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Quinine distribution in pregnant mice with Plasmodium berghei malaria
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 284–290
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Quinine distribution in pregnant mice with Plasmodium berghei malaria Fr´ed´eric Lirussi, Eric Pussard ∗ Hopital de Bicetre, Department de Pharmacology, 94275 Le Kremlin Bicetre, France
a r t i c l e
i n f o
a b s t r a c t
Article history:
Maternal malaria is associated with placental insufficiency that leads to intrauterine growth
Received 16 August 2005
retardation and reduced birth weight. Malaria may impair the exchange of drugs across the
Received in revised form
placenta especially the transmission of antimalarial drugs to the foetus.
16 January 2006
The distribution of quinine and its 3-hydroxymetabolite in blood, tissues and foeto-placental
Accepted 13 March 2006
unit was evaluated on day 18 of pregnancy of mice infected or not with Plasmodium berghei.
Published on line 22 March 2006
During pregnancy, quinine distribution volume increases gradually with the rise of free quinine concentrations in plasma. Quinine concentrations increase in erythrocytes and most
Keywords:
tissues without change in systemic clearance. A maternal-to-foetal gradient of 8:1 limits the
Pregnancy
exposure of foetus to quinine.
Quinine
During malaria, the systemic clearance of quinine and the 3-hydroxyquinine gradually
Placenta
decrease with the rising parasitaemia. Quinine concentrations increase slightly in most
Foetus
of the tissues. The weight of placentas decreases in a parasitaemia-dependant manner and
Mice
is strongly related to the low uptake of quinine by placenta. Foetal weights and quinine
Plasmodium berghei malaria
concentrations in foetus only decrease for the highest parasitaemia. In this experimental model, pregnancy facilitates quinine uptake by erythrocytes and peripheral tissues. Malaria induces a hypotrophy of both placenta and foetus. In placenta, the marked decrease of quinine concentrations may impair the clearance of sequestered parasites. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Plasmodium falciparum infections during pregnancy frequently led to maternal anaemia, abortions, stillbirths, low birth weights and increased infant mortality (McGready et al., 1998; Nosten et al., 2004). Similar observations have been made in rodent pregnancy complicated by Plasmodium berghei malaria (Hioki et al., 1990; Oduola et al., 1982). The pathologic changes in the infected rodent placenta were quite similar to those reported in humans with thickening of the trophoblast and accumulation of parasitized erythrocytes (Andrews and Lanzer, 2002; Galbraith et al., 1980; Oduola et al., 1986; Tegoshi
∗
Corresponding author. Tel.: +33 145213580; fax: +33 145213591. E-mail address: [email protected] (E. Pussard).
0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.03.004
et al., 1992). While intact malaria parasites do not normally pass the placenta, they may nonetheless damage this organ and jeopardize the materno-foetal relationship. Thus, pregnancy like malaria may affect both the distribution of drugs in the dams and the exchange across the placenta to the foetus. Quinine, alone or in combination represents the most frequently available treatment of severe malaria in Africa, especially in pregnant women close to delivery, because there is often no other effective drug available (Nosten et al., 2004). Nevertheless, the persistence of Plasmodium falciparum in the placenta has been described after apparently effective quinine therapy and that might suggest a decrease of drug efficacy
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 284–290
(Menendez et al., 2000; Procop et al., 2001). Quinine pharmacokinetics has been described in the healthy ovine foetus (Czuba et al., 1991) and in pregnant women with severe falciparum malaria (Looareesuwan et al., 1985; Phillips et al., 1986). Nevertheless, the extent of foetal exposure to quinine administered to the mother during malaria is not defined. The aim of this study was to determine whether malaria influences the disposition of quinine during pregnancy in a model of P. berghei-infected mice.
2.
Materials and methods
2.1.
Animals
All experiments were performed according to the official regulations of the French Ministry of Agriculture and were in conformity with the Guiding principles in the use of animals in toxicology adopted by the Society of Toxicology (USA) in July 1989 and revised in 1999. The present study was approved by the animal care committee of our institution (Paris XI University). Twelve-week-old female Swiss OF1 mice (weight 28–30 g) were obtained from Iffa Credo (L’Arbresle, France). Animals were housed 6 per cage, fed ad libitum with standard diet and had free access to tap water. Room temperature was maintained at 21 ± 1 ◦ C, and a 12-h artificial light/dark cycle was programmed. For the gestation of animals, three female were housed with one male and checked for vaginal plug twice a day. The day on which the vaginal plug was determined was classified as day 0 of gestation. Pregnant mice were then placed in separate cages.
2.2.
Experimental infections
The NK65 strain of P. berghei was kindly supplied by Dr. I. Landau (Museum National d’Histoire Naturelle, Paris, France). On day 13 of the gestation period, pregnant mice were infected by intraperitoneal injection of either 104 or 106 parasitized erythrocytes in 100 l of heparinized fresh blood from previously infected mice. Three days after infection, thin smears were made daily from tail blood, stained with Giemsa and observed by light microscopy (oil immersion, magnitude, 1000×).
2.3.
Animal experiments and quinine administration
Non-pregnant control mice, uninfected pregnant mice and pregnant mice with low or high parasitaemia were injected subcutaneously with a 0.1 ml-single dose of quinine saline solution (Quinine hydrochloride, Sigma, France) at a dose of 80 mg/kg of quinine base. Drug administration was made on day 18 of gestation.
5 and 6 h post-injection. Samples were stored at −20 ◦ C until analysis.
2.3.2.
Experiment 2
Quinine distribution in blood and tissues was evaluated in uninfected non-pregnant control (n = 12), in uninfected pregnant mice (n = 12) and in pregnant mice with low [parasitaemia: median (range): 12% (5–17%), n = 12] or high parasitaemia values [parasitaemia: median (range): 44% (37–48%), n = 12]. 2 h after a subcutaneous injection of 80 mg of quinine base per kg of body weight, mice were anaesthetised with 60 mg/kg pentobarbital. About one ml of blood was taken through cardiac puncture. Two aliquots of blood were saved for haematocrit (heparinized capillaries 9 l, Bayer Diagnostics, Bridgend, UK) and quinine whole blood determinations (30 l). After centrifugation at 1500 × g for 15 min, plasma and erythrocytes were separated. Two aliquots of plasma (for total and free quinine measurements), the whole blood and erythrocyte fractions were frozen at −20 ◦ C. Total proteins concentrations in plasma were assayed by the method described by Bradford (1976). The maternal brain, lungs, heart, liver, spleen and kidneys were removed, washed in saline solution (0.9% NaCl) and dried with absorbent paper. Tissues were weighted accurately and stored at −20 ◦ C. The uterine horns were exteriorized and all foetuses and corresponding placenta were removed and weighted. After the amniotic fluid was aspirated, two foetuses and corresponding placentas of each horn were immediately frozen on dry ice.
2.4.
Quinine analysis
After thawing, the tissues were homogenized in 1–3 ml of cold 0.05 N HCl and sonicated on ice with an ultrasonic probe for 1 min. The homogenate was then centrifuged and 25 l of supernatant was used for analysis. Free quinine concentrations were measured with a protein-free ultra filtrate obtained by centrifugation of a 500 l-plasma sample at 2000 × g for 20 min at room temperature (Amicon MPS-1 micro partition system with YMT membranes) (Pussard et al., 1999). Quinine and 3-hydroxyquinine concentrations in blood fractions and tissues homogenates were determined by a high-performance liquid chromatographic method with fluorometric detection as previously described (Pussard et al., 2003). Quinidine was used as internal standard. Standard calibration curves for quinine and 3-hydroxy quinine were prepared in drug-free medium (whole blood, erythrocyte, plasma or supernatant of tissue homogenate fluid) and were linear in the range of concentrations 0.1–100 g/ml. Quinine and 3-hydroxyquinine recoveries were more than 80%. Within-run and betweenrun variabilities were lower than 10% in each biological matrix.
2.5. 2.3.1.
285
Pharmacokinetics analysis
Experiment 1
Quinine pharmacokinetics was evaluated in uninfected nonpregnant mice (n = 12), in pregnant uninfected (n = 12) and in P. berghei-infected pregnant mice [parasitaemia: median (range) 35% (31–40%), n = 12]. Whole blood samples (30 l) were taken from two drops of caudal vein blood and vortexed immediately with solid EDTA. Sampling times were at 0.25, 0.5, 1, 1.5, 2, 3, 4,
Whole blood quinine concentrations were fitted using a 1compartment model. The total area under the whole blood concentration–time curve from time 0 to 6 h (AUC0–6 h ) was calculated by linear trapezoidal rule. Clearance (Cl/F) was determined by dose/AUC0–∞ and volume of distribution (V/F) by dose/(z × AUC0–∞ ) where z is the first order rate constant
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associated with the terminal portion of the curve. The elimination half-life (t1/2 ) was calculated as ln(2)/z . The maximum concentrations of quinine and its 3-hydroxymetabolite in whole blood (Cmax ) and the time to reach Cmax (Tmax ) were observed values.
2.6.
Statistical analysis
Data were reported as means ± standard deviations except parasitaemia expressed as median and range. Bartlett’s test was used to verify the homogeneity of variances. Data were compared using Kruskall–Wallis’s test and Dunn’s procedure for the post hoc multicomparison analysis. The Spearman rank order correlation was used to evaluate the relationship between variables and parasitaemia. A P value <0.05 was accepted as statistically significant.
3.
Results
3.1.
Pharmacokinetic study
The concentrations–time profiles and the pharmacokinetic parameters of quinine and 3-hydroxyquinine in whole blood observed after administration of a single 80 mg/kg subcutaneous injection of quinine were displayed in Fig. 1 and Table 1. Quinine and 3-hydroxyquinine concentrations in blood were higher in pregnant than in control mice. During pregnancy, the V/F and the t1/2 values of quinine increased markedly without any significant change in Cl/F. P. berghei malaria decreased the gain of body weight of the dams at day 18. During malaria, the AUC0–6 h of quinine in blood increased and both quinine Cl/F and 3-hydroxyquinine concentrations in blood simultaneously decreased. Tmax values of both quinine and its metabolite in blood increased. No significant change was observed in the V/F of quinine during malaria.
Fig. 1 – Blood concentrations-time profiles for quinine (A) and 3-hydroxyquinine (B) after a single subcutaneous injection of 80 mg/kg of quinine base in control mice (䊉), uninfected pregnant mice () and pregnant mice infected with P. berghei malaria ().
3.2. Quinine distribution in blood and tissues 2 h after a single 80 mg/kg quinine dose The weights of dams increased by 74% during pregnancy and the gain in body weight only decreased for the highest parasitaemia. During pregnancy, both haematocrit and proteinemia decreased. The concentrations of quinine and its metabolite in all blood fractions were higher in pregnant than in control mice. During malaria, haematocrit values decreased in a parasitaemia-dependant manner and proteinemia remained unchanged. Quinine concentrations in erythrocytes increased with the rise of parasitaemia without any change in other blood fractions. Moreover, 3-hydroxyquinine
Table 1 – Characteristics of the three groups of mice and pharmacokinetic parameters of quinine and 3-hydroxyquinine in blood after a single subcutaneous injection of 80 mg/kg of quinine base Control mice (n = 12) Parasitaemia (%) Gain of body weight (g) Haematocrit (%)
– 0.3 ± 0.1 50.6 ± 1.6
Pregnant mice (n = 12)
Pregnant and infected mice (n = 12)
– 18.7 ± 2.3a 43.8 ± 2.0a
35 (31–40) 14.1 ± 3.2a ,b 37.1 ± 3.5a ,b
Quinine Cmax (mg/l) Tmax (h) AUC0–6 h (mg/l/h) Vd/F (l) Cl/F (l/h) t1/2 (h)
9.7 0.5 21.1 190.4 115.7 1.1
± ± ± ± ± ±
2.4 0.3 4.5 35.6 23.9 0.1
11.7 0.4 30.7 275.3 117.5 1.7
± ± ± ± ± ±
1.2a 0.1 3.8a 25.5a 25.3 0.3a
12.0 ± 2.6a 0.9 ± 0.2a ,b 35.7 ± 4.2a ,b 268.6 ± 29.3a 80.4 ± 25.2a ,b 2.1 ± 0.3a ,b
3-Hydroxyquinine Cmax (mg/l) Tmax (h) AUC0–6 h (mg/l/h) t1/2 (h)
6.7 1.1 22.2 1.4
± ± ± ±
1.6 0.24 4.9 0.5
8.1 1.7 33.7 4.8
± ± ± ±
1.4a 0.5a 3.4a 2.7a
4.1 ± 1.6a ,b 2.5 ± 0.6a 17.4 ± 4.5b 6.2 ± 3.4a ,b
Data were expressed as mean ± S.D. except for parasitaemia (median and range). a b
p < 0.05 vs. values of control group. p < 0.05 vs. values of pregnant group.
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Table 2 – Distribution of quinine and 3-hydroxyquinine in blood fractions of control and pregnant mice 2 h after a single subcutaneous injection of 80 mg/kg of quinine base Control mice
Haematocrit (%) Plasma Proteinaemia (g/l) Total quinine (g/ml) Free quinine (g/ml) Free fraction (%) 3-Hydroxyquinine (g/ml)
Pregnant mice (at D18 of pregnancy) Uninfected
Low parasitaemia
High parasitaemia
45 ± 3a
41 ± 3b
32 ± 4b ,c
49 ± 2 59.8 2.6 0.9 0.34 3.3
± ± ± ± ±
3.4 0.8 0.3 0.06 0.7
51.9 3.8 1.7 0.43 4.2
± ± ± ± ±
4.7a 0.8a 0.8a 0.08a 0.8a
49.7 3.4 1.5 0.43 2.3
± ± ± ± ±
5.3 0.9 0.7 0.12 0.4b
53.2 3.2 1.6 0.51 2.0
± ± ± ± ±
4.2 1.0 0.5 0.11 0.6b
Erythrocyte Quinine (g/ml) 3-Hydroxyquinine (g/ml)
4.8 ± 0.9 6.3 ± 1.5
8.9 ± 4.8a 8.9 ± 2.7a
11.6 ± 4.2b 6.5 ± 1.1b
17.8 ± 6.1b ,c 7.6 ± 1.7
Whole blood Quinine (g/ml) 3-Hydroxyquinine (g/ml)
4.5 ± 1.5 5.9 ± 1.5
7.7 ± 4.3a 8.0 ± 2.0a
6.9 ± 2.7 4.1 ± 1.6b
8.6 ± 2.3 3.7 ± 1.0b
Pregnant mice were on day 18 of pregnancy with or without P. berghei infection. The median (range) of parasitaemia was 12% (5–17) and 44% (37–48) in groups with low and high parasitaemia, respectively. a b c
p < 0.05 vs. values of control group. p < 0.05 vs. values of pregnant group. p < 0.05 vs. values of pregnant group with low parasitaemia.
concentrations in whole blood and plasma decreased in infected mice (Tables 2 and 3). Quinine concentrations only increased in the heart, kidney and lung of pregnant mice. During malaria, quinine concentra-
tions in the heart increased gradually with the parasitaemia without any change in other tissues. In infected groups, 3hydroxyquinine concentrations decreased in all tissues with the rising parasitaemia.
Table 3 – Distribution of quinine in tissues of control and pregnant mice 2 h after a single subcutaneous injection of 80 mg/kg of quinine base Control mice
Pregnant mice (at D18 of pregnancy) Uninfected
Low parasitaemia
High parasitaemia
Brain Quinine (g/g) 3-Hydroxyquinine (g/g)
1.1 ± 0.4 0.3 ± 0.1
1.4 ± 0.4 0.5 ± 0.1a
1.6 ± 0.5 0.3 ± 0.1b
2.1 ± 0.6a 0.3 ± 0.2
Liver Quinine (g/g) 3-Hydroxyquinine (g/g)
14.1 ± 5.6 19.7 ± 5
11.4 ± 4.7 14.7 ± 4.6a
12.3 ± 3.5 10.3 ± 2.1b
9.7 ± 2.9 8.3 ± 1.9b
Spleen Quinine (g/g) 3-Hydroxyquinine (g/g)
99 ± 59 28 ± 10.1
105 ± 34 34.5 ± 12.4a
106 ± 45 23.8 ± 8.4b
113 ± 25 12.9 ± 6.5b ,c
Heart Quinine (g/g) 3-Hydroxyquinine (g/g)
9.8 ± 3.5 6.1 ± 1.4
13.9 ± 3.9a 8.3 ± 1.5a
19.0 ± 5.7a , b 6.9 ± 1.2b
22.9 ± 5.3a ,b ,c 6.2 ± 1.5b
Kidney Quinine (g/g) 3-Hydroxyquinine (g/g)
75 ± 20 35 ± 8.2
124 ± 32a 54.3 ± 12.1a
146 ± 51a 48.2 ± 13.4
132 ± 30a 37.4 ± 13.4b ,c
Lung Quinine (g/g) 3-Hydroxyquinine (g/g)
115 ± 29 41.6 ± 9.3
145 ± 34a 55.2 ± 10.5a
158 ± 43a 41.8 ± 9.0b
167 ± 38a 31.4 ± 8.2b ,c
Pregnant mice were on day 18 of pregnancy with or without P. berghei infection. The median (range) of parasitaemia was 12% (5–17) and 44% (37–48) in groups with low and high parasitaemia, respectively. a b c
p < 0.05 vs. values of control group. p < 0.05 vs. values of pregnant group. p < 0.05 vs. values of pregnant group with low parasitaemia.
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Table 4 – Distribution of quinine and its 3-hydroxy metabolite in the foeto-placental unit of pregnant mice 2 h after a single subcutaneous injection of 80 mg/kg of quinine base Pregnant mice (at D18 of pregnancy) Uninfected
Low parasitaemia
Gain of body weight in Dams (g)
20.1 ± 2.4
19.4 ± 2.7
15.9 ± 3.2b ,c
Placenta Weight (mg) Quinine (g/g) 3-Hydroxyquinine (g/g)
114 ± 6 72 ± 6 30.6 ± 5.1
105 ± 5a 57 ± 11a 20.7 ± 3.2a
93 ± 8a ,b 44 ± 11a ,b 14.8 ± 5.4a ,b
Foetus Weight (mg) Quinine (g/g) 3-Hydroxyquinine (g/g)
1019 ± 166 9.1 ± 2.8 6.2 ± 1.3
980 ± 188 9.3 ± 2.9 4.0 ± 0.7a
641 ± 157a ,b 5.7 ± 3.2a ,b 2.1 ± 0.7a ,b
6.7 ± 2.3 4.1 ± 1.5
4.5 ± 1.6a 1.2 ± 0.5a
4.9 ± 2.1a 2.0 ± 0.6a
Amniotic fluid Quinine (g/ml) 3-Hydroxyquinine (g/ml)
High parasitaemia
Pregnant mice were on day 18 of pregnancy with or without P. berghei infection. The median (range) of parasitaemia was 12% (5–17) group and 44% (37–48) in groups with low and high parasitaemia, respectively. a b
p < 0.05 vs. values of pregnant group. p < 0.05 vs. values of group with low parasitaemia.
3.3.
Quinine distribution in the foeto-placental unit
A negative relationship was observed between the parasitaemia and placenta weight (rs : −0.527, P = 0.005, n = 24). The weights of foetus only decreased in highly infected mice. Quinine concentrations in placenta decreased gradually with the rise of the parasitaemia. A positive relationship was observed between the quinine concentrations and the weight of placenta (rs : 0.498, P = 0.001, n = 24). In foetus, quinine concentrations only decreased for the highest parasitaemia. In the amniotic fluid, quinine concentrations decreased in both groups of parasitized mice. The ratios of foetal to dams whole blood concentrations for quinine were similar in uninfected mice (1.38 ± 0.10) and mice with low parasitaemia (1.20 ± 0.17), but decreased in highly parasitized mice (1.03 ± 0.15, p < 0.05). In all compartments, 3-hydroxyquinine concentrations decreased in infected mice (Table 4).
4.
Discussion
In this study, we reported the effects of the physiological changes that occur at the end of pregnancy on the processes of quinine distribution in mice. As it is done during human treatment, the dose of quinine was adapted to the 60%-increase of body weight of the dam. The distribution volume of quinine increased by only 45% leading to a simultaneous rise of quinine blood concentrations. As a result of plasma volume expansion, both haematocrit and total protein concentrations decreased. The overall effect of this physiological hemodilution is a reduction of quinine binding capacity and, therefore, an increase in free quinine concentrations in plasma. That promotes the uptake of quinine by erythrocyte and most of the tissues. Despite of this increase in free quinine concentrations, the elimination processes of quinine remained unchanged during late pregnancy, as suggested by the similar total clearance of the drug and the comparable quinine-to-metabolite
concentrations ratio in both pregnant and control mice. Nevertheless, quinine uptake by the foetus was much lower than by the placenta and other tissues of the dam. The exposure of the foetus to the drug is limited by a large foeto-placental gradient of about 8:1, similar to that previously reported in the near-term pregnant ewe (Czuba et al., 1991). Despite of anatomical differences, the human fetal placenta is functionally analogous to the murine labyrinth where fetal and maternal blood circulate in close association for physiological exchange (Georgiades et al., 2002). Moreover, trophoblasts of both human and mice placentas expressed transporters like P-glycoprotein that have been shown to be involved in drug efflux and fetal protection from xenobiotics (Audus et al., 2002). During malaria, the metabolic pathway to the 3hydroxymetabolite and the systemic clearance of quinine decrease gradually with the severity of the disease. Although pregnancy and/or malaria may alter Cl/F through out changes in bioavailability of subcutaneous route, this decrease is in agreement with previous clinical and experimental studies showing that malaria impaired the hepatic drug metabolism (Pukrittayakamee et al., 1997; Pussard et al., 2003; Krishna and White, 1996). Moreover, severe P. berghei malaria does not reduce the distribution volume of quinine during pregnancy, in contrast with previous studies in non-pregnant mice. Although the study design cannot allow to evaluate drug uptake and clearance from tissues, quinine concentrations increase slightly in whole blood and in most of the tissues, more specifically in the heart and erythrocytes. Contrasting with these tissues, the foeto-placental unit does not accumulate quinine during malaria. A gradual reduction of the weight of placentas was observed with the rising parasitaemia. During malaria, the placenta is a favoured site for parasites sequestration because of the large sinusoids and the slow blood circulation (Andrews and Lanzer, 2002). Similar histological and ultrastructural alterations have been reported in rodent placentas heavily infected by P. berghei and during
european journal of pharmaceutical sciences
acute P. falciparum malaria in human (Galbraith et al., 1980; Oduola et al., 1982; McGready et al., 2004). Accumulation of parasitized erythrocytes, mononuclear cells and malarial pigment in intervillous spaces and increase of the thickness of the trophoblast basement membrane were observed. This malaria-induced atrophy of placenta is strongly related with a decrease in quinine concentrations. The detrimental consequences of placental parasitization on the growing foetus in utero are characterized by a reduction in foetal weight in highly parasitized mice. Similar growth-retarding effects were previously reported in both rodent and human malaria (Hioki et al., 1990; McGready et al., 1998; Nosten et al., 2004). P. berghei malaria decreased simultaneously quinine and 3hydroxyquinine concentrations in placenta, foetus and amniotic fluid. Moreover, the metabolite-to-quinine concentrations ratios remained similar in the compartments of the foetoplacental unit suggesting that this low quinine uptake is more related to an impairment of drug distribution to the foetoplacental unit than an alteration of the foetal metabolism. During P. falciparum malaria, impaired uteroplacental blood flow is a predictive parameter of the poor perinatal outcome including low birth weight, preterm delivery and perinatal death (Dorman et al., 2002). Reduction of uterine blood flow would first affect the placenta that afterwards undergoes adaptation by decreasing its size. In anaemic dams with placentas affected by malaria, reductions in maternal placental blood flow are likely to jeopardise further the oxygen, nutrient like glucose and may also contribute to the decrease of quinine uptake by fetal tissues. On the other hand, modulation of placental transporters by steroids hormones of pregnancy, inflammatory mediators or quinine itself may be also involved. During pregnancy, quinine concentrations increased in the target erythrocyte and no improvement of the therapeutic regimens appeared to be necessary. Although conclusions drawn from experiments in rodents should only be applied with caution to human malaria infections, the impairment of quinine distribution to the parasitized placenta, if it will be confirm in human, may favour the occurrence of treatment failure with the persistence of parasites even after their clearance from the peripheral blood. Combination of quinine with other drugs may be required to enhance the antimalarial activity in the placenta.
Acknowledgments ´ for their techWe are grateful to Drs. E. Fouquet and P. Bouree nical assistance and helpful discussions.
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Quinine distribution in pregnant mice with Plasmodium berghei malaria Fr´ed´eric Lirussi, Eric Pussard ∗ Hopital de Bicetre, Department de Pharmacology, 94275 Le Kremlin Bicetre, France
a r t i c l e
i n f o
a b s t r a c t
Article history:
Maternal malaria is associated with placental insufficiency that leads to intrauterine growth
Received 16 August 2005
retardation and reduced birth weight. Malaria may impair the exchange of drugs across the
Received in revised form
placenta especially the transmission of antimalarial drugs to the foetus.
16 January 2006
The distribution of quinine and its 3-hydroxymetabolite in blood, tissues and foeto-placental
Accepted 13 March 2006
unit was evaluated on day 18 of pregnancy of mice infected or not with Plasmodium berghei.
Published on line 22 March 2006
During pregnancy, quinine distribution volume increases gradually with the rise of free quinine concentrations in plasma. Quinine concentrations increase in erythrocytes and most
Keywords:
tissues without change in systemic clearance. A maternal-to-foetal gradient of 8:1 limits the
Pregnancy
exposure of foetus to quinine.
Quinine
During malaria, the systemic clearance of quinine and the 3-hydroxyquinine gradually
Placenta
decrease with the rising parasitaemia. Quinine concentrations increase slightly in most
Foetus
of the tissues. The weight of placentas decreases in a parasitaemia-dependant manner and
Mice
is strongly related to the low uptake of quinine by placenta. Foetal weights and quinine
Plasmodium berghei malaria
concentrations in foetus only decrease for the highest parasitaemia. In this experimental model, pregnancy facilitates quinine uptake by erythrocytes and peripheral tissues. Malaria induces a hypotrophy of both placenta and foetus. In placenta, the marked decrease of quinine concentrations may impair the clearance of sequestered parasites. © 2006 Elsevier B.V. All rights reserved.
1.
Introduction
Plasmodium falciparum infections during pregnancy frequently led to maternal anaemia, abortions, stillbirths, low birth weights and increased infant mortality (McGready et al., 1998; Nosten et al., 2004). Similar observations have been made in rodent pregnancy complicated by Plasmodium berghei malaria (Hioki et al., 1990; Oduola et al., 1982). The pathologic changes in the infected rodent placenta were quite similar to those reported in humans with thickening of the trophoblast and accumulation of parasitized erythrocytes (Andrews and Lanzer, 2002; Galbraith et al., 1980; Oduola et al., 1986; Tegoshi
∗
Corresponding author. Tel.: +33 145213580; fax: +33 145213591. E-mail address: [email protected] (E. Pussard).
0928-0987/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2006.03.004
et al., 1992). While intact malaria parasites do not normally pass the placenta, they may nonetheless damage this organ and jeopardize the materno-foetal relationship. Thus, pregnancy like malaria may affect both the distribution of drugs in the dams and the exchange across the placenta to the foetus. Quinine, alone or in combination represents the most frequently available treatment of severe malaria in Africa, especially in pregnant women close to delivery, because there is often no other effective drug available (Nosten et al., 2004). Nevertheless, the persistence of Plasmodium falciparum in the placenta has been described after apparently effective quinine therapy and that might suggest a decrease of drug efficacy
e u r o p e a n j o u r n a l o f p h a r m a c e u t i c a l s c i e n c e s 2 8 ( 2 0 0 6 ) 284–290
(Menendez et al., 2000; Procop et al., 2001). Quinine pharmacokinetics has been described in the healthy ovine foetus (Czuba et al., 1991) and in pregnant women with severe falciparum malaria (Looareesuwan et al., 1985; Phillips et al., 1986). Nevertheless, the extent of foetal exposure to quinine administered to the mother during malaria is not defined. The aim of this study was to determine whether malaria influences the disposition of quinine during pregnancy in a model of P. berghei-infected mice.
2.
Materials and methods
2.1.
Animals
All experiments were performed according to the official regulations of the French Ministry of Agriculture and were in conformity with the Guiding principles in the use of animals in toxicology adopted by the Society of Toxicology (USA) in July 1989 and revised in 1999. The present study was approved by the animal care committee of our institution (Paris XI University). Twelve-week-old female Swiss OF1 mice (weight 28–30 g) were obtained from Iffa Credo (L’Arbresle, France). Animals were housed 6 per cage, fed ad libitum with standard diet and had free access to tap water. Room temperature was maintained at 21 ± 1 ◦ C, and a 12-h artificial light/dark cycle was programmed. For the gestation of animals, three female were housed with one male and checked for vaginal plug twice a day. The day on which the vaginal plug was determined was classified as day 0 of gestation. Pregnant mice were then placed in separate cages.
2.2.
Experimental infections
The NK65 strain of P. berghei was kindly supplied by Dr. I. Landau (Museum National d’Histoire Naturelle, Paris, France). On day 13 of the gestation period, pregnant mice were infected by intraperitoneal injection of either 104 or 106 parasitized erythrocytes in 100 l of heparinized fresh blood from previously infected mice. Three days after infection, thin smears were made daily from tail blood, stained with Giemsa and observed by light microscopy (oil immersion, magnitude, 1000×).
2.3.
Animal experiments and quinine administration
Non-pregnant control mice, uninfected pregnant mice and pregnant mice with low or high parasitaemia were injected subcutaneously with a 0.1 ml-single dose of quinine saline solution (Quinine hydrochloride, Sigma, France) at a dose of 80 mg/kg of quinine base. Drug administration was made on day 18 of gestation.
5 and 6 h post-injection. Samples were stored at −20 ◦ C until analysis.
2.3.2.
Experiment 2
Quinine distribution in blood and tissues was evaluated in uninfected non-pregnant control (n = 12), in uninfected pregnant mice (n = 12) and in pregnant mice with low [parasitaemia: median (range): 12% (5–17%), n = 12] or high parasitaemia values [parasitaemia: median (range): 44% (37–48%), n = 12]. 2 h after a subcutaneous injection of 80 mg of quinine base per kg of body weight, mice were anaesthetised with 60 mg/kg pentobarbital. About one ml of blood was taken through cardiac puncture. Two aliquots of blood were saved for haematocrit (heparinized capillaries 9 l, Bayer Diagnostics, Bridgend, UK) and quinine whole blood determinations (30 l). After centrifugation at 1500 × g for 15 min, plasma and erythrocytes were separated. Two aliquots of plasma (for total and free quinine measurements), the whole blood and erythrocyte fractions were frozen at −20 ◦ C. Total proteins concentrations in plasma were assayed by the method described by Bradford (1976). The maternal brain, lungs, heart, liver, spleen and kidneys were removed, washed in saline solution (0.9% NaCl) and dried with absorbent paper. Tissues were weighted accurately and stored at −20 ◦ C. The uterine horns were exteriorized and all foetuses and corresponding placenta were removed and weighted. After the amniotic fluid was aspirated, two foetuses and corresponding placentas of each horn were immediately frozen on dry ice.
2.4.
Quinine analysis
After thawing, the tissues were homogenized in 1–3 ml of cold 0.05 N HCl and sonicated on ice with an ultrasonic probe for 1 min. The homogenate was then centrifuged and 25 l of supernatant was used for analysis. Free quinine concentrations were measured with a protein-free ultra filtrate obtained by centrifugation of a 500 l-plasma sample at 2000 × g for 20 min at room temperature (Amicon MPS-1 micro partition system with YMT membranes) (Pussard et al., 1999). Quinine and 3-hydroxyquinine concentrations in blood fractions and tissues homogenates were determined by a high-performance liquid chromatographic method with fluorometric detection as previously described (Pussard et al., 2003). Quinidine was used as internal standard. Standard calibration curves for quinine and 3-hydroxy quinine were prepared in drug-free medium (whole blood, erythrocyte, plasma or supernatant of tissue homogenate fluid) and were linear in the range of concentrations 0.1–100 g/ml. Quinine and 3-hydroxyquinine recoveries were more than 80%. Within-run and betweenrun variabilities were lower than 10% in each biological matrix.
2.5. 2.3.1.
285
Pharmacokinetics analysis
Experiment 1
Quinine pharmacokinetics was evaluated in uninfected nonpregnant mice (n = 12), in pregnant uninfected (n = 12) and in P. berghei-infected pregnant mice [parasitaemia: median (range) 35% (31–40%), n = 12]. Whole blood samples (30 l) were taken from two drops of caudal vein blood and vortexed immediately with solid EDTA. Sampling times were at 0.25, 0.5, 1, 1.5, 2, 3, 4,
Whole blood quinine concentrations were fitted using a 1compartment model. The total area under the whole blood concentration–time curve from time 0 to 6 h (AUC0–6 h ) was calculated by linear trapezoidal rule. Clearance (Cl/F) was determined by dose/AUC0–∞ and volume of distribution (V/F) by dose/(z × AUC0–∞ ) where z is the first order rate constant
286
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associated with the terminal portion of the curve. The elimination half-life (t1/2 ) was calculated as ln(2)/z . The maximum concentrations of quinine and its 3-hydroxymetabolite in whole blood (Cmax ) and the time to reach Cmax (Tmax ) were observed values.
2.6.
Statistical analysis
Data were reported as means ± standard deviations except parasitaemia expressed as median and range. Bartlett’s test was used to verify the homogeneity of variances. Data were compared using Kruskall–Wallis’s test and Dunn’s procedure for the post hoc multicomparison analysis. The Spearman rank order correlation was used to evaluate the relationship between variables and parasitaemia. A P value <0.05 was accepted as statistically significant.
3.
Results
3.1.
Pharmacokinetic study
The concentrations–time profiles and the pharmacokinetic parameters of quinine and 3-hydroxyquinine in whole blood observed after administration of a single 80 mg/kg subcutaneous injection of quinine were displayed in Fig. 1 and Table 1. Quinine and 3-hydroxyquinine concentrations in blood were higher in pregnant than in control mice. During pregnancy, the V/F and the t1/2 values of quinine increased markedly without any significant change in Cl/F. P. berghei malaria decreased the gain of body weight of the dams at day 18. During malaria, the AUC0–6 h of quinine in blood increased and both quinine Cl/F and 3-hydroxyquinine concentrations in blood simultaneously decreased. Tmax values of both quinine and its metabolite in blood increased. No significant change was observed in the V/F of quinine during malaria.
Fig. 1 – Blood concentrations-time profiles for quinine (A) and 3-hydroxyquinine (B) after a single subcutaneous injection of 80 mg/kg of quinine base in control mice (䊉), uninfected pregnant mice () and pregnant mice infected with P. berghei malaria ().
3.2. Quinine distribution in blood and tissues 2 h after a single 80 mg/kg quinine dose The weights of dams increased by 74% during pregnancy and the gain in body weight only decreased for the highest parasitaemia. During pregnancy, both haematocrit and proteinemia decreased. The concentrations of quinine and its metabolite in all blood fractions were higher in pregnant than in control mice. During malaria, haematocrit values decreased in a parasitaemia-dependant manner and proteinemia remained unchanged. Quinine concentrations in erythrocytes increased with the rise of parasitaemia without any change in other blood fractions. Moreover, 3-hydroxyquinine
Table 1 – Characteristics of the three groups of mice and pharmacokinetic parameters of quinine and 3-hydroxyquinine in blood after a single subcutaneous injection of 80 mg/kg of quinine base Control mice (n = 12) Parasitaemia (%) Gain of body weight (g) Haematocrit (%)
– 0.3 ± 0.1 50.6 ± 1.6
Pregnant mice (n = 12)
Pregnant and infected mice (n = 12)
– 18.7 ± 2.3a 43.8 ± 2.0a
35 (31–40) 14.1 ± 3.2a ,b 37.1 ± 3.5a ,b
Quinine Cmax (mg/l) Tmax (h) AUC0–6 h (mg/l/h) Vd/F (l) Cl/F (l/h) t1/2 (h)
9.7 0.5 21.1 190.4 115.7 1.1
± ± ± ± ± ±
2.4 0.3 4.5 35.6 23.9 0.1
11.7 0.4 30.7 275.3 117.5 1.7
± ± ± ± ± ±
1.2a 0.1 3.8a 25.5a 25.3 0.3a
12.0 ± 2.6a 0.9 ± 0.2a ,b 35.7 ± 4.2a ,b 268.6 ± 29.3a 80.4 ± 25.2a ,b 2.1 ± 0.3a ,b
3-Hydroxyquinine Cmax (mg/l) Tmax (h) AUC0–6 h (mg/l/h) t1/2 (h)
6.7 1.1 22.2 1.4
± ± ± ±
1.6 0.24 4.9 0.5
8.1 1.7 33.7 4.8
± ± ± ±
1.4a 0.5a 3.4a 2.7a
4.1 ± 1.6a ,b 2.5 ± 0.6a 17.4 ± 4.5b 6.2 ± 3.4a ,b
Data were expressed as mean ± S.D. except for parasitaemia (median and range). a b
p < 0.05 vs. values of control group. p < 0.05 vs. values of pregnant group.
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Table 2 – Distribution of quinine and 3-hydroxyquinine in blood fractions of control and pregnant mice 2 h after a single subcutaneous injection of 80 mg/kg of quinine base Control mice
Haematocrit (%) Plasma Proteinaemia (g/l) Total quinine (g/ml) Free quinine (g/ml) Free fraction (%) 3-Hydroxyquinine (g/ml)
Pregnant mice (at D18 of pregnancy) Uninfected
Low parasitaemia
High parasitaemia
45 ± 3a
41 ± 3b
32 ± 4b ,c
49 ± 2 59.8 2.6 0.9 0.34 3.3
± ± ± ± ±
3.4 0.8 0.3 0.06 0.7
51.9 3.8 1.7 0.43 4.2
± ± ± ± ±
4.7a 0.8a 0.8a 0.08a 0.8a
49.7 3.4 1.5 0.43 2.3
± ± ± ± ±
5.3 0.9 0.7 0.12 0.4b
53.2 3.2 1.6 0.51 2.0
± ± ± ± ±
4.2 1.0 0.5 0.11 0.6b
Erythrocyte Quinine (g/ml) 3-Hydroxyquinine (g/ml)
4.8 ± 0.9 6.3 ± 1.5
8.9 ± 4.8a 8.9 ± 2.7a
11.6 ± 4.2b 6.5 ± 1.1b
17.8 ± 6.1b ,c 7.6 ± 1.7
Whole blood Quinine (g/ml) 3-Hydroxyquinine (g/ml)
4.5 ± 1.5 5.9 ± 1.5
7.7 ± 4.3a 8.0 ± 2.0a
6.9 ± 2.7 4.1 ± 1.6b
8.6 ± 2.3 3.7 ± 1.0b
Pregnant mice were on day 18 of pregnancy with or without P. berghei infection. The median (range) of parasitaemia was 12% (5–17) and 44% (37–48) in groups with low and high parasitaemia, respectively. a b c
p < 0.05 vs. values of control group. p < 0.05 vs. values of pregnant group. p < 0.05 vs. values of pregnant group with low parasitaemia.
concentrations in whole blood and plasma decreased in infected mice (Tables 2 and 3). Quinine concentrations only increased in the heart, kidney and lung of pregnant mice. During malaria, quinine concentra-
tions in the heart increased gradually with the parasitaemia without any change in other tissues. In infected groups, 3hydroxyquinine concentrations decreased in all tissues with the rising parasitaemia.
Table 3 – Distribution of quinine in tissues of control and pregnant mice 2 h after a single subcutaneous injection of 80 mg/kg of quinine base Control mice
Pregnant mice (at D18 of pregnancy) Uninfected
Low parasitaemia
High parasitaemia
Brain Quinine (g/g) 3-Hydroxyquinine (g/g)
1.1 ± 0.4 0.3 ± 0.1
1.4 ± 0.4 0.5 ± 0.1a
1.6 ± 0.5 0.3 ± 0.1b
2.1 ± 0.6a 0.3 ± 0.2
Liver Quinine (g/g) 3-Hydroxyquinine (g/g)
14.1 ± 5.6 19.7 ± 5
11.4 ± 4.7 14.7 ± 4.6a
12.3 ± 3.5 10.3 ± 2.1b
9.7 ± 2.9 8.3 ± 1.9b
Spleen Quinine (g/g) 3-Hydroxyquinine (g/g)
99 ± 59 28 ± 10.1
105 ± 34 34.5 ± 12.4a
106 ± 45 23.8 ± 8.4b
113 ± 25 12.9 ± 6.5b ,c
Heart Quinine (g/g) 3-Hydroxyquinine (g/g)
9.8 ± 3.5 6.1 ± 1.4
13.9 ± 3.9a 8.3 ± 1.5a
19.0 ± 5.7a , b 6.9 ± 1.2b
22.9 ± 5.3a ,b ,c 6.2 ± 1.5b
Kidney Quinine (g/g) 3-Hydroxyquinine (g/g)
75 ± 20 35 ± 8.2
124 ± 32a 54.3 ± 12.1a
146 ± 51a 48.2 ± 13.4
132 ± 30a 37.4 ± 13.4b ,c
Lung Quinine (g/g) 3-Hydroxyquinine (g/g)
115 ± 29 41.6 ± 9.3
145 ± 34a 55.2 ± 10.5a
158 ± 43a 41.8 ± 9.0b
167 ± 38a 31.4 ± 8.2b ,c
Pregnant mice were on day 18 of pregnancy with or without P. berghei infection. The median (range) of parasitaemia was 12% (5–17) and 44% (37–48) in groups with low and high parasitaemia, respectively. a b c
p < 0.05 vs. values of control group. p < 0.05 vs. values of pregnant group. p < 0.05 vs. values of pregnant group with low parasitaemia.
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Table 4 – Distribution of quinine and its 3-hydroxy metabolite in the foeto-placental unit of pregnant mice 2 h after a single subcutaneous injection of 80 mg/kg of quinine base Pregnant mice (at D18 of pregnancy) Uninfected
Low parasitaemia
Gain of body weight in Dams (g)
20.1 ± 2.4
19.4 ± 2.7
15.9 ± 3.2b ,c
Placenta Weight (mg) Quinine (g/g) 3-Hydroxyquinine (g/g)
114 ± 6 72 ± 6 30.6 ± 5.1
105 ± 5a 57 ± 11a 20.7 ± 3.2a
93 ± 8a ,b 44 ± 11a ,b 14.8 ± 5.4a ,b
Foetus Weight (mg) Quinine (g/g) 3-Hydroxyquinine (g/g)
1019 ± 166 9.1 ± 2.8 6.2 ± 1.3
980 ± 188 9.3 ± 2.9 4.0 ± 0.7a
641 ± 157a ,b 5.7 ± 3.2a ,b 2.1 ± 0.7a ,b
6.7 ± 2.3 4.1 ± 1.5
4.5 ± 1.6a 1.2 ± 0.5a
4.9 ± 2.1a 2.0 ± 0.6a
Amniotic fluid Quinine (g/ml) 3-Hydroxyquinine (g/ml)
High parasitaemia
Pregnant mice were on day 18 of pregnancy with or without P. berghei infection. The median (range) of parasitaemia was 12% (5–17) group and 44% (37–48) in groups with low and high parasitaemia, respectively. a b
p < 0.05 vs. values of pregnant group. p < 0.05 vs. values of group with low parasitaemia.
3.3.
Quinine distribution in the foeto-placental unit
A negative relationship was observed between the parasitaemia and placenta weight (rs : −0.527, P = 0.005, n = 24). The weights of foetus only decreased in highly infected mice. Quinine concentrations in placenta decreased gradually with the rise of the parasitaemia. A positive relationship was observed between the quinine concentrations and the weight of placenta (rs : 0.498, P = 0.001, n = 24). In foetus, quinine concentrations only decreased for the highest parasitaemia. In the amniotic fluid, quinine concentrations decreased in both groups of parasitized mice. The ratios of foetal to dams whole blood concentrations for quinine were similar in uninfected mice (1.38 ± 0.10) and mice with low parasitaemia (1.20 ± 0.17), but decreased in highly parasitized mice (1.03 ± 0.15, p < 0.05). In all compartments, 3-hydroxyquinine concentrations decreased in infected mice (Table 4).
4.
Discussion
In this study, we reported the effects of the physiological changes that occur at the end of pregnancy on the processes of quinine distribution in mice. As it is done during human treatment, the dose of quinine was adapted to the 60%-increase of body weight of the dam. The distribution volume of quinine increased by only 45% leading to a simultaneous rise of quinine blood concentrations. As a result of plasma volume expansion, both haematocrit and total protein concentrations decreased. The overall effect of this physiological hemodilution is a reduction of quinine binding capacity and, therefore, an increase in free quinine concentrations in plasma. That promotes the uptake of quinine by erythrocyte and most of the tissues. Despite of this increase in free quinine concentrations, the elimination processes of quinine remained unchanged during late pregnancy, as suggested by the similar total clearance of the drug and the comparable quinine-to-metabolite
concentrations ratio in both pregnant and control mice. Nevertheless, quinine uptake by the foetus was much lower than by the placenta and other tissues of the dam. The exposure of the foetus to the drug is limited by a large foeto-placental gradient of about 8:1, similar to that previously reported in the near-term pregnant ewe (Czuba et al., 1991). Despite of anatomical differences, the human fetal placenta is functionally analogous to the murine labyrinth where fetal and maternal blood circulate in close association for physiological exchange (Georgiades et al., 2002). Moreover, trophoblasts of both human and mice placentas expressed transporters like P-glycoprotein that have been shown to be involved in drug efflux and fetal protection from xenobiotics (Audus et al., 2002). During malaria, the metabolic pathway to the 3hydroxymetabolite and the systemic clearance of quinine decrease gradually with the severity of the disease. Although pregnancy and/or malaria may alter Cl/F through out changes in bioavailability of subcutaneous route, this decrease is in agreement with previous clinical and experimental studies showing that malaria impaired the hepatic drug metabolism (Pukrittayakamee et al., 1997; Pussard et al., 2003; Krishna and White, 1996). Moreover, severe P. berghei malaria does not reduce the distribution volume of quinine during pregnancy, in contrast with previous studies in non-pregnant mice. Although the study design cannot allow to evaluate drug uptake and clearance from tissues, quinine concentrations increase slightly in whole blood and in most of the tissues, more specifically in the heart and erythrocytes. Contrasting with these tissues, the foeto-placental unit does not accumulate quinine during malaria. A gradual reduction of the weight of placentas was observed with the rising parasitaemia. During malaria, the placenta is a favoured site for parasites sequestration because of the large sinusoids and the slow blood circulation (Andrews and Lanzer, 2002). Similar histological and ultrastructural alterations have been reported in rodent placentas heavily infected by P. berghei and during
european journal of pharmaceutical sciences
acute P. falciparum malaria in human (Galbraith et al., 1980; Oduola et al., 1982; McGready et al., 2004). Accumulation of parasitized erythrocytes, mononuclear cells and malarial pigment in intervillous spaces and increase of the thickness of the trophoblast basement membrane were observed. This malaria-induced atrophy of placenta is strongly related with a decrease in quinine concentrations. The detrimental consequences of placental parasitization on the growing foetus in utero are characterized by a reduction in foetal weight in highly parasitized mice. Similar growth-retarding effects were previously reported in both rodent and human malaria (Hioki et al., 1990; McGready et al., 1998; Nosten et al., 2004). P. berghei malaria decreased simultaneously quinine and 3hydroxyquinine concentrations in placenta, foetus and amniotic fluid. Moreover, the metabolite-to-quinine concentrations ratios remained similar in the compartments of the foetoplacental unit suggesting that this low quinine uptake is more related to an impairment of drug distribution to the foetoplacental unit than an alteration of the foetal metabolism. During P. falciparum malaria, impaired uteroplacental blood flow is a predictive parameter of the poor perinatal outcome including low birth weight, preterm delivery and perinatal death (Dorman et al., 2002). Reduction of uterine blood flow would first affect the placenta that afterwards undergoes adaptation by decreasing its size. In anaemic dams with placentas affected by malaria, reductions in maternal placental blood flow are likely to jeopardise further the oxygen, nutrient like glucose and may also contribute to the decrease of quinine uptake by fetal tissues. On the other hand, modulation of placental transporters by steroids hormones of pregnancy, inflammatory mediators or quinine itself may be also involved. During pregnancy, quinine concentrations increased in the target erythrocyte and no improvement of the therapeutic regimens appeared to be necessary. Although conclusions drawn from experiments in rodents should only be applied with caution to human malaria infections, the impairment of quinine distribution to the parasitized placenta, if it will be confirm in human, may favour the occurrence of treatment failure with the persistence of parasites even after their clearance from the peripheral blood. Combination of quinine with other drugs may be required to enhance the antimalarial activity in the placenta.
Acknowledgments ´ for their techWe are grateful to Drs. E. Fouquet and P. Bouree nical assistance and helpful discussions.
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