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WO2006065125A1 - Systemes et procedes permettant de prevoir et/ou d'influencer le transport - Google Patents

Systemes et procedes permettant de prevoir et/ou d'influencer le transport Download PDF

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Publication number
WO2006065125A1
WO2006065125A1 PCT/NL2005/000871 NL2005000871W WO2006065125A1 WO 2006065125 A1 WO2006065125 A1 WO 2006065125A1 NL 2005000871 W NL2005000871 W NL 2005000871W WO 2006065125 A1 WO2006065125 A1 WO 2006065125A1
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WIPO (PCT)
Prior art keywords
compound
bcrp
substrate
milk
breast cancer
Prior art date
Application number
PCT/NL2005/000871
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English (en)
Inventor
Johan Willem Jonker
Antonius Eduard Van Herwaarden
Alfred Hermanus Schinkel
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Het Nederlands Kanter Instituut
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Priority claimed from EP04078414A external-priority patent/EP1672365A1/fr
Application filed by Het Nederlands Kanter Instituut filed Critical Het Nederlands Kanter Instituut
Publication of WO2006065125A1 publication Critical patent/WO2006065125A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

Definitions

  • the invention relates to the field of biology. More specifically, the invention relates to transportation pathways.
  • Milk is the sole food for young mammals and an important constituent of the human diet.
  • adverse compounds such as drugs, carcinogens and environmental toxins are capable of accumulating in milk via poorly understood pathways, posing a health risk to breastfed infants, young suckling non-human mammals and consumers of dairy products in general. It is therefore generally advised to avoid smoking during breast-feeding and to be careful with use of medication during breastfeeding and dairy milk production. It is however not always possible to avoid the intake of compounds involving a health risk to young mammals and/or milk consuming individuals. For instance, the use of medication is sometimes unavoidable.
  • Breast feeding humans and/or lactating non-human mammals suffering from (infectious) disease often need to be treated in order to prevent the disease to become more severe. This results in a health risk for breast-fed infants/ young suckling mammals, and/or in spoilage of milk of dairy mammals since the milk of a mammal receiving medication is often not allowed to be used for consumption.
  • PhIP 2-amino-l- methyl-6-phenylimidazo[4,5-b]pyridine
  • the invention provides a method for determining whether a compound or its metabolite can be transported through active transport by Breast Cancer Resistance Protein into the milk or colostrum of a lactating mammal if said compound or metabolite is circulating within said mammal, the method comprising determining whether said compound or metabolite is a substrate of Breast Cancer Resistance Protein.
  • the invention provides the insight that Breast Cancer Resistance Protein is involved with transport of compounds into the milk or colostrum of a lactating mammal.
  • the Breast Cancer Resistance Protein (BCRP/ABCG2) is an ATP binding cassette (ABC) transmembrane drug transporter.
  • BCRP is a protein of about 72 kDa, localized in the plasma membrane, capable of conferring multidrug resistance to tumor cells.
  • BCRP actively extrudes a wide variety of drugs, carcinogens and dietary toxins from cells. Because of its apical localization in epithelia of the intestine, kidney and placenta and in the bile canalicular membrane, it reduces systemic, tissue and fetal uptake of these xenotoxins, and mediates their extrusion from the body.
  • BCRP is described in more detail in Allen et al. Molecular Cancer Therapeutics (2002) Vol. 1, 427-434. It is to be understood that the term "BCRP" comprises any corresponding gene or expression product thereof in other organisms. The involvement of BCRP with transportation of compounds into milk or colostrum of a lactating mammal was not known before the present invention.
  • BCRP is present in the apical membrane of alveolar mammary gland epithelial cells of mammals during late pregnancy and during lactation.
  • BCRP is capable of transporting a substrate into the milk or colostrum of a lactating mammal.
  • a compound or its metabolite which is a substrate for BCRP has an increased chance of being secreted into the milk or colostrum of a lactating mammal if said compound or metabolite is at least in part circulating within said mammal, as compared to a compound which is not a BCRP substrate.
  • the extent of in vivo transportation of a BCRP substrate depends on the extent of interaction between said substrate and BCRP located in mammary gland epithelial cells.
  • a method of the invention is therefore preferably performed in order to test a compound or metabolite that is capable of circulating in the blood circulation of said mammal, so that said compound or metabolite is capable of being readily contacted with mammary gland epithelial cells.
  • a method of the invention is preferably performed in order to test a compound or metabolite that is not capable of being bound by another component of an animal's body to such extent that transportation by BCRP is essentially inhibited.
  • a method according to the invention is carried out in vitro.
  • a BCRP substrate or a compound whose metabolite is a BCRP substrate, is administered to said lactating mammal, resulting in circulation of said BCRP substrate within said animal, it is at risk of being transported by BCRP into the milk or colostrum of said animal.
  • compounds such as for instance medicaments, pesticides, environmental toxins or carcinogens that are taken up by a lactating mammal and that are a substrate for BCRP are at risk of being transported into the milk or colostrum of said lactating mammal.
  • metabolites of such compounds which metabolites are BCRP substrates are at risk of being transported into the milk or colostrum of said lactating animal. Therefore, if transport into milk is to be avoided, it is preferred to select a compound and/or metabolite which is not a BCRP substrate.
  • a BCRP substrate is modified such that its capability of being transported by BCRP is lost.
  • the insight of the present invention is used for determining whether a candidate compound or its metabolite is at risk of being transported into milk through active transport by BCRP.
  • a screening procedure is performed. It is for instance tested whether a compound that is harmful for a young mammal or for milk consuming individuals is a BCRP substrate. This test is easily performed, preferably in vitro, as outlined below. If said compound appears to be a BCRP substrate, it is preferred to select another compound, or to modify said compound, because a BCRP substrate is at risk of being transported into milk through active transport by BCRP. Hence, a compound which is a BCRP substrate is at risk of accumulating in the milk of a lactating animal.
  • a compound which is not a BCRP substrate is preferably used.
  • One embodiment of the invention therefore provides a method for determining whether a compound or its metabolite can be transported through active transport by Breast Cancer Resistance Protein into the milk or colostrum of a lactating mammal if said compound or metabolite is circulating within said mammal, the method comprising determining whether said compound or metabolite is a substrate of Breast Cancer Resistance Protein and, if said compound appears to be a BCRP substrate, typing said compound as a candidate for secretion into the milk or colostrum of a lactating animal through active transport by BCRP when said compound is provided at the basolateral side of said animal's mammary gland epithelial cells. If said compound appears not to be a BCRP substrate, it is typed as not being a candidate for secretion into the milk or colostrum of a lact
  • a substrate of BCRP is meant herein a compound which is capable of being specifically bound by BCRP and transported across a cell membrane, preferably into the milk or colostrum of a lactating mammal. Said substrate is preferably transported by BCRP from a breast epithelial cell layer into milk.
  • a compound of the present invention comprises any kind of compound which is capable of being administered to a lactating animal.
  • a compound of the present invention preferably comprises an organic molecule of about 50- 3000 Dalton. More preferably said compound comprises a compound of about 150-2000 Dalton, most preferably a compound of about 200-1000 Dalton because BCRP is particularly capable of transporting organic compounds of these sizes.
  • said compound comprises a peptide. Said peptide preferably comprises about 3-20 amino acid residues. More preferably said peptide comprises about 5-15 amino acid residues.
  • a metabolite of a compound is defined as a molecule which is formed when said compound is processed in vivo. After administration of a compound such as for instance a prodrug to an animal, said compound is sometimes altered within said animal. Said compound is for instance cleaved.
  • said compound, or a metabolite thereof is modified by conjugation with an endogenous molecule such as for instance glucuronic acid, glutathione and/or sulfate.
  • a metabolite resulting from such modification may subsequently be cleaved, and/or a cleavage product may subsequently be modified. Any product resulting from in vivo processing of a compound is called herein a metabolite of said compound.
  • Active transport by BCRP means transportation of a BCRP substrate across a membrane.
  • Said BCRP substrate and BCRP specifically bind, after which BCRP transports this substrate from one side of a cell, or a membrane, to another side of said cell or membrane.
  • said substrate is transported from a basolateral side of an epithelial cell to an apical side of said cell.
  • a lactating mammal is defined herein as a mammal that produces milk or colostrum, or a mammal that starts producing milk or colostrum while said compound or a metabolite thereof is circulating within said mammal.
  • a method of the invention is suitable for determining whether a compound or its metabolite will be secreted into the milk or colostrum of a mammal upon administration of said compound to a mammal that lactates or starts lactating after said compound has been administered, and while at least part of said compound or metabolite is still circulating within said mammal.
  • a mammal of the invention preferably comprises a human individual or a dairy animal.
  • a mammal of the invention for instance comprises a female mammal during late pregnancy and/or a female mammal who is lactating after birth of her baby.
  • a mammal of the invention also comprises a mammal without a baby which mammal is nevertheless lactating, such as a dairy animal that continues lactating.
  • Said dairy mammal preferably comprises a cow, a sheep, a goat, a yak, a camel, a llama, a mare or a rabbit, since these mammals produce milk that is suitable for further use by humans.
  • Colostrum is a milk-like substance produced by a lactating mammal in the early days of lactating. This special milk is often high in carbohydrates, protein and antibodies.
  • the basolateral side of an epithelial cell is defined as the side of said cell that is normally adjacent to the extracellular matrix in vivo. If said epithelial cell is used in vitro it is often still possible to determine what side is the basolateral side, because epithelial cells are polarized. For instance, the nucleus is often present in the basolateral half of the cell.
  • the apical side of an epithelial cell is defined as the side of said cell which is normally exposed to a compartment of an animal such as the alveolar lumen which is considered the exterior.
  • the basolateral side of epithelial cells lining an alveolar lumen of a mammary gland is directly or indirectly exposed to the blood stream, while the apical side of said epithelial cells is exposed to the alveolar lumen.
  • a functional part of a Breast Cancer Resistance Protein is defined as a part which has the same transportation properties in kind, not necessarily in amount.
  • transportation properties is meant the capability to transport a BCRP substrate across a cell membrane.
  • a functional part is for instance produced by deleting at least one amino acid residue of BCRP such that a transportation capability - in kind, not necessarily in amount - is maintained.
  • a derivative of a BCRP is defined as a proteinaceous molecule which has been altered such that the transportation properties of said molecule are essentially the same in kind, not necessarily in amount.
  • a derivative is provided in many ways, for instance through conservative amino acid substitution. Conservative substitution comprises a substitution of one amino acid residue with another residue with generally similar properties (size, hydrophobicity, etc), such that the overall functioning is not seriously affected.
  • analogue of a BCRP. This is for instance done through screening of a peptide library.
  • Such analogue has essentially the same immunogenic properties of said protein in kind, not necessarily in amount.
  • said analogue comprises non natural residues, such as D-amino acid residues and/or modified amino acid residues.
  • a functional part of a BCRP gene is meant a part of said gene, at least 30 base pairs long, preferably at least 200 base pairs long, comprising at least one expression characteristic (in kind not necessarily in amount) as BCRP.
  • said part encodes a proteinaceous molecule capable of at least in part transporting a BCRP substrate.
  • An analogue of a BCRP gene is defined herein as a molecule with at least one characteristic (in kind, not necessarily in amount) as a BCRP gene. Said analogue for instance comprises peptide nucleic acid (PNA).
  • a functional part or an analogue of a BCRP gene preferably encodes BCRP, or a functional part, derivative and/or analogue thereof.
  • a compound is circulating in a mammal if said compound is spreading within at least part of said mammal such that different kinds of locations within said mammal are reached. Said compound is preferably circulated within the blood circulation of said mammal. While circulating, said compound reaches a mammary gland epithelial cell layer comprising BCRP, and/or is transported into said layer. If a compound that is a BCRP substrate is contacted with BCRP of a mammary gland it is actively transported by BCRP into the milk or colostrum of a lactating mammal.
  • said compound's BCRP binding properties remain essentially the same — in kind, not necessarily in amount — during circulation in vivo, so that the result of an in vitro assay determining whether said compound is a BCRP substrate remains indicative for said compound circulating in vivo.
  • In vivo modifications, or in vivo binding of said compound to another molecule, resulting in diminished or lost BCRP specificity is preferably avoided in case transport of a beneficial compound into milk is desired.
  • said metabolite(s) is/are preferably tested with a method of the invention in order to determine whether said metabolite(s) is/are a BCRP substrate. If a metabolite appears to be a BCRP substrate, it is at risk of being secreted into the milk or colostrum of a lactating mammal once said metabolite is circulating within said mammal.
  • a compound or its metabolite is circulating within said mammal as a result of administration of said compound to said mammal. It is possible to administer a compound to a mammal in various ways. Said compound is for instance administered enterally, parenterally, intrapulmonary and/or via dermal application.
  • an in vitro inside-out vesicle uptake experiment is used.
  • a vesicle comprising BCRP, or a functional part, derivative and/or analogue thereof, is used which is oriented such that a BCRP substrate is transported into the vesicle.
  • After incubation with at least one candidate compound it is determined whether said candidate compound is accumulating in the vesicle, indicating that it has been transported into the vesicle by said BCRP or functional part, derivative and/or analogue, which in turn indicates that said compound is a BCRP substrate.
  • a control is used in order to confirm that said candidate compound is accumulating in said vesicle in a BCRP-dependent manner.
  • BCRP-dependence is preferably established by comparison with an analogous inside-out vesicle not comprising BCRP or a functional part, derivative and/or analogue thereof.
  • This assay is particularly suitable for testing hydrophilic compounds since hydrophilic compounds essentially do not diffuse into/through a hydrophobic vesicle membrane.
  • a transwell experiment is performed for determining whether a compound is a substrate of Breast Cancer Resistance Protein.
  • basolateral to apical transport by BCRP is determined as follows.
  • a transwell membrane preferably a polycarbonate filter, is incubated with BCRP comprising cells, or cells comprising a functional part, derivative and/or analogue of BCRP, preferably epithelial cells.
  • the cells are allowed to grow, preferably into a confluent layer.
  • the cell coated membrane is placed into a well such that the cells separate two compartments, preferably an upper (apical) and lower (basolateral) compartment.
  • a candidate compound is added, for instance to the basolateral side of the cells.
  • a candidate compound is added to the apical side of said cells. If passive transport of said candidate compound to the basolateral side of said cells occurs to some extent, said compound will be present at the basolateral side after a while. If said compound is a BCRP substrate, it will be, at least in part, transported back by BCRP to the apical side. Hence, when cells comprising BCRP or a functional part, derivative and/or analogue thereof are used, and when passive transport occurs to some extent, a BCRP substrate will be present at the basolateral side to a lower extent as compared to an experiment wherein cells are used that do not comprise BCRP or a functional part, derivative and/or analogue thereof.
  • a BCRP substrate will be present at the basolateral side to a lower extent as compared to a compound which is not a BCRP substrate.
  • a first compound is added to the apical side of the cells, and when said first compound appears at the basolateral side of the cells to a lower extent than is observed when cells without BCRP or functional part, derivative or analogue thereof are used, it indicates that said first compound is a BCRP substrate.
  • a transwell experiment is particularly suitable for testing hydrophobic compounds because hydrophobic compounds are capable of entering an epithelial cell layer. If a hydrophilic compound is tested it is preferred to use a transport system capable of transporting said hydrophilic compound into the epithelial cell layer. More preferably, however, a hydrophilic compound is tested with an inside-out vesicle uptake experiment. In yet another embodiment transport of a candidate compound by cells comprising BCRP, or a functional part, derivative and/or analogue thereof, preferably compared with cells not comprising BCRP or a functional part, derivative or analogue thereof, is determined.
  • said cells comprising BCRP or a functional part, derivative and/or analogue thereof and said cells not comprising BCRP or functional part, derivative or analogue thereof are the same kind of cells, such as for instance epithelial cells.
  • a reduced accumulation of a candidate compound inside cells comprising BGRP, or a functional part, derivative and/or analogue thereof, as compared to the accumulation of said candidate compound inside cells not comprising BCRP or a functional part, derivative and/or analogue thereof, is also a good indication that said compound is a BCRP substrate.
  • a reduced accumulation inside cells comprising BCRP, or a functional part, derivative and/or analogue thereof, indicates that said compound is transported out of the cell by BCRP, or by said functional part, derivative and/or analogue thereof.
  • an inhibitor of BCRP is capable of influencing transport of a candidate compound by cells comprising BCRP, or a functional part, derivative and/or analogue thereof.
  • Cells comprising BCRP or a functional part, derivative and/or analogue thereof are incubated with a candidate compound, either with or without the presence of a BCRP inhibitor. If a candidate compound is transported by said cells in the absence of a BCRP inhibitor, and if said candidate compound is less, or essentially not, transported by said cell in the presence of said inhibitor, it indicates that said candidate compound is transported by BCRP or a functional part, derivative and/or analogue thereof and, hence, that said compound is a substrate of BCRP.
  • BCRP inhibitors are known in the art, such as for instance Fumitremorgin C, Kol43, Kol34, (described in Allen et al., Molecular Cancer Therapeutics, VoI 1, 417-425, April 2002) GF120918 (iV- ⁇ 4-[2-(l,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)-ethyl]-phenyl ⁇ -9,10- dihydro-5-methoxy-9-oxo-4-acridine carboxamide, described in Hyafil F et al. (1993) Cancer Res.53:4595-4602), CI1033, Iressa, novobiocin, flavopiridol, reserpine and XR9576.
  • inhibitor GF120918 is used.
  • a compound is a substrate of BCRP, using a non-human animal.
  • said candidate compound is also administered to a non-human animal that does not, or to a lesser extent, express BCRP in its mammary gland cells. If less compound accumulates in the milk of said non-human animal as compared to the amount of compound that accumulates in the milk of an animal expressing BCRP, it indicates that said compound is a BCRP substrate.
  • a non-human animal provided with human BCRP or a functional part, derivative and/or analogue thereof.
  • human BCRP includes human BCRP or a functional part, derivative and/or analogue thereof.
  • Said human BCRP is preferably present in the apical membrane of alveolar mammary gland epithelial cells of said non -human animal.
  • a transgenic non-human animal expressing human BCRP in its mammary glands is used.
  • said non-human animal comprising human BCRP is provided with a candidate compound.
  • said candidate compound or at least one metabolite thereof appears to be transported into the milk of said transgenic animal, it suggests that said compound or metabolite is a substrate of human BCRP.
  • an animal which normally expresses endogenous BCRP in its mammary glands such as a mouse
  • said endogenous BCRP gene is at least in part deleted and/or substituted via homologous recombination.
  • endogenous BCRP is exchanged for human BCRP.
  • an endogenous BCRP gene is rendered inactive, for instance via site directed mutagenesis resulting in an inactive gene or by administration of a nucleic acid molecule comprising an antisense sequence such as for instance small interfering RNA (siRNA).
  • siRNA small interfering RNA
  • a BCRP knockout mutant preferably a BCRP knockout mouse
  • a BCRP knockout mutant is an animal that has essentially lost its ability of expressing endogenous BCRP in its mammary glands.
  • a BCRP knockout mutant is produced in various ways known in the art, such as for instance via homologous recombination, site directed mutagenesis or by administration of an antisense nucleic acid or small interfering RNA, as outlined above.
  • said compound is administered to said non- human animal in the same way as it is normally acquired by a mammal.
  • a medicament that is normally administered orally to a patient is preferably also orally administered to a non-human animal in a method of the present invention.
  • said compound or metabolite is administered orally, intrapulmonary, via dermal application or via intracutaneous, subcutaneous or intramuscular injection.
  • a method of the invention comprises determining in vitro whether a compound or metabolite is a substrate of BCRP, because an in vitro test avoids the risk of suffering of non-human animals and it is often cheaper.
  • An in vitro test is therefore preferably performed during early stages of investigation.
  • an in vitro test is performed in order to select one, or several, promising candidate compounds, after which an in vivo test is performed with at least one selected compound in order to study its in vivo characteristics in more detail.
  • a method of the invention is suitable for testing a wide variety of compounds.
  • a compound is at risk of accumulating in milk, its administration is sometimes undesired, for instance in case of administration of a pesticide, medicament or vaccine.
  • administration of a compound that is at risk of accumulating in milk is desired, for instance in case of administration of a nutrient or a medicament which is beneficial for the health of a human infant or non-human suckling young mammal.
  • a nutrient appears to be a BCRP substrate, it has a higher chance of being secreted into the milk or colostrum of said lactating animal, as compared to a compound which is not a BCRP substrate.
  • Accumulation in milk is sometimes beneficial to a young mammal or to a milk consuming individual, in which case said nutrient is preferably administered to a lactating animal.
  • transportation of a medicament into the milk of a lactating animal is desired when a dairy animal suffers from an infection affecting its mammary glands, such as mastitis. If a dairy animal suffers from such disease, administration of a medicament that is primarily transported to mammary gland epithelial cells and the milk is preferred. In this case it is preferred to administer a medicament that is a BCRP substrate.
  • a method of the invention is used for testing a medicament, a vaccine, a pesticide, a toxin and/or a carcinogen.
  • a vaccine e.g., a pesticide, a toxin and/or a carcinogen.
  • One embodiment thus provides a method of the invention wherein said compound comprises a nutrient, a medicament, a vaccine, a pesticide, a toxin and/or a carcinogen.
  • a method of the invention is particularly suitable for designing and/or selecting a compound which is not, or to a small extent, secreted into milk and/or colostrum. This is for instance desired when a medicament, vaccine or pesticide is designed and/or selected since a medicament, vaccine or pesticide is beneficial for a lactating animal but often - albeit not always -unfavourable for young suckling mammals and/or milk consumers.
  • Said medicament or vaccine for instance comprises a medicament or vaccine capable of at least treating or preventing an infectious disease.
  • An example of said medicament is an antibiotic.
  • a pesticide is often beneficial for a lactating animal but harmful for milk consuming individuals.
  • said pesticide comprises a composition capable of at least in part counteracting the presence of lice, fleas, insects and/or mites on the skin and/or fur of said animal.
  • Such composition is often applied to the skin/fur of said animal, for instance by spraying.
  • said pesticide is administered otherwise such as for instance orally or via injection.
  • a pesticide is often harmful to milk consuming individuals.
  • a compound such as a medicament, vaccine or pesticide is therefore preferably tested with a method of the invention.
  • a plurality of candidate compounds is tested with a method of the invention.
  • a candidate compound or its metabolite is found not to be a BCRP substrate, it has a smaller chance, as compared to a BCRP substrate, of being transported into the milk of a lactating animal.
  • a compound which is not a substrate of BCRP is therefore preferably used for administration or application to a lactating animal.
  • a compound such as a medicament, vaccine or pesticide is selected and/or developed that is safely administered or applied to lactating animals such as breast feeding human individuals and dairy animals.
  • a method of the invention is therefore provided further comprising selecting a compound which is essentially not a substrate of Breast Cancer Resistance Protein.
  • a compound appears to be a BCRP substrate, and if said compound is unfavourable for young suckling mammals or milk consumers, it is advisable not to administer said compound to a lactating mammal.
  • said compound is sometimes advantageous, or even indispensable, for a lactating animal.
  • a lactating animal sometimes needs a medicament which is contra-indicated for young suckling mammals or milk consumers.
  • a pesticide such as an anti-lice, anti-flea composition or an insect repellent is often highly advantageous for the well-being of an (adult) animal. If there is no suitable alternative medicament/pesticide available, one often has to provide said lactating animal with said compound, despite its accumulation into milk.
  • This is for instance performed by modifying a BCRP binding site of said compound, resulting in impaired transport or, preferably, complete loss of transport by BCRP.
  • a compound is also deprived of its property of being secreted into milk or colostrum by altering its conformation such that a BCRP substrate is no longer capable of binding to BCRP, for instance because of sterical hindrance.
  • a compound is preferably modified such that at least one desired property, such as for instance medical, prophylactic and/or pesticide activity, is maintained, provided or enhanced.
  • a BCRP substrate is altered by addition to said substrate, and/or deletion from said substrate, of at least one hydroxyl group, amino group, sulphate group, glucuronide group and/or gluthathione.
  • the addition and/or deletion of at least one of such groups is particularly suitable for modifying a compound such that at least one characteristic is altered, whereas at least one other characteristic is retained.
  • addition and/or deletion of at least one of such groups is particularly suitable for depriving a compound of its property of being secreted into milk or colostrum, while at the same time maintaining another, beneficial, property such as for instance a therapeutic activity.
  • at least one sulphate group is added to, and/or removed from, a BCRP substrate. Most preferably at least one sulphate group is removed from a BCRP substrate resulting in a diminished capability of said former BCRP substrate of being secreted into milk or colostrum.
  • An altered compound according to the invention is preferably tested in order to determine whether said altered compound is a BCRP substrate, for instance using any one of the methods described herein before.
  • said altered compound appears to be a BCRP substrate as well, it is preferred to further modify said altered compound, and/or to modify the original BCRP substrate in an alternative way, in order to produce at least one further modified compound.
  • Said further modified compound is preferably also tested in order to determine whether it is a BCRP substrate, et cetera. Once a modified compound is found not to be a BCRP substrate — or to a significant lesser extent as compared to an original BCRP substrate — it is preferably determined whether said modified compound has maintained at least one desired property of interest, such as for instance a therapeutic activity. If this is the case, said modified compound is preferably selected for further use, instead of the original BCRP substrate.
  • a harmful compound is preferably altered such that it is essentially not a BCRP substrate anymore, before it is administered to a lactating animal.
  • the invention thus provides a method for at least in part depriving a Breast Cancer Resistance Protein substrate of its property of being secreted into the milk of a lactating mammal if said compound is circulating within said mammal, the method comprising altering said substrate such that the resulting compound is essentially not a Breast Cancer Resistance Protein substrate.
  • the invention furthermore provides a transgenic non-human mammal wherein an endogenous BCRP gene is operably linked to a heterologous regulatory element which is active in a mammary gland.
  • said regulatory element is specific for a mammary gland.
  • a heterologous regulatory element is a sequence which is normally not operably linked to said gene in said animal and which is capable of regulating expression of said gene.
  • Said heterologous regulatory element preferably comprises a promoter and/or an enhancer.
  • a gene and a regulatory element are "operably linked" when said regulatory element is capable of influencing expression of said gene.
  • said regulatory element resides upstream of said gene.
  • said regulatory element resides downstream of said gene.
  • Said regulatory element is preferably located within 3000 base pairs of said gene. More preferably, said regulatory element is located within 1000 base pairs of said gene, most preferably within 500 base pairs of said gene.
  • a regulatory element is active in a mammary gland when said regulatory element is capable of influencing expression of a gene when present in a mammary gland cell.
  • a regulatory element is specific for a mammary gland if said regulatory element is capable of influencing expression of a gene only when it is present in a mammary gland cell. Said regulatory element is essentially not, or to a significantly lesser extent, capable of influencing expression of a gene when it is present in cell of other tissue.
  • a transgenic non-human animal of the invention is suitable for influencing transport of a BCRP substrate into the milk or colostrum of said animal.
  • an animal comprising an endogenous BCRP gene operably linked to a heterologous promoter and/or enhancer has an enhanced capability of transporting a BCRP substrate into milk. This is for instance desired if a beneficial compound is collected via the milk of a dairy animal.
  • the invention also allows for at least partial inhibition of secretion of a BCRP substrate into milk or colostrum. This is for instance desired if a harmful compound is administered to said animal.
  • Inhibition of transport of a BCRP substrate is in one embodiment performed by reducing expression of BCRP in the mammary glands, for instance by operably linking a BCRP gene to a repressor.
  • Expression of BCRP in a mammary gland is preferably regulated with an inducible regulatory element such as an inducible promoter, enhancer or repressor. This way, expression of BCRP is temporarily enhanced when a beneficial compound is administered to a lactating animal, and/or temporarily diminished if a harmful compound is administered to a lactating animal.
  • An example of an inducible promoter, enhancer or repressor is the tetracycline repressor-operator system (e.g. Gossen M, Freund Kunststoff S, Bender G, Muller G, Hillen W, Bujard H (1995) Transcriptional activation by tetracyclines in mammalian cells, Science 268:1766-1769).
  • BCRP is preferably enhanced. This is for instance performed by enhancing expression of an endogenous BCRP gene.
  • expression of BCRP is enhanced by providing a non-human animal with a heterologous BCRP gene, or a functional part or analogue thereof, operably linked to a regulatory element which is active in a mammary gland.
  • said regulatory element is specific for a mammary gland.
  • the invention thus furthermore provides a transgenic non-human mammal comprising a heterologous BCRP gene, or a functional part or analogue of said gene, operably linked to a regulatory element which is specific for a mammary gland.
  • Said heterologous BCRP or functional part, derivative or analogue thereof preferably has an altered specificity as compared to endogenous BCRP.
  • Said heterologous BCRP or functional part, derivative or analogue thereof is for instance capable of transporting more different beneficial compounds as compared to endogenous BCRP.
  • said heterologous BCRP or functional part, derivative or analogue thereof has a higher affinity for at least one beneficial compound, and/or a lower affinity for at least one adverse compound, as compared to endogenous BCRP.
  • a non-human animal comprising human BCRP is provided.
  • said animal preferably expresses human BCRP in mammary gland epithelial cells.
  • a non-human animal comprising human BCRP is particularly suitable for determining whether a compound is a substrate of human BCRP. Accumulation of a candidate compound in the milk of said animal indicates that said compound is a substrate of human BCRP, as outlined above. If an animal of the invention is used for this purpose, expression of endogenous BCRP is preferably diminished, more preferably essentially abolished.
  • the invention thus provides a transgenic non-human animal which essentially does not express endogenous BCRP in its mammary gland cells, as well as a use of a lactating non- human animal comprising human BCRP for determining whether a candidate compound is a BCRP substrate.
  • a lactating non- human animal comprising human BCRP for determining whether a candidate compound is a BCRP substrate.
  • it is determined whether said candidate compound is a BCRP substrate by determining whether said candidate compound accumulates in the milk of said lactating non-human animal.
  • a candidate compound is administered to a non- human animal comprising (endogenous and/or heterologous) BCRP in its mammary gland cells, and to a non-human animal which essentially does not comprise BCRP in its mammary gland cells.
  • Said animal which essentially does not comprise BCRP preferably comprises a knock out mutant of the same species as said animal comprising BCRP.
  • a wild type mouse and a knock out mouse are used.
  • a mouse, preferably a knock out mouse, provided with human BCRP is used, together with an endogenous BCRP knockout mouse which is not provided with human BCRP.
  • a candidate compound appears to accumulate in the milk of said animal comprising BCRP, or a functional part, derivative and/or analogue thereof, in its mammary gland cells, while said candidate compound does not, or to a significantly lesser extent, accumulate in the milk of said animal which does not comprise BCRP, or a functional part, derivative and/or analogue thereof, in its mammary gland cells, it indicates that said candidate compound is a BCRP substrate.
  • a second compound is capable of at least in part inhibiting accumulation of said BCRP substrate in the milk of said animal comprising BCRP, or a functional part, derivative and/or analogue thereof, in its mammary gland cells, it indicates that said second compound is a BCRP inhibitor.
  • a non-human lactating animal according to the invention expressing (endogenous and/or heterologous) BCRP is used for producing a compound which is beneficial for human beings, and for transporting said compound into the milk.
  • Said compound is preferably specifically adapted to human use.
  • said compound need not be beneficial for said non-human animal.
  • Said compound is preferably essentially not used by said non-human animal. Instead, said non-human animal is preferably used for production and collection of a compound which is to be used by human individuals.
  • a BCRP inhibitor is used for at least in part avoiding secretion of a BCRP substrate into milk or colostrum by BCRP or by a functional part, derivative or analogue of BCRP.
  • a BCRP inhibitor is defined as a compound which is at least in part capable of counteracting transportation of a BCRP substrate by BCRP, and/or by a functional part, derivative and/or analogue of BCRP. BCRP inhibitors are known in the art.
  • Said BCRP inhibitor for instance comprises an organic molecule capable of binding BCRP. More preferably said inhibitor comprises GF120918 (iV- ⁇ 4-[2-(l,2,3,4-tetrahydro-6,7-dimethoxy-2- isoquinolinyl)-ethyl]-phenyl ⁇ -9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide) (Hyafil F et al (1993) Cancer Res.53:4595-4602) or Kol43 (3-(6- Isobutyl-9-methoxy-l,4-dioxo-l,2,3,4,6,7,12,12a-octahydro- pyrazino[l ) ,2':l,6]pyrido[3,4-b]indol-3-yl)-propionic acid tert-bntyl ester) (Allen JD et al (2002) Molecular Cancer Therapeutics. 1:4
  • BCRP is capable of transporting BCRP substrates into milk
  • a first compound is capable of influencing accumulation of a second compound in milk. If a certain compound is capable of inhibiting BCRP, it is capable of at least in part inhibiting active transport of a BCRP substrate by BCRP, and/or by a functional part, derivative and/or analogue of BCRP. In one embodiment it is therefore determined whether a compound is a BCRP inhibitor. If said compound appears to be a BCRP inhibitor, it is administered to a lactating human individual or animal in order to at least in part prevent a BCRP substrate from accumulating in the milk.
  • a medicament, vaccine or pesticide is a BCRP substrate
  • it is preferably administrated together with a BCRP inhibitor to a lactating human individual or animal in order to at least in part prevent accumulation of said medicament, vaccine or pesticide in milk.
  • accumulation of a compound such as a nutrient or a medicament in milk is desired.
  • compounds are preferably screened for BCRP inhibition activity.
  • the invention thus provides an assay for determining whether a compound is a BCRP inhibitor, characterized in that a mammary gland epithelial cell is used. According to the present invention, mammary gland epithelial cells are suitable for a BCRP inhibition test.
  • a use of a mammary gland epithelial cell for determining whether a compound is a BCRP inhibitor is therefore also provided.
  • One embodiment provides a method for determining whether a candidate compound is an inhibitor of BCRP, comprising determining whether - and if so, to what extent- a BCRP substrate in the presence of said candidate compound is capable of accumulating in milk. Preferably the result of this test is compared with the extent of milk accumulation of said BCRP substrate in the absence of said candidate compound. If less BCRP substrate is capable of accumulating in milk in the presence of said candidate compound as compared to the extent of accumulation of said BCRP substrate in milk in the absence of said candidate compound, it indicates that said candidate compound is a BCRP inhibitor.
  • the invention furthermore provides a use of an assay capable of determining whether a compound is a BCRP inhibitor for determining whether said compound is suitable for co-administration with a second compound to a lactating human individual or animal. If accumulation of a compound in milk is desired, co- administration of a BCRP inhibitor is preferably avoided. However, if accumulation of a compound in milk is undesired, co-administration of a BCRP inhibitor is preferred.
  • a BCRP inhibitor is separately administered to a lactating animal in order to at least partially inhibit secretion of a BCRP substrate into the milk or colostrum.
  • a BCRP inhibitor and a BCRP substrate are administered at the same time in order to at least in part prevent secretion of said substrate into milk or colostrum.
  • a mixture of said inhibitor and said BCRP substrate is administered.
  • a BCRP inhibitor is coupled to a BCRP substrate. Upon administration of such inhibitor-BCRP substrate molecule, said molecule is cleaved in vivo and the resulting inhibitor at least partly prevents secretion of said BCRP substrate into milk or colostrum.
  • the invention thus provides a use of an inhibitor of BCRP for at least in part inhibiting secretion of a BCRP substrate into the milk or colostrum of a lactating mammal.
  • a compound selectable or obtainable by a method of the invention which is essentially not a substrate of BCRP is preferably used for preparing a composition which is beneficial for lactating animals but which is unfavourable for suckling young mammals or milk consumers.
  • Said composition for instance comprises a nutrient, medicament, pesticide and/or vaccine.
  • said composition comprises a pesticide.
  • a pesticide which is essentially not a BCRP substrate is preferably used for protecting vegetation that is taken up by lactating dairy animals, since said pesticide does not accumulate in the milk or colostrum of said lactating animals through active transport by BCRP.
  • a use of a compound selected by or obtainable by a method of the invention, for preparing a nutrient, medicament, vaccine and/or pesticide is therefore also herewith, provided.
  • the invention furthermore provides a use of Breast Cancer Resistance Protein, or a functional part, derivative or analogue thereof, for at least in part enhancing secretion of a Breast Cancer Resistance Protein substrate by a mammary gland cell.
  • an increased amount of BCRP, or a functional part, derivative or analogue thereof, in the mammary glands allows for increased secretion of BCRP substrates into milk or colostrum of a lactating animal through active transport by BCRP.
  • said mammary gland cell is cultured ex vivo.
  • a mammary gland cell of the invention preferably comprises a heterologous nucleic acid sequence encoding a Breast Cancer Resistance Protein substrate.
  • Said BCRP substrate is produced by said mammary gland cell and exported by BCRP or by a functional part, derivative or analogue thereof, preferably into the milk or colostrum of a lactating non- human animal or into a culture medium.
  • a BCRP substrate This is in one embodiment performed by providing cells capable of producing a BCRP substrate with BCRP or with a functional part, derivative or analogue thereof. Said cells capable of producing a BCRP substrate are cultured, BCRP substrate is produced by said cells and transported out of the cell by BCRP or by a functional part, derivative or analogue thereof.
  • the invention thus provides a method for obtaining a Breast Cancer Resistance Protein substrate, comprising providing a cell capable of producing said substrate with a Breast Cancer Resistance Protein, or with a functional part, derivative and/or analogue thereof, and culturing said cell under culture conditions that allow the production of said substrate by said cell.
  • This embodiment is particularly suitable when cells are used that do not comprise a significant amount of endogenous, active, BCRP. Such cells will normally not secrete a significant amount of BCRP substrate into the environment. Once such cells have been provided with BCRP, or with a functional part, derivative and/or analogue thereof, they will secrete a BCRP substrate into the environment.
  • One embodiment thus provides a method according to the invention wherein said cell capable of producing said substrate does not comprise a significant amount of endogenous, active, BCRP.
  • Said cell preferably does not comprise any endogenous BCRP.
  • said substrate is harvested from the culture medium of said cell. Said cells are therefore preferably cultured under conditions that allow extrusion of said substrate by said cell, allowing harvesting of said substrate from the culture medium.
  • said cells are cultured under conditions that are favourable for growth and/or production of said BCRP substrate, but that are less favourable for extrusion and/or harvesting of said BCRP substrate. Said conditions are subsequently changed in order to provide more favourable conditions for extrusion and/or harvesting of said substrate. In this embodiment conditions are adapted to a desired stage of a production process.
  • vitamin B2 is a substrate of BCRP. Riboflavin is converted in vivo to the essential coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which participate in many key enzymatic redox reactions in the body. Riboflavin is amongst other things needed for growth and for the production of erythrocytes and antibodies. Riboflavin cannot be synthesized by mammals, and there is only limited, short term storage capacity of this vitamin in the liver. Human individuals existing on diets lacking dairy products and meat often have a deficiency of vitamin B2.
  • FMN flavin mononucleotide
  • FAD flavin adenine dinucleotide
  • vitamin B2 is usually administered artificially to the diet of these people.
  • Vitamin B2 is currently produced by micro organisms such as yeast. These micro organisms are however not capable of extruding riboflavin into the culture medium. This implicates that the producing cells need to be lysed in order to obtain riboflavin, after which a fresh culture should be started.
  • yields are limited because of intracellular inhibition as a result of accumulation of said BCRP in the cells.
  • vitamin B2 is produced by providing a cell capable of producing vitamin B2 with BCRP, or a functional part, derivative and/or analogue thereof, and culturing said cell under culture conditions that allow the production of vitamin B2 by said cell.
  • vitamin B2 is harvested from the culture medium of said cell.
  • Micro-organisms such as bacteria, fungi or yeast
  • many protocols are available for genetically modifying and culturing micro-organisms and harvesting products produced by said micro-organisms.
  • a preferred embodiment thus provides a method of the invention wherein said cell comprises a cell of a micro-organism, preferably a bacterium, fungus or yeast.
  • the invention furthermore provides methods for providing a compound with the property of being secreted into the milk or colostrum of a lactating mammal.
  • Secretion of a beneficial compound into milk or colostrum is desired at various occasions.
  • a medicament or vaccine which is beneficial for a mother is sometimes also beneficial for her baby.
  • HIV medication received by an HIV infected mother is also transferred to her (possibly) infected baby.
  • beneficial compounds such as antiretroviral drugs do not always accumulate in the milk or colostrum of a breastfeeding mother or lactating non-human animal. Therefore, it is preferably determined whether a beneficial compound such as an antiviral drug is a BCRP substrate.
  • a BCRP substrate is preferably selected.
  • such beneficial compounds are provided with a BCRP substrate in order to provide them with the capability of being secreted into the milk or colostrum of a lactating animal through active transport by BCRP.
  • a beneficial compound Once a beneficial compound has been provided with a BCRP substrate, it is determined whether the resulting compound is a substrate of BCRP. If a resulting beneficial compound is a BCRP substrate, it is preferably selected and used for administration to a breast feeding mother and/or lactating non-human animal.
  • the invention thus provides a method for providing a compound with the capability of being secreted into the milk or colostrum of a lactating mammal through active transport by BCRP if said compound is circulating within said mammal, the method comprising providing said compound with a substrate of Breast Cancer Resistance Protein.
  • Said compound preferably comprises a nutrient, a vaccine and/or a medicament, preferably an antiviral drug, since these kinds of compounds are in particular beneficial for young suckling mammals at various occasions.
  • a compound is provided with a BCRP substrate.
  • the invention thus provides a use of a Breast Cancer Resistance Protein substrate for transporting a compound into milk of a lactating mammal.
  • said mammal comprises a non-human animal. It is also possible to modify a compound such that it becomes a BCRP substrate.
  • a BCRP substrate selected with a method of the invention, or produced with a method of the invention, is preferably used for preparing a nutrient, medicament and/or a vaccine.
  • the present invention provides various modifications of a compound.
  • a BCRP substrate is converted into a compound which is not a BCRP substrate, but which is readily metabolized in vivo to yield a BCRP substrate.
  • Said compound is converted into a BCRP substrate in vivo and transported into the milk of a lactating animal.
  • a beneficial compound which is not a BCRP substrate is converted into a prodrug which is a BCRP substrate.
  • Said prodrug is administered to a lactating human individual or non-human animal and subsequently transported into the milk of said human individual or animal.
  • Said prodrug is subsequently converted into said beneficial compound in the milk.
  • the invention provides means and methods for modifying a compound such that said compound's capability of being a BCRP substrate is altered or essentially lost.
  • a method for altering the capability of a compound of being secreted into the milk of a lactating mammal if said compound is circulating within said mammal, the method comprising altering at least part of said compound such that said compound's capability of being a substrate of Breast Cancer Resistance Protein is altered, is therefore also provided.
  • a method of the invention preferably comprises inducing a modification in said substrate of Breast Cancer Resistance Protein.
  • the structure of said compound is altered such that the BCRP specificity of said compound is altered.
  • Topotecan and 14 C-topotecan were from GlaxoSmithKline (King of Prussia, PA).
  • PhIP and 14 C-PhIP (10 Ci mol" 1 ) were from Toronto Research Chemicals Inc. (Ontario, Canada); 3 H-folic acid (26.2 Ci mmoH) and 14 C- acyclovir (56 Ci moH) were from Moravek Biochemicals, Inc.
  • mice were housed and handled as described (Jonker, J. W., et al. Proc. Natl. Acad. ScL USA 99, 15649-15654 (2002)).
  • 5 ⁇ l drug solution per g body weight was injected into the tail vein of mice lightly anesthetised with methoxyflurane.
  • Levels of radioactivity in plasma and milk were determined by liquid scintillation counting. Measurements of drug secretion into the milk were done in lactating dams with pups of approximately 10 days old.
  • oxytocin 200 ⁇ l of an 1 LU. ml- 1 solution
  • Mrpl, Mrp2 and P-glycoprotein were probed with Mabs MRPrI (1:1000) (Flens, M.J., et al. Am. J.Pathol. 148, 1237- 1247 (1996)), M 2 III-5 (1:50) (Scheffer, G.L., et al Cancer Res. 60, 5269-5277
  • Vesicular transport assays were performed as described (Ozvegy, C, et al. Functional characterization of the human multidrug transporter, ABCG2, expressed in insect cells. Biochem. Biophys. Res. Commun. 285, 111-117 (2001); Zelcer, N., et al. Evidence for two interacting ligand-binding sites in human MRP2 (ABCC2). J. Biol. Chem. 278, 23538-23544 (2003)), using vesicles from Sf9 cells infected with BCRP-expressing or wild -type baculovirus, in the presence or absence of 4 mM ATP. ATP-dependent transport was calculated by subtracting the uptake in absence of ATP from that in the presence of ATP.
  • Samples were protected from light during the whole sample pre-treatment. Methanol (4 times the sample volume) was added to plasma and milk samples to precipitate proteins. After mixing, samples were centrifuged 10 min at 10,500 g. Clear supernatants were evaporated to dryness at 40 0 C under a stream of nitrogen. Samples were reconstituted in 100 ⁇ l of methanol — 50 mM ammonium acetate pH 5 (30:70, v/v) and 25 ⁇ l aliquots were injected. The chromatographic system consisted of a Perkin Elmer 200 series pump and ISS 200 autosampler (Perkin-Elmer (PE) 3 Norwalk, CT).
  • PE Perkin-Elmer
  • Chromatographic separation was performed on a Zorbax SB-C18 column (150 x 4.6 mm ID, 3.5 ⁇ m particle size) (Rockland Technologies Inc., Newport, DE).
  • the mobile phase consisted of a mixture of 50 mM ammonium acetate pH 5 and methanol and the following gradient was used: 0-5 min 30% to 90% methanol, 5-7 min 90% methanol, 7-8 min 90% to 30% methanol and 8-12 min 30% methanol.
  • the flow was 1.0 ml min" 1 and the detection was performed fluorimetrically using a FP920 Intelligent Fluorescence Detector (Jasco International Co. Ltd., Tokyo, Japan) with excitation/emission wavelengths set at 372/520 nm and 40 nm bandwidth.
  • the capacity of the flow-cell of the fluorescence detector was 16 ⁇ l. Riboflavin, FMN, and FAD were eluting after approximately 5.3, 3.9 and 2.5 min, respectively. Processed samples were stable in the autosampler for at least 24 h. Calibration concentrations were between 0.001 and 10 ⁇ g ml-1. Calibration curves were calculated by least-squares linear regression using a weighting factor of the reciprocal of the concentration.
  • Bcrpl the murine ortholog of BCRP
  • Bcrpl is highly expressed in mouse mammary gland epithelium during late pregnancy.
  • Bcrpl was detected primarily in the apical membrane of alveolar epithelial cells, but not in main ducts. During involution following cessation of lactation, expression declined rapidly.
  • BCRP/Bcrpl is thus responsible for pumping vitamin B2 into milk.
  • milk is a primary source of riboflavin and a deficiency is often endemic in human populations that exist on diets lacking dairy products and meat.
  • GF120918 was suspended at 5 mg/ml in water for injection and concentrated stock vehicle (1:1).
  • the concentrated stock vehicle consisted of hydroxypropyl methyl cellulose (10 g/1) and 2% Tween 80 in water for injection.
  • Animals were administered GF120918 (250 mg/kg; 10 ⁇ l of drug solution/g body weight) by gavage into the stomach at 2 hours before [ 14 C]- topotecan injection.
  • [ 14 C] -topotecan (1 mg/kg) was injected into the vein of mice lightly anesthetized with methoxyflurane.
  • oxytocin 200 ⁇ l of 1 1.
  • UJmI solution was administered subcutaneously to lactating dams at 15 min before each milk sample was collected. At indicated time points (15, 30, 60 and 120 min after [ 14 C] -topotecan injection), 50 ⁇ l of milk was collected from the mammary glands by gentle vacuum suction. Immediately after milk collection, a small blood sample was collected from the tail. At the last sampling point, animals were sacrificed by cardiac puncture after anesthesia with methoxyflurane.
  • Example 3 shows milk [ 14 C] topotecan concentration and M/P ratio versus time curves in wild -type and Bcrpl -knockout mice, with or without Bcrp
  • Heterocyclic amines are mutagenic and carcinogenic compounds formed during the heating of protein-containing foods such as meat and fish. HAs can be detected in the urine of healthy volunteers on a normal diet, and exposure occurs on a daily basis.
  • IQ 2-amino-3- methylimidazo[4,5-f]quinoline
  • Trp-P-1 3-amino-l,4-dimethyl-5H-pyrido[4,3- bjindole
  • IQ 2-amino-l-methyl-6-phenylimidazo[4,5- b]pyridine
  • Trp-P-1 increases the incidence of liver carcinomas (Eisenbrand et al, 1993).
  • HAs are present in nanogram per gram quantities in cooked meat, this exposure is often chronic.
  • the incremental cancer risk due to dietary intake of HAs in humans in the developed world has been estimated at 1.1 x lO" 4 .
  • mice were housed and handled according to institutional guidelines complying with Dutch legislation. Animals used were Bcrpl '- and wild-type mice, all of >99% FVB genetic background, between 9 and 14 weeks of age. Animals were kept in a temperature-controlled environment with a 12-hour light / 12-hour dark cycle. They received a standard diet (AM-II, Hope Farms, Woerden, The Netherlands) and acidified water ad libitum.
  • Transport assay In the transwell transport assay we used the previously described polarized pig kidney epithelial cell line LLC-PKl and human multidrug resistance (MDR)I, murine Mdrla- and Bcrpl- transfected LLC- PKl subclones, the polarized canine kidney cell line MDCK-II and murine Bcrpl- , human multidrug resistance associated protein (MRP)2- and human BC ⁇ P-transduced MDCK-II subclones (van Herwaarden et al, 2003; Pavek et al, 2005).
  • MDR multidrug resistance
  • MRP human multidrug resistance associated protein
  • LLC-PKl cells and derivatives are especially suitable to study P-glycoprotein-mediated transport due to the very low endogenous porcine P-glycoprotein in these cells, whereas MDCK-II cells and derivatives are very suitable to study Bcrpl/BCRP-mediated transport due to their negligible endogenous BCRP-like transport activity (Jonker et al, 2000; van Herwaarden et al, 2003). Transport assays and cell culture conditions were carried out as previously described (van Herwaarden et al, 2003).
  • Radiolabeled compounds were administered by intravenous (i.v.) injection into the tail vein or by gavage into the stomach of mice lightly anesthetized with methoxyflurane.
  • IQ or Trp-P-1 was formulated in 20% (v/v) DMSO, 4% (w/v) D-glucose solution.
  • Aflatoxin Bl or DEHP was formulated in Cremophor EL - ethanol (1:1, w/v) and diluted in 5% (w/v) D-glucose solution (1:3).
  • Trp-P-1 is transported by Bcrpl, and that both human BCRP and murine Bcrpl transport IQ.
  • the hepatocarcinogen aflatoxin Bl is also a transported substrate of murine Bcrpl and human BCRP.
  • human MEP2, human MDRl or murine Mdrla do not efficiently transport these carcinogens.
  • Plasma [ 14 C]IQ and [ 14 C]Trp-P-l were 1.7-fold and 2.3-fold increased in Bcrp ⁇ '- mice compared to the wild-type 30 min after oral administration, and 1.8-fold and 2.2-fold 30 min after i.v. administration (Figure 10).
  • the AUC 7 .5-3o for i.v. administration was 1.8-fold higher in Bcrpl- f - compared to wild-type mice, further supporting that Bcrpl activity substantially decreases the systemic exposure of its substrates (278 ⁇ 8.2 vs. 155 ⁇ 5.0, h*ng/ml, P ⁇ 0.01; Figure 11).
  • Bcrpl activity substantially decreases the systemic exposure of aflatoxin Bl, IQ and Trp-P-1, but not of DEHP.
  • tissue distribution of [ 14 C]IQ and [ 3 H]aflatoxin Bl further substantiates that these compounds are substrates of Bcrpl, and that Bcrpl affects the systemic exposure to these carcinogens in vivo.
  • [ 14 C]IQ, [ 14 C]Trp-P-l and [ 3 H]aflatoxin Bl are transported into breast milk by Bcrpl
  • Xenotoxin and carcinogen accumulation in breast milk and dairy products is a major public health concern.
  • Bcrpl/BCRP is expressed in the mammary gland.
  • [ 14 C]IQ, [i 4 C]Trp-P-l, [ 3 H]aflatoxin Bl or [ 14 C]DEHP (i.v., 1 mg/kg) to female lactating wild-type and Bcrpl '- mice. 30 min after i.v. administration milk and maternal plasma samples were obtained and milk-to-plasma (M/P) concentration ratios were calculated.
  • Nitrofurantoin (l-[(5-nitro-2-furanyl)methylene]amino-2,4-imidazolidinedione) is a nitrofuran-derivative antibacterial agent widely used in human and veterinary medicine. In humans, it is mainly used to treat urinary tract infections, which are among the most common bacterial infections. Patients receiving nitrofurantoin may have rare but serious side effects such as chronic liver disease, cholestatic hepatitis, or hemolytic anemia in glucose-6-phosphate dehydrogenase-deficient patients. Moreover, nitrofurantoin has been shown to be mutagenic and carcinogenic in animal models. Further knowledge about the factors affecting the pharmacokinetics of nitrofurantoin is therefore of clinical and toxicological importance.
  • Nitrofurantoin is also prescribed to lactating women. Inadvertent transfer of drugs administered to the mother to milk is always a matter of concern in view of possible adverse effects in the infant. Nitrofurantoin is an inexpensive antibiotic that is often used in developing countries where formula feeding is not an alternative for breastfeeding. However, preliminary results from a continuous breeding study with mice revealed that chronic nitrofurantoin treatment of lactating mice resulted in decreased pup growth rate.
  • mice were housed and handled according to institutional guidelines complying with Dutch legislation. Animals used were male or lactating female Bcrpl- ⁇ and wild -type mice, all of >99% FVB genetic background between 9 and 14 weeks of age. Animals were kept in a temperature-controlled environment with a 12-h light/12-h dark cycle and received a standard diet (AM-II; Hope Farms, Woerden, The Netherlands) and acidified water ad libitum. Chemicals. Nitrofurantoin, furazolidone and xylazine were from Sigma Chemical Co.
  • MDCK-II Human MDRl-, MRP2-, BCRP- and murine Bcrpl -transduced MDCK-II subclones were described previously (Jonker et al., 2000; Evers et al., 1998; Pavek et al., 2005).
  • the MDCK-II cells and transduced subclones were cultured in DMEM supplied with glutamax (Life Technologies, Inc.) and supplemented with penicillin (50 units/ml), streptomycin (50 ⁇ g/ml), and 10% (v/v) fetal calf serum (Life Technologies, Inc.) at 37 0 C in the presence of 5% CO2.
  • the cells were trypsinized every 3 to 4 days for subculturing.
  • Transport assays were carried out as described earlier (Huisman et al., 2001), with minor modifications.
  • Cells were seeded on microporous membrane filters (3.0 ⁇ m pore size, 24 mm diameter; Transwell 3414; Costar, Corning, NY) at a density of 1.0 x 10 6 cells per well. Cells were grown for 3 days, and medium was replaced every day. Two hours before the start of the experiment, medium at both the apical and basolateral side of the monolayer was replaced with 2 ml of Optimem medium (Life Technologies, Inc.), without serum, either with or without 5 ⁇ M Kol43.
  • Optimem medium Life Technologies, Inc.
  • the appearance of nitrofurantoin in the opposite compartment was measured by HPLC as described below, and presented as the fraction of total nitrofurantoin added at the beginning of the experiment.
  • the tightness of the monolayer was measured by monitoring the paracellular flux of [ 3 H]inulin to the opposite compartment, which had to remain ⁇ 1.5% of the total radioactivity/hour.
  • Pharmacokinetic experiments For oral administration of nitrofurantoin (10 mg/kg), 3.3 ⁇ l of drug solution [appropriate concentration in 50% (v/v) ethanol, 50% (v/v) polyethyleneglycol 400]/g body weight were dosed by gavage into the stomach. For i.v.
  • nitrofurantoin 5 mg/kg
  • drug solution [appropriate concentration in 10% (v/v) ethanol, 40% (v/v) polyethyleneglycol 400, 50% PBS]/g body weight
  • Animals were sacrificed by cardiac puncture after anesthesia with methoxyflurane, and blood was collected. Heparinized blood samples were centrifuged immediately at 3000 x g for 15 min and plasma collected and stored at -2O 0 C until the time of HPLC analysis. 3-5 animals were used for each time point.
  • Milk secretion experiments For milk experiments, pups of approximately 10 days old were separated from the mother approximately 4 hours before starting the experiment. To stimulate milk secretion, oxytocin (200 ⁇ l of 1 I.U./ml solution) was administered subcutaneously to lactating dams. Nitrofurantoin (5 mg/kg) was injected into the tail vein at 30 min before milk was collected. At the indicated time, 50 ⁇ l of milk was collected from the 4 th and 5 th pairs of mammary glands by gentle vacuum suction. Immediately after milk collection, animals were sacrificed by cardiac puncture after anesthesia with methoxyflurane, and heparinized blood was collected. Milk and plasma were stored at — 20°C until the time of HPLC analysis. 3-4 animals were used for each group.
  • the mixture was vortexed vigorously, and 50 ⁇ l of methanol at — 20°C was added for protein precipitation. Extraction was carried out by vigorously shaking the reaction tube for 60 s and incubating at - 30°C for 15 min.
  • the organic and water phases were separated by centrifugation at 16000 x g for 5 min at 4 0 C and 50 ⁇ l of the organic phase was injected into the HPLC system. Samples from the transport assays were not processed and 100 ⁇ l of the culture media were directly injected into the HPLC system.
  • the system consisted of a Waters 616 pump, a Waters 717plus autosampler and a UV detector (model UV2000, ThermoSeparation products).
  • nitrofurantoin was consistenly translocated somewhat more efficiently in the basolateral direction than in the apical direction (Fig. 14A), suggesting low endogenous basally directed transport.
  • apically directed translocation was highly increased and basolaterally directed translocation drastically decreased (Fig. 14, B and D).
  • the selective Bcrpl inhibitor Kol43 was used (Allen et al., 2002)
  • the Bcrpl/BCRP mediated transport was completely inhibited (Fig. 14, C and E), resulting in a vectorial translocation pattern equal to that of the MDCK-II parental cell line.
  • MDRl- and MRP2-transduced MDRl- and MRP2-transduced
  • MDCK-II cell lines the vectorial translocation was similar to the MDCK-II parental cell line (Fig. 14, F and G).
  • nitrofurantoin secretion of nitrofurantoin into the milk in Bcrpl-'- and wild-type mice.
  • nitrofurantoin 5, mg/kg was administered i.v. to lactating Bcrpl-/- and wild-type females, and 30 min after administration, milk and blood were collected. The data obtained from the analysis of milk and plasma are shown in Fig. 16. Despite the higher plasma level (5-fold), the concentration of nitrofurantoin was substantially lower in the milk of Bcrpl ⁇ '' ⁇ mice (almost 15- fold).
  • FIG. 1 Expression of Bcrpl/BCBP in the mammary gland, a, Western analysis on crude membrane fractions from mouse mammary glands at different stages (10 ⁇ g protein per lane). b, Western analysis of Mrpl, Mrp2, and P-glycoprotein in mammary glands of virgins and 2-wk lactating female wild-type mice, c-h, Immunohistochemical detection (xlOO) in (c) 14-wk old virgin mouse, (d) 1-wk lactating mouse, (e) virgin cow, (f) lactating cow, (g) nonlactating human, Qa) lactating human.
  • PhIP PhIP
  • l ⁇ C-topotecan (c-d) l ⁇ C-topotecan
  • e-f ⁇ H-cimetidine.
  • Bcrpl transports riboflavin (vitamin B2) and mediates its active secretion into milk.
  • a, b Endogenous riboflavin concentrations in (a) plasma and milk of wild-type and Bcrpl-/- mice, and (b) M/P ratios in lactating dams; Data represent the mean ⁇ s.d., n - 3-4, **P ⁇ 0.01 (two-tailed unpaired Student's t-test).
  • Bcrpl mediates secretion of [ 3 H]aflatoxin Bl, [ 14 C]IQ and [ 14 C]Trp- P-I, but not [ 14 C]DEHP into the milk.
  • Radiolabeled compounds (1 mg/kg) were intravenously administered to lactating wild-type or Bcrpl- ; - females and after 30 min, plasma and milk concentrations and milk/plasma (MIP) ratios were determined for (A) [ 14 C]IQ, (B) [ 14 C]Trp-P-l, (C) [ 3 H]aflatoxin Bl and (D) [ 14 C]DEHP.
  • FIG. 14 Transepithelial transport of nitrofurantoin (10 ⁇ M) in MDCKII (parent) (A), MDCKII-Bcrpl (B and C), MDCKII-BCRP (D and E), MDCKII- MDRl (F) and MDCKII-MRP2 (G) monolayers.
  • Hyafil F Vergely C, Du Vignaud P, Grand-Perret T (1993) In vitro and in vivo reversal of multidrug resistance by GF120918, an acridonecarboxamide derivative. Cancer Res.53:4595-4602
  • Palmiter RD Brinster RL
  • Hammer RE Trumbauer ME
  • Rosenfeld MG Birnberg NC
  • Evans RM Dramatic growth of mice that develop from eggs microinjected with r ⁇ etallothionein-growth hormone fusion genes. Nature 300:611-615, (1982)

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Abstract

L'invention concerne un procédé permettant de déterminer si un composé ou son métabolite peut être sécrété dans le lait ou le colostrum d'un mammifère en lactation par un transport actif via la protéine de résistance au cancer du sein, si le composé ou son métabolite circule dans le mammifère. Le procédé consiste à déterminer si le composé ou son métabolite est un substrat de la protéine considérée. De préférence, ce composé comprend un nutriment, un médicament, un vaccin, un pesticide, une toxine et/ou un carcinogène. On décrit un procédé de modification de la capacité d'un composé à être sécrété dans le lait d'un mammifère en lactation si ce composé circule dans le mammifère, lequel consiste à modifier au moins une partie du composé de sorte la capacité de ce composé à être un substrat de la protéine considérée soit modifiée.
PCT/NL2005/000871 2004-12-16 2005-12-16 Systemes et procedes permettant de prevoir et/ou d'influencer le transport WO2006065125A1 (fr)

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WO2000069390A2 (fr) * 1999-05-17 2000-11-23 Cancer Research Ventures Limited Procede pour ameliorer la biodisponibilite de medicaments administres par voie orale, procede pour selectionner des activateurs d'une telle biodisponibilite et nouvelles compositions pharmaceutiques pour medicaments a administration par voie orale
WO2001036477A2 (fr) * 1999-11-18 2001-05-25 The Government Of The United States, As Represented By The Secretary Of The Department Of Health And Human Services Inhibition de transporteurs de cassette de liaison atp (abc) par des analogues de domaine transmembranaire
US6521635B1 (en) * 2000-01-20 2003-02-18 The United States Of America As Represented By The Department Of Health And Human Services Inhibition of MXR transport by acridine derivatives
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