Folate (folic acid, vitamin B9, pteroylmonoglutamate, PteGlu) is used as tetrahydrofolate (THF) derivatives (folylpolyglutamates) in metabolic pathways as a carrier of one-carbon fragments in methylation reactions, and is essential for cell proliferation. Folate pool reducing molecules are some of the oldest anticancer drugs. 5-methyltetrahydrofolate (MTHF, 5-Me-THF) is the most abundant of folates, and as a methyl-group carrier it is used for synthesis of methionine from homocysteine. MTHF can bind to and inhibit glycine-N-methyltransferase (GNMT).
THF is reduced from dihydrofolate by enzyme dihydrofolate reductase (DHFR). Aminopterin, an old anticancer drug, functions by inhibiting DHFR.
Folates are hydrophilic molecules and anions at physiological pH, and therefore do not diffuse well across cell membranes.
The reduced folate carrier (RFC1, FOLT1, SLC19A1) is the most important transporter for folates; it is expressed ubiquitously in mammalian tissues, including intestinal and colonic epithelia, basolateral membrane of renal proximal tubules, and blood-brain barrier, and although usually it is considered to be a facilitative transporter, in case of negatively charged folates it can work strongly against concentration gradient. RFC1 is the major target for antifolate drugs in cancer chemotherapy because of its abundance, but antifolates usually target also other folate transporters.
Proton-coupled folate transporter (PCFT, SLC46A1) is mainly located in the upper (acidic) part of the intestine, renal tubules, and also elsewhere in the body, also in several solid tumours. It can transport both reduced and oxidized folates. Because its activity is higher in low pH, it may be suitable target in tumours with acidic microenvironment (Hou et al., 2017).
Multidrug resistance-associated proteins (MRPs) and organic anion transporters (OATs) transport folates in addition to many other substrates, mostly in epithelial tissues. In the liver OATs mediate the uptake of folic acid and release into the bile, enabling the extensive enterohepatic circulation of folates. Liver stores folates as polyglutamates, and releases folates into the bile or blood when necessary, mainly as 5-methyl tetrahydrofolate monoglutamate. Intestinal bacteria synthesize and utilize folates.
The high-affinity folate binding protein (FBP) exists in humans in four isoforms, folate receptors (FRα and FRβ, or FOLR1 and FOLR2) that are attached to the outer cell surface by glycosyl-phosphatidyl-inositol (GPI) link, and FRγ (FOLR3) that is a secretory protein, and expressed at much lower levels than FRα and FRβ. FRδ (Juno, FOLR4) does not bind folic acid or reduced folates. Folate receptors FRα and FRβ bind folate and its reduced forms, and are important in folate tissue uptake, and FRα especially in renal reabsorption. FRα and FRβ accumulate folates via endocytotic process, and this can be utilized in delivery of therapeutic macromolecules into cells (Leamon and Low, 1991). FRα is normally expressed in secretory epithelial cells, and it, but not FRβ, is overexpressed on the basolateral surface of tumour cells. FRα is abundant in kidneys, lungs, and salivary and bronchial glands. FRβ is normally expressed in hematopoietic tissues (bone marrow, spleen, thymus), and also in some leukaemia cells. FRβ is the main transporter for methotrexate (MTX), next to the RFC1. FRβ is expressed on activated macrophages at the site of inflammation. FRγ is found in blood plasma and intracellularly in neutrophil granulocytes.
Folates are carried in the blood mostly in red blood cells, where the concentration of polyglutamated forms of folate is tens of times higher than concentration of folates in plasma (Perna et al., 2013). Most of the folates are accumulated as red blood cell mature in the bone marrow. Erythrocytes transport also methotrexate, with similar concentration as in serum, but mature cells do not store it as polyglutamates (Weigand et al., 2000). FBPs are not involved in folate transport in mature RBCs, but at least ABCC1 (MRP1) and possibly RFC transports folates across the RBC membrane. Transport is however relatively slow (Branda and Anthony, 1979; Antony et al., 1989). Haemoglobin is a low-affinity/high-capacity binding protein for folate and folate polyglutamates.
Folate receptors are frequently overexpressed on activated macrophages and on many tumour cell types, offering a target for therapy of cancer and inflammation (Paulos et al., 2004; Müller & Schibli, 2011; 2013; Chandrupatla et al., 2019).
FRα is overexpressed in certain types of cancer, and FRβ is expressed on activated macrophages in sites of inflammation and in autoimmune diseases. FRα and FRβ selective PET radiopharmaceuticals are therefore much needed tools in clinical imaging. A non-selective SPECT radiopharmaceutical [99mTc]EC20 is already in use in clinics, and has been used to select patients for FR-targeted therapies.
FRα is normally expressed on the luminal side of the epithelial cells, and therefore not accessible by intravenously administrated PET radiopharmaceuticals. Kidneys are an exception to this, since FRα is expressed in proximal kidney tubules. Folate tracer uptake in kidneys is usually high, because of the active reabsorption via FRα, and usually also high in the liver and intestine because of metabolism, storage, and excessive enterohepatic circulation of folates. Kidney uptake can be reduced by adding an albumin-binding entity to the radiopharmaceutical (Fischer et al., 2013; Farkas et al., 2016). Generally, folate receptor radiopharmaceutical should not be too close in structure to folate, because that could lead to normal tissue uptake via RFC1 and PCFT. Conjugation of folate via its alpha or gamma carboxylate allows it still to bind to folate receptors, but prevents the facilitated transport (Mathias et al., 2003). Hydrophilic polyethylene glycol (PEG) linker has reduced the radiopharmaceutical uptake into gallbladder, liver, and lungs (Kularatne et al., 2013).
Several 18F and 68Ga labeled folate derivatives, that bind to FRα and FRβ with similar affinities, have been developed, including [18F]FDG-folate (Al Jammaz et al., 2012; Fischer et al., 2012), 2’-[18F]fluorofolic acid (Ross et al., 2010), 3’-Aza-2’-[18F]fluorofolic acid (Betzel et al., 2013), [18F]fluoro-PEG-folate (Gent et al., 2013; Chandrupatla et al., 2017 and 2018), 4-[18F]fluorophenylfolate (Kularatne et al., 2013), [18F]AlF-NOTA-folate (Chen et al., 2016; Silvola et al., 2018), [68Ga]DOTA-folate conjugates (Fani et al., 2011; Kularatne et al., 2013), [68Ga]NOGADA-folate conjugates (Fani et al., 2012), and [68Ga]NOTA-folate conjugates (Aljammaz et al., 2014; Jain et al., 2016; Brand et al., 2017), which have enabled good visualization of folate receptor positive tumours and sites of inflammation, including atherosclerotic plaques, in animal models. 124I-labelled folate conjugates have also been studied in animal tumour models (Aljammaz et al., 2014). These radiopharmaceuticals are internalized into folate receptor positive cells, or stay on the cell surface bound to the FRs and possibly RFC1. The fraction of internalized radiopharmaceutical usually increases over time, and is dependent on the radiopharmaceutical; for example only small fraction of [68Ga]DOTA-folate conjugates were internalized (Fani et al., 2011). Binding and internalization can be blocked with excess of folic acid. Optimal scan time may be quite late, 120-150 min p.i. in mice for 3’-Aza-2’-[18F]fluorofolic acid (Betzel et al., 2013), but one hour has been found to be sufficient with 68Ga-labelled folate tracers in animal models.
FRα-specific PET radiopharmaceuticals are not yet available, but a reduced and alkylated form of folic acid, DMTHF, is selective for FRα, and it has been labelled with 99mTc (Vaitilingam et al., 2012). 89Zr-labelled FRα-targeted antibody has been used in preclinical study (Brand et al., 2018).
Circulating folates, such as 5-methyltetrahydrofolate, may lead to competitive inhibition of FR tracer uptake in physiological concentrations. Specific FR binding can be blocked using glucosamine folate (Gent et al., 2013).
Plasma vs blood
Although folates are found in erythrocytes, the transport is slow. Plasma-to-blood ratio was 1.5 in [18F]folate-NOTA-AlF and 1.7 in [99mTc]EC20 mouse study, 90 min p.i. (Chen et al., 2016), ∼1.4-1.5 in [18F]AlF-NOTA-folate study in mice, 120 min p.i. (Silvola et al., 2018), and about 1.9 in [18F]fluoro-PEG-folate rat study at 60 min pi.i (Chandrupatla et al., 2017), suggesting that these radioligands did not pass the RBC membrane.
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Updated at: 2019-06-05
Created at: 2017-04-26
Written by: Vesa Oikonen