vivo, OCTN2 has not been reported as a target for drug–drug interactions to date. However, multiple drugs have been observed to cause carnitine deficiency through various mechanisms. Administration of the anticonvulsant valproic acid and the antibiotic pivalic acid causes reduced plasma carnitine levels and, in some cases, clinically relevant carnitine deficiency. Multiple mechanisms have been proposed. One possibility is that valproate and pivalate directly inhibit the binding pocket for carnitine in OCTN2. Other studies have suggested that rather than inhibit OCTN2 directly, these drugs form carnitine conjugates and are likely effluxed out of the kidney with poor reabsorption, resulting in carnitine wasting and depletion. Alternatively, the valproyl‐ and pivaloyl‐carnitine esters could block reabsorption of free carnitine at OCTN2. Regardless of mechanism, these cases of drug‐induced carnitine deficiency have been fatal in patients with carnitine transporter deficiency who already have reduced systemic carnitine levels.
In recent years, OCTN2 has become a target of drug delivery optimization strategies. Multiple properties make it an attractive drug target. First, it is theorized to increase oral bioavailability of targeted drugs due to high expression in the small intestine. Second, it has the potential to increase blood–brain barrier permeability of substrates due to expression at the BBB. Third, it allows for the targeting of drugs to the kidney. And fourth, it has been hypothesized to increase delivery of asthma therapeutics to the lung [90]. Multiple carnitine‐conjugated prodrugs have been developed, including butyrate used in treatment for gut inflammation, nepotic acid used to treat seizures, and the chemotherapeutic drug, gemcitabine. Carnitine‐conjugated gemcitabine exhibits 5‐fold bioavailability over gemcitabine alone. In addition, nanoparticles are being explored for targeted delivery via OCTN2 for other cancer drugs like paclitaxel.
2.9.2 Regulation
Transcriptional regulation of OCTN2 is mediated in part by the peroxisome proliferator‐activated receptor α (PPARα). PPARα plays an important role in the regulation of genes involved in lipid metabolism and energy homeostasis and is highly expressed in tissues that use fatty acid oxidation as a primary energy source, including heart muscle, skeletal muscle, and kidney [82]. Notably, OCTN2 is expressed highly in these tissues as well. Tentative PPAR response elements (PPREs) are found in the promoter and intronic regions of SLC22A5 in several species. In rats, treatment with the PPARα agonist clofibrate leads to increased transcription of OCTN2 in the liver and small intestine, but not the kidney or muscle tissue. Aligned with the upregulation of OCTN2 in rat liver, hepatic concentrations of carnitine are also increased by PPARα activation. These findings are supported by the downregulation of OCTN2 and overall reduction in systemic carnitine levels in PPARα‐null mice. Upregulation of OCTN2 by PPARα has also been demonstrated in pigs. In addition to fibrates, PPARα‐mediated regulation of OCTN2 is affected by cisplatin. Cisplatin is hypothesized to inhibit DNA binding to the PPARα/RXR complex, resulting in an overall downregulation of OCTN2 and an increase in urinary carnitine wasting in mice [91]. The PPARγ/RXRα complex also modulates OCTN2 expression in the large intestine. Human colonocytes and mouse colon exhibit altered expression of OCTN2 in response to PPARγ, but not PPARα [92]. In a mouse model of IBD, proinflammatory cytokines interact with the PPARγ/RXRα complex to reduce OCTN2 expression, contributing to disease pathology. Treatment with the PPARγ agonist luteolin to rescue OCTN2 expression results in the reduction of colonic inflammation [93].
OCTN2 is upregulated by the estrogen receptor (ER) in breast cancer cells and tumor tissue, an effect attributed to the identification of a novel estrogen‐responsive element (ERE) in an intronic region of SLC22A5 [2].
OCTN2 is further regulated by PDZ domain‐containing proteins [83]. PDZK1 colocalizes with OCTN2 at the apical membrane of renal tubule cells. PDZK1 stimulates carnitine uptake via OCTN2 by increasing the V max of the transporter, although cell‐surface expression of OCTN2 is unchanged suggesting PDZK1 stimulates translocation of carnitine. The four terminal amino acids at the carboxyl end of OCTN2 serve as a PDZ binding motif, and deletion or substitution of these residues eliminates PDZK1 stimulation of OCTN2. PDZK2 also increases the transport capacity of OCTN2, but through a different mechanism, increasing localization of OCTN2 to the plasma membrane [83].
Lastly, OCTN2 expression is regulated by heat shock transcription factor 1 (HSF1) [2]. The promoter variant −207G>C disrupts a consensus sequence for an HSF binding element. Cells with the −207G wild‐type promoter containing the intact HSF1 binding site have higher expression of OCTN2 after heat‐shock compared to cells with the −207C variant, which results in disrupted HSF1 binding.
2.9.3 Animal Models
Octn2 −/− knockout mice have been characterized as a model of carnitine deficiency, resulting from a point mutation that causes a change from the amino acid leucine to arginine at residue 352. This substitution causes complete OCTN2 loss‐of‐function in vitro and in vivo. The mice, deemed juvenile visceral steatosis (jvs) mice, present with growth retardation and enlarged abdomen due to hepatic steatosis, as well as hyperammonia and hypoglycemia [94]. Pharmacokinetics in jvs mice reveal drastically altered carnitine parameters, including reduced bioavailability, decreased volume of distribution, decreased tissue‐to‐plasma concentration ratios, and increased clearance of carnitine compared with wild‐type mice [95]. Furthermore, jvs mice display spontaneous intestinal apoptotic phenotypes including ulceration and gut perforation, and an immune response involving macrophage and lymphocyte infiltration [96]. Inflammation and intestinal apoptosis are reduced when mice are treated with carnitine supplementation.
2.9.4 Human Genetic Studies
Biallelic loss‐of‐function mutations in OCTN2 result in a Mendelian disease known as carnitine transporter deficiency (CTD, also referred to as primary carnitine deficiency, systemic carnitine deficiency, or carnitine uptake defect; OMIM #212140) [67]. Almost 200 OCTN2 mutations have been identified among patients, the majority of which are missense, followed in frequency by nonsense, frameshift, and noncoding mutations that affect splice sites or regulatory regions [88]. Systemically, patients display extremely low plasma carnitine levels caused by reduced dietary carnitine absorption and excessive carnitine wasting in the urine due to loss of reabsorption by OCTN2 [97]. The disorder varies in time to onset and disease severity and/or presentation, with common symptoms including cardiomyopathy, cardiac arrhythmias, hepatic encephalopathy, and hypoglycemia. Missed diagnosis can be fatal in infants, thus many developed countries screen infants for CTD, among other disorders, at birth. Diagnosis in individuals with less severe disease can be delayed well into adulthood, exemplified by the identification of maternal carnitine deficiency from low carnitine levels in the newborn during screening. CTD is treated with supplemental carnitine at high doses, up to 200 mg/kg multiple times per day, with decent outcomes. Lack of adherence to treatment has resulted in sudden death in at least one report. Incidence of CTD varies globally, affecting 1:120,000 individuals in Australia, 1:75,000 in the United States, 1:40,000 in Japan, 1:27,000 in China, and up to 1:300 individuals in the Faroe Islands [97]. In at least one case, CTD has manifested as intellectual disability and autism spectrum disorder [98].
Extensive functional genomic studies have been conducted for OCTN2. The common promoter variant −207G>C is well characterized, known to decrease transcription of OCTN2 due to disruption of HSF1 transcription factor binding. Another variant in the 5′‐UTR of the gene (−149G>A) creates an early ATG translation start site, reducing the translation of wild‐type OCTN2 and decreasing carnitine transport [99]. This variant has been found repeatedly in CTD patients for whom no or only one known deleterious variant is detected. Many studies have functionally characterized OCTN2 variants associated with CTD. Loss‐of‐function results from multiple mechanisms, including altered kinetic parameters decreasing carnitine affinity or capacity for transport, decreased affinity for sodium, and reduced plasma membrane localization.
