1 Pathogen Molecular Biology and Biochemistry Unit, Department of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel St., London WC1E 7HT, UK; 2 Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143-00446, USA
Received 6 September 2002; returned 25 January 2003; revised 6 February 2003; accepted 6 May 2003
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Abstract |
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Keywords: hydroxychloroquine, chloroquine, amodiaquine, drug-resistance, enantiospecificity
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Introduction |
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Both CQ and HCQ are chiral (Figure 1), and their metabolism and excretion in man are chirally specific.68 A chiral drug structure contains at least one asymmetric carbon atom, and in synthetic drugs, two mirror images of the drug [(+) and () enantiomeric forms] are present in the racemic (±) pharmaceutical preparation. If the enantiomers have different intrinsic activities, differential metabolism and excretion can complicate predictions of in vivo efficacy. Whereas there is no difference in the intrinsic antiplasmodial activities of enantiomers and racemic CQ against CQ-sensitive and resistant strains of P. falciparum in vitro,9 information is lacking for HCQ, where a method of separation of the enantiomers has recently been developed.10
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This study therefore reports the antiplasmodial activity of the enantiomers and the racemate of HCQ against CQ-sensitive and -resistant P. falciparum, and the physicochemical parameters involved in these processes (ionization constants and partition coefficients at the appropriate pH values) for HCQ in comparison with CQ and desethyl chloroquine (DCQ) its major metabolite and a contributor to its action in vivo, and with AQ and DAQ.
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Materials and methods |
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Drugs were tested in triplicate for ability to inhibit the proliferation of a chloroquine-resistant (K1) isolate,23 and a chloroquine-sensitive (T9-96)24 cloned isolate of P. falciparum, from Kanchanaburi and from Mae Sod, Thailand, respectively, cultured in vitro, using a [3H]hypoxanthine incorporation procedure.25 Briefly, the parasites were grown in A+ human erythrocytes, with 10 mL AB serum (National Blood Service, UK) added per 100 mL RPMI medium. Before use for drug assay, parasites were synchronized twice using sorbitol lysis.26 An initial 1% parasitaemia at 5% haematocrit, with >95% of parasites in the growing young trophozoite stage was prepared. After preparation of stock drug solutions in serum-free medium, serial dilutions were prepared in 96-well flat-bottomed microtitre plates and then an equal volume of the infected blood suspended in medium was added to give a final 2.5% haematocrit at 1% parasitaemia. After incubation in a sealed vessel with 3% O2, 4% CO2, 93% N2 at 37°C for 24 h, [3H]hypoxanthine, of specific activity 611 GBq (16.5 Ci)/mmol (Amersham, UK) was added as 10 µL of a 20 µCi/mL solution to each well, re-gassed and incubated for a further 1824 h.
Red cells were harvested from each well (Filtermate 196, Packard: Perkin Elmer Life Sciences, Boston, USA) on to Unifilter 96-well plates (Packard) followed by water washing for lysis, removal of unincorporated isotope and of other soluble components. After drying at 56°C, the undersides of the plates were sealed and scintillation fluid added to each well before sealing the top of the plate. Tritium disintegrations per minute (dpm) were determined in a Packard Topcount scintillation spectrometer, and the data were collected on a floppy disc. Percentage inhibitions of incorporation were calculated using the following equation.
% inhibition = 100 [((dpm test mean dpm uninfected cells)/(mean dpm infected cells mean dpm uninfected cells)) x 100]
These values were used to plot doseresponse curves from which the IC50 values were derived (EXCEL add-in XL-fit program, ID Business solutions, Guildford, UK). Mean, standard error and 95% confidence intervals were calculated.
Physicochemical data
The accumulation of an ionizable drug in cells is determined (apart from special transport mechanisms) by two major parameters: the partition coefficient P, usually expressed as log P (measured in the octanolwater system for the un-ionized compound), and its modification by the ionization constant (pKa) of the drug. For partially ionized compounds, the partition coefficient between two phases at any fixed pH is called the distribution coefficient D, usually expressed as log D. In carrying out these calculations, the assumption is routinely made that only the un-ionized species partitions from the aqueous to the organic phase27 (this has been confirmed experimentally for chloroquine and the red cell membrane28). The difference between log P and log D is therefore determined by the percentage of the drug ionized at different pH values and is given29,30 by Equation 1 for a base.
log D = log P log [1 + antilog (pKa pH)] (Equation 1)
or, log D = log P log [1 + 10(pKapH)]
When the difference between pKa and pH is more than 1.0 log unit, subtraction of this difference from log P will furnish log D.31 This makes it possible to calculate32 log D from a knowledge of log P, pKa and pH. The ClogP program33 provides a useful method of computing approximate log P values for compounds of known structure, but does not take into account some differences due to conformational isomerism.34 Therefore, where possible, we have experimentally measured these parameters for the drugs examined in this study.
In the case of a drug possessing two basic centres, as these 4-aminoquinoline drugs all do, the correction needed in log P to obtain log D at a given pH involves the additive contribution of both ionized species, and Equation 1 must be modified to Equation 2 (It should be noted that for diacidic bases, pKa1 > pKa2 in Equation 2).35
log D = log P log [1 + 10(pKa1 pH) + 10(pKa1 + pKa2 2pH)] (Equation 2)
Log D at the physiological pH of 7.4 was obtained experimentally from the plot of log D versus pH. This was compared with the calculated value of log D, obtained from Equation 2. Log D at the probable pH of the digestive vacuole or lysosome was calculated from log P by substituting pH 5.2 for 7.4 in Equation 2.
Dissociation constants and partition coefficients were determined by Robertson Microlit Laboratories (Madison, NJ, USA) at 25°C using the Sirius GLpK automated computerized potentiometric system.36
Titration employed water containing 0.15 M KCl in an argon atmosphere. The pKa values were determined in triplicate with a S.E. of ±0.20. For one compound (AQ) the base precipitated above pH 8. The titration was carried out with methanol as a co-solvent, using three different ratios of methanol to water. The aqueous pKa was determined by extrapolation to 0% methanol using the method of Yasuda-Shedlovsky37,38 which gives a linear fit.
Partition coefficients between octan-1-ol and water were measured by dual-phase potentiometric titration using various amounts of water-saturated octanol. Titrant addition was carried out with vigorous stirring of the assay solution. Three different ratios of octanol/water were employed for each compound. The log P values were obtained from the difference between the aqueous pKa of the species and the apparent pKa determined from the dual phase titration.39,40 Measurements were carried out in triplicate with a S.E. of ±0.40. The potentiometric method was validated by comparison with results obtained by the standard shake-flask technique.41
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Results |
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Little difference was reported in antiplasmodial activity in vitro between the enantiomers of CQ on P. falciparum, in both CQ-resistant and -sensitive isolates9 and similar results were obtained for the activity of the enantiomers and the racemic form of HCQ in vitro. The activities of CQ and HCQ were also similar against the sensitive parasites, but whereas CQ was about 14 times less active against the resistant isolate, the factor for HCQ was in the region of 79. Molecule for molecule, HCQ was 1.6 times less active than CQ against the CQ-sensitive, but 8.8 times less active than CQ against the resistant isolate (Table 1). This result was confirmed in further assays on a different occasion (data not shown).
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Using CQ as reference compound for the ionization constants, pKa2 could be clearly identified with the aromatic amidinium ion by comparison with the parent substance, 4-amino-7-chloroquinoline (pKa 8.23).42 In HCQ hydrogen bonding of the terminal OH with the side chain tertiary N lowered pKa1 similar to the drop from triethylamine (10.74) to 2-diethylamino ethanol (9.7).43 DCQ has a side-chain resembling diethylamine (pKa 10.98) and shows a similar enhanced pKa due to a reduction in steric hindrance to protonation of the N. Both DAQ and AQ possess an aromatic substituent attached to the quinoline 4-N, causing a drop in pKa2 by 1 unit, and a similar reduction in pKa1 since the alkylamine side-chain has been replaced by a benzylamine group. All three isomers of HCQ showed the expected enhanced hydrophilic properties in terms of the experimental log P (3.84 ± 0.2) compared with CQ (4.72), whereas the secondary amine metabolite DCQ revealed a smaller but significant decrease in log P (4.35). Measured log P values for all five compounds were in good agreement with those calculated using ClogP. For AQ, the experimental log P (4.26) was 1.2 log units lower than the calculated ClogP value (5.47); such a difference is frequently observed in compounds which may undergo either intramolecular H-bonding or actual bending of the molecule.34 The same is true for the desethyl metabolite DAQ. Its higher polarity is reflected in the decrease in log P to 3.31, again 1.2 log units lower than the calculated value (4.5).
The distribution coefficient log D at pH 7.4 affords some insight into the potential membrane transfer capabilities of these drugs. All three isomers of HCQ show an experimental log D (pH 7.4) of 0.62 ± 0.04 reflecting increased hydrophilicity, whereas CQ is the most hydrophobic (0.96) and DCQ the least hydrophobic (0.06) of the DCQ, HCQ, CQ group. The greatly increased lipophilicity of AQ (compared with CQ) is demonstrated by its high log D (2.83), and for DAQ the decrease in hydrophobic character is reflected in the drop of log D to 1.67, similar to the drop of 1 log unit from CQ to DCQ.
It is relevant to examine phase transfer behaviour at pH 5.2. In most cases, it was possible to determine log D at this pH experimentally, and the values agreed well with those calculated from Equation 2. In the CQ series, the drop in pH from 7.4 to 5.2 results in log D values between 3.4 and 4.6, which accounts for the trapping of the (now diprotonated) drug in the lysosome. It should be noted that AQ and DAQ, while possessing the highest log D values at pH 7.4 (where log D 2.83 represents a favourable lipid/water phase transfer ratio of 700:1) also have the least negative values at pH 5.2; this, as discussed later, may be related to retention of activity against CQ-resistant strains (Table 2).
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Discussion |
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On the view that PfCRT protein resembles an aqueous ion channel, it is illuminating to consider that the drugs like furosemide which inhibit membrane chloride channels,50 possess at least one negatively-charged anionic group which is attracted to the chloride gate within the membrane, while having a lipophilic tail which binds strongly to hydrophobic groups in the channel lining, effectively preventing access of anions to the channel. By analogy with this, 4-aminoquinolines with higher hydrophobicity would not only bind to the lining of the mutated export channel, but also have difficulty passing through it, allowing the development of the high drug concentrations within the lysosome required for lethal interaction with haematin.
All the enantiomers of HCQ have lower log D values at pH 7.4 than CQ, reflecting an increase in hydrophilic character. Whereas the IC50 values of HCQ on a molar basis for the sensitive isolate are only 1.6 times higher than CQ, they are 8.8 times higher than CQ against the resistant isolate. CQ prophylaxis uses an adult dose of 300 mg base per week, and the maximum safe dose of HCQ is 309.6 mg base per day,4 the molar equivalent of 295.0 mg CQ base/day and approximately seven times the 300 mg/week used in CQ prophylaxis of malaria.3 To give a daily dose of HCQ likely to have a similar limited protective effect to chloroquine against CQ-resistant P. falciparum would demand daily doses of HCQ above the recommended safe level. These observations contraindicate the use of HCQ in prophylaxis against CQ-resistant strains. A feasible approach in circumstances where CQproguanil is the only prophylactic possible for a patient on HCQ would be to use prophylactic proguanil and replace the anti-inflammatory HCQ with a suitable CQ regimen [maximum adult daily dose 150 mg base (200 mg sulphate)].
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Note added during revision |
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Acknowledgements |
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Footnotes |
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