Photoreactivity of biologically active compounds

European Journal of Pharmaceutical Sciences, 5 (1997) 139–146

Photoreactivity of biologically active compounds XI. Primaquine and metabolites as radical inducers a, b a Solveig Kristensen *, Leonid Grinberg , Hanne Hjorth Tønnesen a

Department of Pharmaceutics, Institute of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, N-0316 Oslo, Norway b Hadassah Medical Organization, P.O. Box 24035, Il-91240 Jerusalem, Israel Received 4 April 1996; accepted 29 November 1996

Abstract Reduction of ferricytochrome C and oxidation of haemoglobin was used to examine redox properties of primaquine, metabolites and photodegradation products of the drug. The influence of oxygen radicals (O 2 ?2 and OH?) were studied by the addition of oxygen radical scavengers. Photodecomposition of primaquine (80 mW/ cm 2 , xenon lamp, 290–800 nm) prior to dark-incubation resulted in a substantial accelerated drug-induced O 2 ?2 formation and haemoglobin oxidation. Formation of OH? (dark reaction) could be detected after photochemical degradation of primaquine. In the presence of erythrocytes the formation of oxygen radicals induced by the photodecomposition products was even more pronounced. A high oxygen content in the medium during irradiation accelerated the photodecomposition-rate of primaquine. The metabolite 6-desmethyl primaquine was a more potent O 2 ?2 producer and haemoglobin oxidizer than primaquine (dark reactions). During irradiation (80 mW/ cm 2 , 290–800 nm) primaquine formed more O 2 ?2 and produced a detectable level of OH? compared to the dark reactions. Keywords: Primaquine; Photoreactivity; Phototoxicity; Redox properties; Radical induction

1. Introduction Primaquine is an 8-aminoquinoline with antimalarial activity. The drug eliminates persistent liver forms of Plasmodium ovale and P. vivax. Primaquine has no effect on the erythrocytic stages of Plasmodia unless toxic doses are administered. Severe adverse effects (haemolysis and methaemoglobinaemia) are observed after administration of primaquine, manifested particularly in patients with glucose-6-phosphate dehydrogenase deficiency (WHO, 1988). This enzyme is part of the anti-oxidative defence system, protecting the erythrocytes against peroxidative reactions (Chiu et al., 1989). Thus primaquine is not suitable for general malaria prophylaxis, although it might have to be given prophylactically together with *Corresponding author: Tel.: 147 22 854218; fax: 147 22 857494; e-mail: [email protected] 0928-0987 / 97 / $32.00  1997 Elsevier Science B.V. All rights reserved PII S0928-0987( 97 )00268-6

a blood schizontocide (e.g. chloroquine) in areas where P. ovale and P. vivax are endemic (Reynolds, 1989). Primaquine absorbs light in the UV-visible region of the spectrum. Light-exposure of the compound in an aqueous oxygen containing medium causes various structural changes in the side chain of the molecule. Eight major and several minor decomposition products are formed (Kristensen et al., 1993). Primaquine acts as a photosensitizer in vitro, inducing photohaemolysis and photopolymerization of proteins (Kristensen et al., 1994, 1995). The compound oxidizes haemoglobin and NADPH by formation of superoxide, hydroxyl radicals and hydrogen peroxide in vitro (Summerfield and Tudhope, 1978; Thornalley et al., 1983). The haematologic adverse effects caused by primaquine might be due to the formation of radicals in vivo. Radical formation is further important with

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respect to drug stability and formation of degradation products which may be toxic. This work was undertaken to obtain further information on the in vitro formation of radicals from primaquine during light exposure and from the photodecomposition products and most important metabolites of primaquine in the absence of light. 2. Experimental procedures

2.1. Materials Primaquine diphosphate (PQ; .99% pure, Aldrich, Germany), ferricytochrome C (cytC; from Bovine heart, 99% pure, ferrocytochrome C content ,5%), superoxide dismutase (SOD; from Bovine erythrocytes, 98% pure), D-mannitol (MAN), 6-methoxy quinoline (6MQ), 8-amino quinoline (8AQ) and quinoline (QUI; 98% pure), all Sigma, USA, were used as received. The metabolites of PQ (6-desmethyl primaquine hydrobromide (DPQ), carboxy primaquine (CPQ), N-acethyl primaquine (APQ) and 8-amino-6-methoxy quinoline (AMQ)) were kindly provided by professor J.D. McChesney, School of Pharmacy, The University of Mississippi, USA. The molecular structures of the metabolites were verified by electron impact (EI) mass spectrometry via direct inlet (Prospec, Fisons Instruments / VG, ion source temperature 2208C, ionization potential 70 eV). All other chemicals were of p.a. grade. Stock solutions of SOD (3 mg / ml) in PBS (phosphate buffered saline; NaCl 75 mM 1 Na 2 HPO 4 / NaH 2 PO 4 75 mM, pH 7.4) were stored at 2208C. Stock solutions of PQ (0.4 mM) and cytC (6.2 mg / ml) in PBS were prepared ex temporae. The stock solution of DPQ (5 mM) was prepared in PBS. Due to low solubilities in PBS stock solutions (5 mM) of the other metabolites (CPQ, APQ and AMQ) and derivatives (6MQ, 8AQ and QUI) were prepared in dimethyl sulfoxide (DMSO) and stored at 2208C before further dilution in PBS. Human blood, obtained from one volunteer, was stored refrigerated (148C) in EDTA-tubes. All other solutions were kept refrigerated or on ice during the experiments. The final samples were prepared in PBS containing 5.6 mM glucose. Incubations were carried out dark under atmospheric conditions at 378C in a water bath, the tubes were regularly shaken. Each experiment was performed in duplicate.

2.2. Preparation of RBC Immediately before the experiment, 3 ml blood was centrifuged for 5 min at 1500 rpm (Hettich Universal Centrifuge) and plasma was removed. The red blood cells (RBC) were washed three times with 5 ml PBS and centrifuged for 5 min at 2000 rpm between each washing procedure. The RBC were suspended in PBS and the concentration (20% v / v) was determined by haematocrit measurements (LIC Lars Ljungberg Microhaematocrit 4, rotation for 3 min).

2.3. Photochemical degradation of PQ Irradiated samples of PQ containing a mixture of photodecomposition products and the parent drug were obtained by irradiation of PQ (0.4 mM in PBS) at 290–800 nm for 10, 20, 40, 60 or 80 min under constant stirring. A 1.8 kW xenon burner (Suntest CPS, Heraeus GmbH, Hanau, Germany) equipped with a quartz-glass dish with infrared (IR) reflective coating and a UV-filter (transmission 290–800 nm) was used as a light source to simulate the light conditions outdoors. The energy output was adjusted to 80 mW/ cm 2 , quantified by a thermopile voltmeter (200 mV, Applied Photophysics Ltd.) calibrated against a black-body radiator. To study the influence of the oxygen content on formation of reactive photodecomposition products, 20 ml samles of PQ (0.4 mM in PBS) were flushed with helium gas (He) for 15 min prior to 40 min irradiation (290–800 nm) or with oxygen gas (O 2 ) for 15 min prior to 2, 5 or 10 min irradiation (290–800 nm). The solutions were continously stirred during irradiation. Non-irradiated PQ was kept in the dark at 378C (water bath) to correct for the temperature rise in the Suntest-cabinet (up to 358C).

2.4. Reduction of ferricytochrome C (cytC) Ferricytochrome C (620 mg / ml) was used to detect formation of reducing radicals. References were prepared without drug present. The reduction of cytC is manifested by a rise in the absorbance at 534–564 nm (maximum at 550 nm). The molar absorptivity of ferri-cytC (Fe 31 ) and ferrocytC (Fe 21 ) at 550 nm are 0.89?10 4 and 2.99?10 4

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M 21 cm 21 , respectively (Fridovich, 1985). Absorption spectra (500–600 nm) were recorded using a Shimadzu UV-2101PC spectrophotometer. The area under the absorption curve (AUC; 534–564 nm) was calculated to quantitate the reduction.

2.5. Reduction in the absence of light PQ (0.2 mM, non-irradiated or irradiated for 2-80 min under atmospheric oxygen pressure or after flushing with O 2 or He), derivatives and metabolites of PQ (0.2 mM) were tested. RBC (2% v / v) was added to one series of each of the compounds investigated. To detect the influence of O 2 ?2 or OH?, respectively, samples containing radical scavengers (SOD; 60 mg / ml or MAN; 1 mM) were used as blanks. References without drug were prepared with and without DMSO (4% v / v) to correct for the effect of this solvent which was present in some of the samples. After incubation for 60 min under atmospheric conditions, the tubes were stored on ice for 10 min to stop the reaction. The samples containing RBC were centrifuged at 2000 rpm for 12 min prior to absorption measurements of the solutions / supernates and calculation of the AUC. The AUC of the reference (no drug added) was substracted from the AUC of the sample to correct for the reactions not induced by the drug, resulting in the corrected area.

2.6. Reduction during irradiation Test samples (with PQ at 0.2 mM) and reference samples (no PQ) were irradiated (80 mW/ cm 2 , 290– 800 nm) for 2, 5, 10, 20, 30, 40, 60 and 80 min with constant stirring (thickness of the sample layer51 cm). Identical samples stored in the dark at 378C were used as blanks to correct for the reaction not induced by the light and for the temperature rise in the Suntest-cabinet during irradiation. Absorption spectra were recorded, and the AUC subsequently calculated, resulting in the corrected area. Further, the formation of reactive oxygen species during irradiation of PQ was studied by the addition of SOD or MAN to the blank samples. All samples (thickness of the sample layer52 mm) were irradiated (290–800 nm) for 10 min The AUC of the references (no drug added) were substracted, resulting in the corrected area.

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2.7. Oxidation of haemoglobin ( Hb) in the absence of light RBC (2% v / v) were mixed with PQ (0.22 mM, non-irradiated or irradiated for 2–80 min under atmospheric oxygen pressure or after flushing with O 2 or He) or derivatives or metabolites of PQ (0.22 mM). Samples to be used as blanks were prepared without RBC. References without drug were made with and without DMSO (4.4% v / v). The samples were incubated for 2 h under atmospheric conditions. 200 ml was mixed with 1 ml purified water and 40 ml Triton-X (2.5%) and kept on ice for 10 min to allow haemolysis to complete. Subsequently the absorption spectra (500–700 nm) were recorded. The oxidation of haemoglobin (Hb(Fe 21 )) to methaemoglobin (Hb(Fe 31 )) is manifested by a rise in the absorbance above 600 nm (maximum 630 nm) parallel to a decrease in the absorbance at 540 and 576 nm (Caughey and Watkins, 1985). The absorbance at 630 nm was measured, and the absorbance of the reference (no drug added) was substracted, resulting in the corrected absorbance.

2.8. HPLC analysis The HPLC system was kindly provided by Weifa A / S, Norway, and used after optimization. A Spectra-Physics SP 8700 liquid cromatograph equipped with a Shimadzu SIL-6A auto injector, a Shimadzu C-R3A Chromatopac integrator and a Schimadzu SPD-6AV UV-vis spectrophotometric detector (357 nm) was used. The analysis were carried out at ambient temperature. The stationary phase was Nova-Pak C18 (Waters, Millipore, USA), particle size 4 mm, pre-packed in a 150 mm33.9 mm i.d. column. The mobile phase consisted of 15% v / v methanol, 7.5% v / v acetonitrile, 0.5% v / v acetic acid (conc.) and 5 mM heptane sulphonic acid in purified water (PIC Reagent B7, Waters, Millipore, USA). To one litre of eluent was added 2.8 ml triethylamine and 2 ml dimethyl-octyl-amine. The reproducibility (n510, 10 mM PQ) was 2.2% (relative standard deviation). The lower detection limit was 1 mM (calculated as three times the noise of the baseline). The correlation coefficient calculated on the basis of six dilutions of the sample in the concentration range 5 mM to 50 mM (n53) was 0.9998.

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The photochemical degradation products of PQ (isolated by the methode described by Kristensen et al., 1993) were not found to influence the analysis of PQ in this HPLC system.

3. Results and discussion The structural formulas of PQ and the derivatives and metabolites of the drug are illustrated in Fig. 1. Each result (Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 and the results given in the text) is the mean of two measurements. 74% of the parallels deviate less than 10% from the mean, 16% deviate 10–20% and 10% deviate more than 20% from the mean.

3.1. Photodecomposition products of PQ as source of O2?2 and Hb-oxidizers in the absence of light The PQ-induced formation of O 2 ?2 is accelerated by irradiation of the drug prior to incubation with cytC (Fig. 2). Pre-irradiation under atmospheric conditions leads to an optimum effect on O 2 ?2 formation after 40 min exposure. At this point 59% of PQ is decomposed (Fig. 3). Flushing with oxygen gas prior to irradiation accelerates the photodecomposition of PQ (Fig. 3) and increases the subsequent

Fig. 1. Structural formulas of the compounds studied (PQ5primaquine, AMQ58-amino-6-methoxy quinoline, APQ5N-acethyl primaquine, CPQ5carboxy primaquine, DPQ56-desmethyl primaquine, 6MQ56methoxy quinoline, 8AQ58-amino quinoline and QUI5quinoline).

Fig. 2. Formation of superoxide (O 2 ?2) in the absence of light as a function of pre-irradiation time of PQ (290–800 nm, 80 mW/ cm 2 ). PQ5primaquine irradiated under atmospheric conditions, PQ1O 2 5 primaquine-samples flushed with oxygen prior to irradiation, PQ1He5 primaquine-samples flushed with helium prior to irradiation.

formation of O 2 ?2 (Fig. 2), while flushing with helium gas retards the photodecomposition (Fig. 3) and extensively lowers the subsequent O 2 ?2 formation (Fig. 2). In the presence of RBC the formation of O 2 ?2 initiated by the photoproduct(s) is even more pronounced (Fig. 2). It is previously demonstrated that formation of H 2 O 2 from O 2 ?2 does not affect PQ-induced cytC reduction (Summerfield and Tudhope, 1978). Therefore, catalase was not added to the samples in the present work. The drug-induced oxidation of Hb is also more pronounced after pre-irradiation of PQ (Fig. 4). The effect reaches an optimum after 40 min pre-irradia-

Fig. 3. Photochemical degradation of PQ as a function of irradiation time (290–800 nm, 80 mW/ cm 2 ). PQ5primaquine irradiated under atmospheric conditions, PQ1O 2 5primaquine-samples flushed with oxygen prior to irradiation, PQ1He5primaquine-samples flushed with helium prior to irradiation.

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Fig. 6. Reduction of cytC as a function of irradiation time of PQ (290–800 nm, 80 mW/ cm 2 ). Ref. is the reference without drug added. The calculated area is corrected for the dark reaction. Fig. 4. Oxidation of haemoglobin (Hb) in the absence of light as a function of pre-irradiation time of PQ (290–800 nm, 80 mW/ cm 2 ). PQ5primaquine irradiated under atmospheric conditions, PQ1O 2 5 primaquine-samples flushed with oxygen prior to irradiation, PQ1He5 primaquine-samples flushed with helium prior to irradiation.

tion under atmospheric conditions. Flushing with oxygen gas prior to irradiation increases the subsequent oxidation of Hb, while flushing with helium gas lowers Hb-oxidation. This is consistent with the observed O 2 ?2 formation.

There seems to be a linear correlation between % PQ decomposed during irradiation (atmospheric conditions) and the formation of O 2 ?2 during the subsequent incubation with cytC (Fig. 5). Thus photoproducts with higher reducing capability than PQ seem to accumulate during irradiation of the drug. When RBC are present, however, the resulting curve indicates a stepwise reaction. Initially, photo-

Fig. 5. Formation of superoxide (O 2 ?2; continuous curves) and oxidation of haemoglobin (Hb; dotted curves) in the absence of light as a function of photodecomposition of PQ (290–800 nm, 80 mW/ cm 2 ). PQ5primaquine irradiated under atmospheric conditions, PQ1O 2 5primaquine-samples flushed with oxygen prior to irradiation.

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product(s) with high RBC-interaction seems to be formed, resulting in a rapid increase in the formation of O 2 ?2. This can be observed up to 11% PQ decomposed. Beyond this point the curve changes, running parallel with the curve representing samples without RBC. This can be due to the formation and accumulation of new photoproduct(s) which interact less with RBC. It is also possible that the initially formed photoproduct(s) decompose during further irradiation, or that the biochemical test system is inactivated in some way. Since oxidation of Hb runs parallel with reduction of cytC in the presence of RBC (Fig. 5), these two reactions seem to be related in the present test system. The oxygen level in the medium is important for the reaction rate of PQ-photodecomposition (Fig. 3). However, a high oxygen level seems to have little influence on the amount of reactive photoproduct(s) formed relative to PQ decomposed. The plots of O 2 ?2 formation (RBC present / absent) and Hb-oxidation during the subsequent incubations as a function of % PQ photodecomposed after flushing with oxygen are about identical with the plots recorded after irradiation under atmospheric conditions (Fig. 5).

3.2. Photodecomposition products of PQ as source of OH? in the absence of light PQ was not a source of OH? in the absence of light under the experimental conditions used. However, after irradiation of the drug (for 40 min under atmospheric oxygen pressure) prior to incubation the formation of this radical was detected by a scavenging reaction with mannitol (corrected area (534–564 nm) was 0.0660.01 in the absence of RBC and 0.6860.06 in the presence of RBC). Thus one or several of the photodecomposition products of PQ likely seems to have the potential to form OH? (to a greater extent than PQ), a property accelerated by the presence of RBC. The low level of OH? detected can be explained by the presence of glucose in the samples (scavenging OH?, k510 9 M 21 s 21 , Halliwell and Gutteridge, 1985) which was added to simulate physiological conditions. Besides, MAN is less effective than cytC in competing for OH? (k510 9 and 1.4?10 10 M 21 s 21 , respectively; Halliwell and Gutteridge, 1985; Koppenol and Butler, 1984). Even if OH? is a strong

oxidizer, the radical is known to reduce cytC through hydrogen abstraction from the surface with a subsequent intramolecular electron transfer resulting in reduction of the haem (yield of reduction: 25–55%). OH? can further react with ferro-cytC (k.10 10 M 21 s 21 ). The yield of this reaction is only 5%, therefore it is not likely to influence the results to any extent (Koppenol and Butler, 1984). However, the reaction rates and the reaction yields will depend on the experimental conditions.

3.3. Derivatives and metabolites of PQ as source of O2?2 and Hb-oxidizers in the absence of light Of the metabolites and derivatives studied (Baty et al., 1975; Mihaly et al., 1984)(Fig. 1), only the metabolite DPQ was observed to be a a more potent O 2 ?2 inducer than PQ (corrected area (534–564 nm) for DPQ was 1.5760.11 in the absence of RBC and 1.1960.12 in the presence of RBC, compared to 0.2260.02 and 0.2760.17 for PQ). Thus the presence of RBC was observed to lower the formation of O 2 ?2 induced by DPQ. DPQ is also a more potent oxidizer of Hb than PQ (corrected absorbance at 630 nm is 12.0?10 23 61.5?10 23 for DPQ compared to 5.7?10 23 60.6?10 23 for PQ). DMSO was used as a cosolvent for the other metabolites / derivatives. DMSO is known to be a scavenger of OH? (Halliwell and Gutteridge, 1985), but is not reported to react with O 2 ?2. The derivative 6MQ (Fig. 1) produces O 2 ?2 to a slight extent (corrected area (534–564 nm) in the absence of RBC is 0.1060.01 compared to 0.1360.01 for PQ in the presence of DMSO), but no oxidation of Hb was observed (the corrected absorbance (630 nm) for PQ 23 23 is 22.5?10 61.0?10 in the presence of DMSO). The other metabolites / derivatives do not induce O 2 ?2 production to any extent or show any other oxidative properties in the test system used.

3.4. Reducing effect on cytC in the presence of light PQ reduces cytC during exposure to light (Fig. 6), and the effect is increased by an increase in irradiation time. The reduction detected is the total effect caused by reducing species (oxygen radicals and radicals of PQ) formed during irradiation of PQ. The reference samples (no drug added) have slightly

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negative corrected areas, caused by an accelerated reduction of cytC in the blanks (dark references stored at 378C) compared to the irradiated samples. Both O 2 ?2 and OH? seem to be formed during irradiation of PQ. When SOD was used as a scavenger the corrected area (534–564 nm) was 0.1160 in the dark and 0.3560.05 after irradiation for 10 min. When MAN was used as a scavenger, no radical formation was detected in the dark, while the corrected area was 0.1160 after irradiation. None of the scavengers absorb light above 290 nm, i.e. the detected inhibition is not due to a filter effect. Thus light exposure seems to accelerate the formation of oxygen radicals induced by PQ, and catalyze the formation of reducing radicals formed by the drug. In addition to a direct reaction between OH? and cytC, the OH? induced reduction of cytC can be promoted by abstraction of a hydrogen atom from an organic molecule (e.g. PQ), leading to the formation of a radical which will reduce cytC through electron transfer (Koppenol and Butler, 1984). Many of the photodecomposition products of PQ have one or several carbon–carbon double bonds in the side chain (Kristensen et al., 1993), which may have been formed by OH? induced hydrogen abstraction. Thus the formation of PQ(–H)? reducing radicals during irradiation of PQ seems likely. Thornalley et al. (1983) and Bisby (1988) have previously suggested formation of a strongly reducing PQH? or PQ?2 radical after reaction with NADPH / NADH. This radical may possibly also be formed during irradiation of PQ.

4. Conclusion One (or several) of the photochemical degradation products formed during irradiation of PQ is a more potent inducer of oxygen radicals (O 2 ?2 and OH?) and a more powerful Hb-oxidant than the parent drug in the absence of light. The oxygen content in the sample affects the photodecomposition-rate of PQ. Hence, PQ exposed to light seems to form decomposition product(s) more toxic than the drug itself. The presence of RBC was observed to accelerate the O 2 ?2 and OH? production induced by the photodecomposition product(s), an effect which may be of importance in vivo. One of the metabolites of PQ (DPQ) is also a more

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potent inducer of O 2 ?2 and a more powerful Hboxidant than PQ. Thus the metabolite may be a more toxic compound than the parent drug. However, the main in vivo metabolite of PQ in human plasma is CPQ (Mihaly et al., 1984), a compound which was not found to have in vitro redox properties to any extent. Light exposure was found to accelerate the formation of oxygen radicals (O 2 ?2 and OH?) induced by PQ. Besides, formation of several radicals of the drug itself is likely. Light above 320 nm is penetrating Caucasian skin (Megaw and Drake, 1986). Thus light induced reactions caused by the photolabile drug PQ may also take place in vivo, since PQ absorbs light up to 430 nm under physiological conditions. Acknowledgments The authors thank Henrik Schultz, Weifa A / S, Norway, and Professor J.D. McChesney, University of Mississippi, USA, for providing the HPLC methode and the metabolites of PQ, respectively, John Vedde, Institute of Chemistry, University of Oslo, Norway, for assistance with the MS analysis, and Anne-Lise Orsteen, Institute of Pharmacy, University of Oslo, Norway, for drawing figures. References Baty, J.D., Price Evans, D.A. and Robinson, P.A. (1975) The identification of 6-methoxy-8-aminoquinoline as a metabolite of primaquine in man. Biomed. Mass Spectrom. 2, 304–306. Bisby, R.H. (1988) One-electron reduction of the antimalarial drug primaquine, studied by pulse radiolysis. Free Radic. Res. Commun. 5, 117–124. Caughey, W.S. and Watkins, J.A. (1985) Oxy radical and peroxide formation by hemoglobin and myoglobin. In Greenwald, R.A. (Ed.), CRC Handbook of Methods for Oxygen Radical Research, CRC Press, Florida, pp. 95–104. Chiu, D., Kuypers, F. and Lubin, B. (1989) Lipid peroxidation in human red cells. Semin. Hematol. 26, 257–276. Fridovich, I. (1985) Cytochrome c. In Greenwald, R.A. (Ed.), CRC Handbook of Methods for Oxygen Radical Research, CRC Press, Florida, pp. 121–122. Halliwell, B. and Gutteridge, J.M.C. (1985) Free Radicals in Biology and Medicine, Clarendon Press, Oxford. Koppenol, W.H. and Butler, J. (1984) The radiation chemistry of cytochrome c. Isr. J. Chem. 24, 11–16. Kristensen, S., Grislingaas, A-L., Greenhill, J.V., Skjetne, T., Karlsen J. and Tønnesen, H.H. (1993) Photochemical stability of biologically active compounds: V. Photochemical degradation of primaquine in an aqueous medium. Int. J. Pharm. 100, 15–23. Kristensen, S., Karlsen, J. and Tønnesen, H.H. (1994) Photoreactivity of

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Reynolds, J.E.F. (1989) Martindale, The Extra Pharmacopoeia, 29th edn. The Pharmaceutical Press, London, pp. 506–518. Summerfield, M. and Tudhope, G.R. (1978) Studies with primaquine in vitro: Superoxide radical formation and oxidation of haemoglobin. Br. J. Clin. Pharmacol. 6, 319–323. Thornalley, P.J., Stern, A. and Bannister, J.V. (1983) A mechanism for primaquine mediated oxidation of NADPH in red blood cells. Biochem. Pharmacol. 32, 3571–3575. WHO (1988) WHO Drug Information, Editorial. (1988) Essential Drugs. Malaria 2, 79–93.