active than chloroquine in vitro 3. Hastings IM. The origins of antimalarial drug resistance. Trends Parasitol. against the resistant strains with a lower RI value, the PK 4. Maltha J, Jacobs J. Clinical practice. Eur J Pediatr. 2011;170:821–9.
properties were not improved relative to chloroquine. 5. Fitch CD. Involvement of heme in the antimalarial action of chloroquine. Compound 1 was less active in vitro against the resist- Trans Am Clin Climatol Assoc. 1998;109:97–105.
ant strains than compound 2 and chloroquine with poor 6. Fitch CD. Ferriprotoporphyrin IX, phospholipids, and the antimalarial actions of quinoline drugs. Life Sci. 2004;74:1957–72.
oral bioavailability, but it proved to be an effective anti- 7. Orjih AU. On the mechanism of hemozoin production in malaria para- malarial compound in vivo when dosed intravenously. sites: activated erythrocyte membranes promote β-hematin synthesis. Nevertheless, chloroquine remained more effective Exp Biol Med. 2001;226:746–52.
8. Combrinck JM, Mabotha TE, Ncokazi KK, Ambele MA, Taylor D, Smith P, in vivo than both compounds and this means that further et al. Insights into the role of heme in the mechanism of action of anti- modifications on the chemical structure could improve malarials. ACS Chem Biol. 2012;8:133–7.
PK properties, such as oral bioavailability, thus improving 9. Egan TJ, Ross DC, Adams PA. Quinoline anti-malarial drugs inhibit spontaneous formation of β-haematin (malaria pigment). FEBS Lett. 10. Loria P, Miller S, Foley M, Tilley L. Inhibition of the peroxidative degrada- tion of haem as the basis of action of chloroquine and other quinoline antimalarials. Biochem J. 1999;339:363–70.
AUC: area under the curve; CL: clearance; CQ: chloroquine; CQR: chloroquine 11. Ferrer P, Tripathi AK, Clark MA, Hand CC, Rienhoff HY, Sullivan DJ. Antima- resistant; CQS: chloroquine sensitive; Da: Daltons; FP-IX: ferriprotoporphy- larial iron chelator, FBS0701, shows asexual and gametocyte Plasmodium rin IX; HEPES: 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid; HPO: falciparum activity and single oral dose cure in a murine malaria model. hydroxypyridones; IV: intravenously; LC–MS/MS: liquid chromatography–mass PLoS One. 2012;7:e37171.
spectrometry; LOQ: limit of quantitation; MRM: multiple reaction monitoring; 12. Lytton SD, Mester B, Libman J, Shanzer A, Cabantchik Z. Mode of PBS: phosphate buffered saline; PK: pharmacokinetics; pLDH: lactate dehydro- action of iron (III) chelators as antimalarials: II. Evidence for differen- genase; RBC: red blood cells; RI: resistance index; T tial effects on parasite iron-dependent nucleic acid synthesis. Blood. 1/2: half-life; UFLC: ultra-fast liquid chromatography; Vss: volume of distribution.
13. Andayi WA, Egan TJ, Gut J, Rosenthal PJ, Chibale K. Synthesis, antiplasmo- dial activity and β-hematin inhibition of hydroxypyridone-chloroquine ND participated in research design, conducted in vitro and in vivo experi- hybrids. ACS Med Chem Lett. 2013;4:642–6.
ments, interpreted the data and wrote the manuscript. PS, KC and LW partici- 14. Hershko C, Theanacho E, Spira D, Peter H, Dobbin P, Hider R. The effect pated in research design and contributed to the writing of the manuscript. AE of N-alkyl modification on the antimalarial activity of 3-hydroxypyridin- developed and validated the LC–MS/MS assay for the quantitative determina- 4-one oral iron chelators. Blood. 1991;77:637–43.
tion of compounds 1 and 2 in mouse blood. DT performed in vivo efficacy 15. Trager W, Jensen JB. Human malaria parasites in continuous culture. Sci- experiments. JN assisted with the evaluation of the PK-properties using PK- summit software. AA and TE synthesized the compounds. All authors read and 16. Makler MT, Ries JM, Williams JA, Bancroft JE, Piper RC, Gibbins BL, et al. approved the final manuscript.
Parasite lactate dehydrogenase as an assay for Plasmodium falciparum drug sensitivity. Am J Trop Med Hyg. 1993;48:739–41.
17. Makler MT, Piper RC, Milhous WK. Lactate dehydrogenase and the diag- 1 Division of Clinical Pharmacology, Department of Medicine, University nosis of malaria. Parasitol Today. 1998;14:376–7.
of Cape Town, Observatory, Cape Town 7925, South Africa. 2 Department 18. Austin JC, du Toit D, Fraser N, Lloyd P, Mansfield D, Macleod A, et al. 2004. of Chemistry, University of Cape Town, Rondebosch, Cape Town 7700, South Guidelines on ethics for medical research: use of animals in research and training. South African Medical Research Council.
19. Peters W. The chemotherapy of rodent malaria, XXII. The value of drug- resistant strains of P. berghei in screening for blood schizontocidal activity. The authors acknowledge Mr. Trevor Finch for assisting with animal handling. Ann Trop Med Parasitol. 1975;69:155–71.
The University of Cape Town, Clinical Infectious Diseases Research Initiative 20. Jun Tang C, Redmond W. Comparison of serial and parallel blood sam- and National Research Foundation, South African Medical Research Council, pling techniques in mouse pharmacokinetics study. 2nd Asian Pacific and South African Research Chairs Initiative of the Department of Science ISSX Meeting; 2008.
and Technology, administered through the South African National Research 21. Nzila A, Mwai L. In vitro selection of Plasmodium falciparum drug-resistant Foundation are gratefully acknowledged for support.
parasite lines. J Antimicrob Chemother. 2010;65:390–8.
Dambuza et al. Malar J (2015) 14:505 22. Ekoue-Kovi K, Yearick K, Iwaniuk DP, Natarajan JK, Alumasa J, de Dios A, 24. Patel K, Batty KT, Moore BR, Gibbons PL, Bulitta JB, Kirkpatrick CM. et al. Synthesis and antimalarial activity of new 4-amino-7-chloroquinolyl Mechanism-based model of parasite growth and dihydroartemisinin amides, sulfonamides, ureas and thioureas. Bioorg Med Chem Lett. pharmacodynamics in murine malaria. Antimicrob Agents Chemother. 23. Kaushik NK, Sharma J, Sahal D. Anti-plasmodial action of de novo- 25. Salako LA. Pharmacokinetics of antimalarial drugs; their therapeutic and designed, cationic, lysine-branched, amphipathic, helical peptides. Malar toxicological implications. Ann Ist Super Sanità. 1985;21:15–25.
Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries
• Our selector tool helps you to find the most relevant journal
• We provide round the clock customer support
• Convenient online submission
• Thorough peer review
• Inclusion in PubMed and all major indexing services
• Maximum visibility for your research
Submit your manuscript at
2001 · 2:142–149 © Springer-Verlag 2001 J. Rovira1 · R.Tremosa1 · A. Gilabert2 · M.Torralba21 Grup de Recerca en Economia de la Politica Social,Universitat de Barcelona,Spain2 Servei Català de la Salut,Unitat de Planificació Farmacèutica,Generalitat de Catalunya,Spain The role of prices in drug expenditure analysis