Folates are molecules that have a pteridine ring that can be reduced or oxidized, a para-aminobenzoic acid (PABA) linker that together with the pteridine ring binds 1C units, and a variable chain length polyglutamate tail that serves to localize the molecule within the cell. 5-methyltetrahydrofolate (MTHF, 5-MTHF, 5-Me-THF) is the most abundant of natural folates. Vitamin B9 (pteroylmonoglutamate, PteGlu) is reduced to dihydrofolate and further to tetrahydrofolate (THF). THF derivatives (folylpolyglutamates) are used in metabolic pathways as carriers of one-carbon (1C) units in methylation reactions, and are essential for amino acid homeostasis, redox defence, and cell proliferation (Ducker and Rabinowitz, 2017; Zheng and Cantley, 2019). Folylpoly-γ-glutamate synthetase (FPGS) catalyzes the addition of multiple glutamates to THF, and is needed for retaining folates intracellularly. Cytosol and mitochondria contain their own isoforms of FPGS, as folylpolyglutamates cannot traverse mitochondrial membranes.
Folate pool reducing molecules are some of the oldest anticancer drugs. One of these, aminopterin, functions by inhibiting dihydrofolate reductase (DHFR), which reduces dihydrofolate into THF. Methotrexate (MTX, amethopterin) inhibits DHFR and enzymes involved in purine metabolism. Involvement in purine metabolism leads to extracellular release of adenosine, which reduces inflammation. MTX is used for cancer chemotherapy and as disease-modifying treatment for autoimmune diseases, including rheumatoid arthritis, psoriasis, and sarcoidosis. Inhibitors of bacterial folate synthesis can be used as antibiotics.
Oxidative decomposition of folates is one of the numerous endogenous sources of formaldehyde. Alcohol dehydrogenase 5 detoxifies formaldehyde to formate, which is used as a source of 1C units in normal metabolism.
Folates are hydrophilic molecules and anions at physiological pH, and therefore do not diffuse well across cell membranes.
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 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. Reduced folate carrier is constitutively expressed on immune cells, including the non-proliferating macrophages. RFC1 is the major target for antifolate drugs in cancer chemotherapy because of its abundance, but antifolates usually target also other folate transporters.
Mitochondrial folate transporter (MFT, SLC25A32) is present in the inner mitochondrial membrane, facilitating the transport of folate and folate monoglutamate between cytosol and mitochondrial matrix (Lawrence et al., 2014; Ducker and Rabinowitz, 2017).
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).
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. Conjugation of proteins and liposomes to folate does not necessarily prevent the receptor-mediated endocytosis, and this can be utilized in delivery of therapeutic macromolecules into cells (Leamon and Low, 1991). Reduced folate carrier does not transport folate conjugates (Leamon et al., 2002), increasing the specificity of folate-based therapeutics aimed for treating inflammation or tumours. In cell lines, multivalent folate conjugates internalize the same rate as monovalent conjugates, although through different endocytic pathway (Bandara et al., 2014).
FRα is normally expressed on the apical surfaces of secretory epithelial cells, being inaccessible to folates from the blood. FRα, but not FRβ, is overexpressed on the basolateral surface of tumour cells of epithelial origin, and therefore readily accessible to folate tracers and pharmaceuticals from the blood. 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, next to the RFC1. Modulation of MTX terminal groups could increase its transport through FRα system (Nogueira et al., 2018). FRβ is expressed on activated macrophages at the site of inflammation, but its folate transport capacity is much less than that of the RFC1. 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., 2004a; 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; Chen et al., 2017). The expression of folate receptors can differ in different species, and is for instance much higher in the lungs, heart, and liver in humans than in mice and rats (Parker et al., 2005).
Several 18F and 68Ga labelled 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. First-in-human PET imaging has been performed with [68Ga]EC2115 in patients with COPD (Cohen et al., 2019). 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). The rate of folate α receptor recycling is variable but slow, generally less than 5%/h, and the release of folate conjugates from tumours or kidney is even slower (Paulos et al., 2004b). The recycling frequency of FRβ in inflamed tissue is much faster, ∼10-20 min, but the release of folate conjugate is slow from FR-positive tissues (Varghese et al., 2014). 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). 18F-labelled 5-MTHF conjugates have shown promising initial results for FRα imaging (Boss 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). Bandara et al (2014) did not observe any impact of FOLR saturation by folate or conjugates does to the rate of internalization in cell lines. Blocking reduces folate receptor tracer uptake especially in heart and skeletal muscle, but also in other tissues including lung, spleen, and liver (Chen et al., 2017).
Retrieving blood curve from dynamic PET image may be difficult because of high uptake into muscles, including myocardial muscle, which can cause marked spill-in effect into the blood pools. In mice studies, ex vivo heart/blood ratio at 90 min was ∼4 for [18F]AlF-NOTA-folate, and ∼9 for [18F]AlF-PEG12-NOTA-folate (Chen et al., 2017). Muscle uptake may be relatively slow; heart/blood ratio for [18F]FDG-folate was ∼1.1 at 30 min, ∼2.6 at 60 min, and ∼3.2 at 90 min (Fischer et al., 2012). Initial few time frames after tracer administration can however be used to locate the blood pools in the image.
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. This is consistent with the findings that folate conjugates are not transported by folate carriers.
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Updated at: 2019-09-03
Created at: 2017-04-26
Written by: Vesa Oikonen