Chloroquine accumulates in breast-milk cells: potential impact in the prophylaxis of postnatal mother-to-child transmission of HIV-1 (2001)

Each day, an estimated 1600 infants acquire HIV-1, mainly by mother-to-child transmission (MTCT) in resource-poor countries, where breastfeeding is customary and is essential in preventing infant morbidity/mortality from infectious diseases and malnutrition [1–7]. However, breastfeeding accounts for one-third to one-half of HIV-1 MTCT from breastfeeding mothers [2,3,8]. Several approaches to prevent breastfeeding-related MTCT have been proposed, alone or in combination, mainly: (i) modified feeding practices, such as formula feeding or exclusive breastfeeding with abrupt weaning after 3–6 months; (ii) maternal or child antiretroviral prophylaxis [2,3,5–7]. Here, we examined to what extent chloroquine, which has anti-HIV activity [9–13], accumulates in breast-milk cells, where it may become available for inhibiting HIV replication. We studied chloroquine accumulation in cells most relevant to HIV-1 replication in the breast, i.e. macrophages and possibly mammary epithelial cells [14,15]. The following cell lines were used: macrophage-like THP-1 (ECACC 88081201), mammary epithelial MEC-1, isolated from women undergoing reductive mammoplasty [14] and fibroblast MRC-5 (ECACC 97112601). Moreover, cells were isolated from human colostrum (first post-partum week); milk was mixed with phosphate-buffered saline (PBS) and iced; cells were pelletted, allowing the recovery of approximately 106 cells/ml, and resuspended in RPMI-1640 plus fetal bovine serum, amphotericin B, vancomycin and gentamicin. A total of 103 cells/cm2 were cultivated in six-well microplates containing basal defined medium [16], fetal bovine serum and insulin. After one week, adhering cells were incubated for 4 h in 1 ml serum-free basal defined medium containing 1 μM chloroquine, rinsed with ice-cold PBS and detached in hydrochloric acid. Chloroquine and protein were assayed, the latter with bovine serum albumin as standard. Chloroquine was assayed by a modified, previously reported high performance liquid chromatography method [17]. After 4 h incubation with 1 μM chloroquine, THP-1 cells accumulated 2.43 ± 0.05, MEC-1 cells 0.85 ± 0.06, and MRC-5 fibroblasts (positive control) 3.08 ± 0.16 pM chloroquine/μg cell protein. Similarly, colostrum cells accumulated 2.99 ± 0.15 pM/μg cell protein. Assuming a cell volume : cell protein ratio of 3.66 μl/mg cell protein [18], intracellular : extracellular chloroquine concentration ratios at 4 h were: 664 (THP-1), 231 (MEC-1), 841 (MRC-5) and 817 (colostrum cells). In order to confirm in vivo these results obtained in vitro, 20 Burkinabè mothers of unknown HIV status taking chloroquine as antimalarial prophylaxis (300 mg/week) during pregnancy volunteered to modify their chloroquine scheme to 100 mg/day during the last 10 days of pregnancy and first 10 days postpartum. Within the latter period, for three consecutive days they provided one milk sample, which was collected at the end of suckling, stored at 4°C for 2 h and separated into three fractions: full milk, cell-rich and cell-poor fractions obtained by centrifugation. Cell-rich fractions were washed in PBS. One full blood sample was taken over 12 h after chloroquine intake and stored at −20°C. This study was approved by the institutional ethical committees at Brugge and Bobo-Dioulasso. Table 1 shows the chloroquine concentrations obtained. The cell-poor milk and blood concentrations were similar. The cell-rich milk fractions contained higher concentrations, with a high variability, probably caused by a lack of homogeneity of several samples. Colostrum contained a mean of 50% macrophages (measured diameter 30 μm, calculated volume 14.1 pl) and also smaller cells such as polymorphonuclear granulocytes (diameter 15 μm, calculated volume 1.8 pl). Taking these cell distribution and volumes into account, the median chloroquine concentration in colostrum cells was 253 μM, being 243-fold higher than the blood concentration.Table 1: In-vivo concentration of chloroquine in human blood, colostrum and colostrum cells. Chloroquine and hydroxychloroquine are endowed with anti-HIV-1 activity both in vitro, with a mean effective dose (ED50) on macrophages of 0.1–1 μM and in HIV-infected patients, probably by a posttranslational effect on gp120 [9–13]. We were unsuccessful in infecting colostrum cells with HIV-1 in vitro, possibly because of the residual coating of the target cells for HIV by inhibitory milk substances, and we could therefore not study the anti-HIV-1 effect of chloroquine on these cells. The weak base chloroquine accumulates in cellular acid vesicles by ion-trapping [19], the extent of accumulation varying according to tissue/cell types. The known high accumulation of the drug in monocyte macrophages [20] is confirmed here in colostrum cells that have a high macrophage content and that accumulated chloroquine 817-fold in vitro. Furthermore, colostrum cells from Burkinabè mothers taking chloroquine 100 mg a day accumulated chloroquine 243-fold compared with blood. In conclusion, our work provides preliminary results indicating that chloroquine accumulates greatly in milk cells in vitro and in vivo. We hypothesize that the high accumulation of chloroquine in mammary cells actively replicating HIV-1 should allow the drug to inhibit HIV replication. Therefore, maternal chloroquine administration during the breastfeeding period may decrease milk viral load, and therefore lower the risk of breastfeeding-related transmission. This warrants further study as a strategy additional to antiretroviral prophylaxis in order to decrease the risk of HIV-1 transmission by breastfeeding. Chloroquine combines the advantages of carrying no social stigma, of widespread distribution, having low toxicity at prophylactic dosages and a very limited cost. We are currently setting up a controlled study in sub-Saharan Africa on the effects of postpartum chloroquine administration to HIV-1-positive breastfeeding mothers on milk HIV-1-RNA and cell-associated DNA levels. Acknowledgements The authors would like to thank nursing mothers, Mrs Baes and maternity nurses, Algemeen Ziekenhuis St-Jan, Brugge, for providing milk samples; Burkinabè nursing mothers for study participation; Pr Antonio Toniolo, University of Pavia, Pavia, Italy, for providing MEC-1 cells; and Pr Jack Levy, St-Pierre University Hospital, Brussels, Belgium, for helpful discussions. Johan R. Boelaerta Seydou Yarof Patrick Augustijnsc Nicolas Medaf Yves-Jacques Schneidere Dominique Scholsd Raf Molsc Emmanuel A. De Laereb Philippe Van de Perref