6.5 Transporters

Transporters are vital membrane proteins that move molecules across biological barriers. They play a crucial role in drug absorption, distribution, and elimination, impacting how medications work in the body.

Understanding transporter types, structures, and functions is key to drug development. This knowledge helps predict how drugs interact with the body, their effectiveness, and potential side effects, guiding the creation of safer, more targeted medications.

Types of transporters

• Transporters are membrane proteins that facilitate the movement of molecules across biological membranes, playing a crucial role in drug absorption, distribution, and elimination
• They can be classified based on their energy requirements (passive vs active), the direction of transport (influx vs efflux), and the number and type of substrates they transport (uniporter vs symporter vs antiporter)

Passive vs active transport

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• Passive transport occurs down the concentration gradient without the requirement of energy input and includes simple diffusion and facilitated diffusion (via channels or carriers)
• Active transport moves molecules against their concentration gradient by utilizing energy in the form of ATP hydrolysis (primary active transport) or by coupling with the movement of another molecule down its concentration gradient (secondary active transport)
• Examples of passive transport: Glucose transport via GLUT1, drug diffusion across cell membranes
• Examples of active transport: P-glycoprotein (P-gp) efflux of chemotherapeutic agents, sodium-potassium ATPase pump

Uniporter vs symporter vs antiporter

• Uniporters transport a single substrate in one direction, either into or out of the cell (GLUT1 glucose transporter)
• Symporters co-transport two or more substrates in the same direction, often coupling the movement of one substrate down its concentration gradient with the uphill transport of another substrate (sodium-glucose co-transporter SGLT1)
• Antiporters, also known as exchangers, transport two or more substrates in opposite directions across the membrane (sodium-calcium exchanger NCX)

Influx vs efflux transporters

• Influx transporters facilitate the movement of molecules into the cell, such as the uptake of nutrients, ions, or drugs (organic cation transporter OCT1)
• Efflux transporters move molecules out of the cell, often serving as a protective mechanism against xenobiotics or endogenous toxins (breast cancer resistance protein BCRP)
• The balance between influx and efflux transporters can significantly impact the intracellular concentration and pharmacokinetics of drugs

Structure of transporters

• Transporters are integral membrane proteins that span the lipid bilayer, typically consisting of multiple transmembrane domains, substrate binding sites, and regions that undergo conformational changes during the transport process
• Understanding the structure-function relationship of transporters is crucial for designing targeted drugs and predicting drug-transporter interactions

Transmembrane domains

• Transporters contain multiple alpha-helical transmembrane domains (TMDs) that traverse the lipid bilayer, forming a channel or pathway for substrate translocation
• The number of TMDs varies among different transporter families, ranging from 6 to 12 or more (P-gp has 12 TMDs, while GLUT1 has 12)
• TMDs are essential for substrate recognition, binding, and translocation, as well as maintaining the overall structural integrity of the transporter

Substrate binding sites

• Transporters possess specific binding sites that recognize and interact with their substrates, determining the selectivity and affinity of the transport process
• Substrate binding sites are typically located within the transmembrane regions or at the interface between TMDs and can be highly specific or accommodate a broad range of structurally diverse compounds (P-gp has multiple binding sites for various substrates)
• Mutations or polymorphisms in substrate binding sites can alter transporter function and lead to changes in drug disposition and response

Conformational changes during transport

• Transporters undergo conformational changes during the transport cycle, alternating between inward-facing and outward-facing states to allow substrate translocation across the membrane
• These conformational changes are often driven by the binding and hydrolysis of ATP (in primary active transporters) or the movement of ions down their electrochemical gradient (in secondary active transporters)
• Examples include the "alternating access" mechanism in ABC transporters and the "rocker-switch" mechanism in major facilitator superfamily (MFS) transporters

Function of transporters

• Transporters play a vital role in regulating the absorption, distribution, and elimination of drugs, nutrients, and endogenous substances, making them important targets for drug development and understanding drug disposition
• They are expressed in various tissues and organs, including the intestine, liver, kidney, and blood-brain barrier, where they can significantly impact drug pharmacokinetics and pharmacodynamics

Role in drug absorption

• Transporters expressed in the intestinal epithelium can facilitate or limit the absorption of orally administered drugs
• Uptake transporters (PEPT1, OATP2B1) can enhance drug absorption by mediating the active uptake of drugs from the intestinal lumen into enterocytes
• Efflux transporters (P-gp, BCRP) can reduce drug absorption by pumping drugs back into the intestinal lumen, limiting their systemic exposure

Impact on drug distribution

• Transporters expressed in tissue barriers (blood-brain barrier, blood-testis barrier) and various organs (liver, kidney) can influence the distribution of drugs to their target sites
• Efflux transporters (P-gp, BCRP) at the blood-brain barrier can restrict the entry of drugs into the central nervous system, while uptake transporters (OAT1, OAT3) in the kidney can facilitate the renal secretion of drugs
• The interplay between uptake and efflux transporters determines the tissue-specific distribution and accumulation of drugs

Significance in drug elimination

• Transporters expressed in the liver and kidney play a crucial role in the elimination of drugs and their metabolites from the body
• Hepatic uptake transporters (OATP1B1, OATP1B3) mediate the uptake of drugs into hepatocytes, where they can undergo metabolism or biliary excretion
• Renal uptake transporters (OAT1, OCT2) facilitate the secretion of drugs into the urine, while efflux transporters (P-gp, MRP2) in the kidney can also contribute to drug elimination
• Transporter-mediated drug elimination can significantly impact the pharmacokinetics and half-life of drugs

Transporter-mediated drug interactions

• Drug-drug interactions involving transporters can occur when one drug alters the function of a transporter, leading to changes in the absorption, distribution, or elimination of another drug
• These interactions can result in increased or decreased drug exposure, potentially affecting drug efficacy and safety
• Understanding and predicting transporter-mediated drug interactions is crucial for optimizing drug therapy and avoiding adverse effects

Inhibition of transporters

• Drugs can inhibit the function of transporters by competing for binding sites or by allosteric modulation, leading to reduced transport activity
• Inhibition of efflux transporters (P-gp) can increase the absorption and systemic exposure of substrate drugs, while inhibition of uptake transporters (OATP1B1) can decrease drug uptake and clearance
• Examples of transporter inhibitors: Cyclosporine (P-gp inhibitor), rifampin (OATP1B1 inhibitor)

Induction of transporters

• Drugs can induce the expression of transporters by activating transcription factors or by stabilizing transporter mRNA, leading to increased transport activity
• Induction of efflux transporters (P-gp) can decrease the absorption and systemic exposure of substrate drugs, while induction of uptake transporters (OATP1B1) can increase drug uptake and clearance
• Examples of transporter inducers: Rifampin (P-gp inducer), phenytoin (MRP2 inducer)

Consequences for drug pharmacokinetics

• Transporter-mediated drug interactions can significantly alter the pharmacokinetics of drugs, affecting their absorption, distribution, metabolism, and elimination (ADME) properties
• Inhibition of efflux transporters can lead to increased drug absorption and bioavailability, while inhibition of uptake transporters can result in decreased drug uptake and clearance
• Induction of efflux transporters can cause decreased drug absorption and increased clearance, while induction of uptake transporters can enhance drug uptake and clearance
• These changes in pharmacokinetics can impact drug efficacy, toxicity, and dosing requirements, emphasizing the need for careful consideration of transporter-mediated interactions in drug development and clinical practice

Clinically relevant transporters

• Several transporter families have been identified as clinically relevant due to their significant impact on drug disposition, efficacy, and safety
• These transporters are often involved in drug-drug interactions, genetic polymorphisms, and interindividual variability in drug response
• Understanding the role of these transporters in drug disposition and their tissue-specific expression patterns is essential for optimizing drug therapy and predicting potential drug-related issues

ABC transporters (P-gp, BCRP, MRPs)

• ATP-binding cassette (ABC) transporters are a large family of efflux transporters that use ATP hydrolysis to pump substrates out of cells
• P-glycoprotein (P-gp, ABCB1) is expressed in the intestine, liver, kidney, and blood-brain barrier, and plays a key role in limiting drug absorption and distribution (digoxin, loperamide)
• Breast cancer resistance protein (BCRP, ABCG2) is expressed in the intestine, liver, and blood-brain barrier, and can affect the disposition of various drugs (topotecan, rosuvastatin)
• Multidrug resistance-associated proteins (MRPs, ABCCs) are involved in the efflux of drugs and their conjugated metabolites (methotrexate, SN-38 glucuronide)

SLC transporters (OATPs, OCTs, OATs)

• Solute carrier (SLC) transporters are a diverse group of influx transporters that mediate the uptake of drugs and endogenous substrates into cells
• Organic anion transporting polypeptides (OATPs) are expressed in the liver, intestine, and blood-brain barrier, and facilitate the uptake of various drugs (statins, rifampin)
• Organic cation transporters (OCTs) are expressed in the liver and kidney, and mediate the uptake of cationic drugs (metformin, cisplatin)
• Organic anion transporters (OATs) are expressed in the kidney and brain, and are involved in the renal secretion and distribution of anionic drugs (antibiotics, antivirals)

Tissue-specific expression patterns

• The expression of transporters varies across different tissues and organs, contributing to the selective distribution and accumulation of drugs
• Intestinal transporters (P-gp, BCRP, PEPT1) play a crucial role in determining oral drug absorption and bioavailability
• Hepatic transporters (OATP1B1, OATP1B3, OCT1) are involved in the uptake and clearance of drugs in the liver, impacting first-pass metabolism and systemic exposure
• Renal transporters (OAT1, OAT3, OCT2, P-gp) mediate the secretion and reabsorption of drugs in the kidney, influencing drug elimination and potential nephrotoxicity
• Blood-brain barrier transporters (P-gp, BCRP, OATP1A2) restrict the entry of drugs into the central nervous system, affecting drug distribution to the brain

Transporter polymorphisms

• Genetic variations in transporter genes can lead to altered transporter expression, function, and substrate specificity, contributing to interindividual variability in drug response and toxicity
• Polymorphisms in transporter genes can result in reduced, increased, or absent transporter activity, affecting drug absorption, distribution, and elimination
• Identifying and characterizing transporter polymorphisms is crucial for personalized medicine and optimizing drug therapy based on an individual's genetic profile

Genetic variations in transporter genes

• Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation in transporter genes, resulting in amino acid changes or altered gene expression
• Copy number variations (CNVs) and insertions/deletions (indels) can also occur in transporter genes, leading to changes in transporter function and expression
• Examples of transporter polymorphisms: ABCB1 (P-gp) C3435T, SLCO1B1 (OATP1B1) c.521T>C, ABCG2 (BCRP) c.421C>A

Impact on drug response and toxicity

• Transporter polymorphisms can significantly affect drug pharmacokinetics, leading to altered drug exposure and response
• Reduced function variants can lead to increased drug absorption and systemic exposure, potentially increasing the risk of adverse effects (SLCO1B1 c.521T>C and simvastatin-induced myopathy)
• Increased function variants can result in decreased drug absorption and reduced efficacy (ABCG2 c.421C>A and reduced response to allopurinol)
• Transporter polymorphisms can also influence the distribution of drugs to target tissues, affecting drug efficacy and toxicity (ABCB1 polymorphisms and CNS side effects of antipsychotics)

Pharmacogenomics of transporters

• Pharmacogenomics aims to understand how genetic variations in transporters influence drug response and to develop personalized treatment strategies based on an individual's genetic profile
• Genotyping of transporter polymorphisms can help predict drug response and guide dose adjustments to optimize therapy and minimize adverse effects
• Incorporating transporter pharmacogenomics into drug development and clinical practice can improve drug safety and efficacy, and reduce healthcare costs associated with adverse drug reactions
• Examples of transporter pharmacogenomics in clinical practice: SLCO1B1 genotyping for simvastatin dosing, ABCG2 genotyping for allopurinol dose adjustment

Transporter-based drug delivery strategies

• Exploiting the expression and function of transporters can be a promising approach for targeted drug delivery and overcoming drug resistance
• Transporter-based drug delivery strategies aim to enhance drug absorption, distribution, and accumulation at target sites while minimizing off-target effects and toxicity
• These strategies involve the design of prodrugs, transporter inhibitors, and targeted drug conjugates that can selectively interact with specific transporters

Targeted drug delivery via transporters

• Targeting drugs to specific tissues or cell types can be achieved by exploiting the tissue-specific expression of transporters
• Drug conjugates or nanoparticles can be designed to interact with uptake transporters expressed in target tissues, enhancing drug accumulation and efficacy (folate receptor-targeted drug delivery in cancer cells)
• Inhibiting efflux transporters in target tissues can also increase drug retention and therapeutic effects (P-gp inhibitors for enhancing chemotherapy efficacy in multidrug-resistant tumors)

Prodrugs and transporter-mediated uptake

• Prodrugs are inactive compounds that are converted to active drugs by metabolic or enzymatic processes in the body
• Designing prodrugs that are substrates for uptake transporters can enhance drug absorption and bioavailability (valacyclovir, a prodrug of acyclovir, is a substrate for PEPT1)
• Prodrugs can also be designed to target specific tissues or cell types by exploiting transporter expression patterns (HMG-CoA reductase inhibitor prodrugs targeting hepatic OATP1B1)

Overcoming multidrug resistance

• Multidrug resistance (MDR) is a major obstacle in cancer chemotherapy, often mediated by the overexpression of efflux transporters (P-gp, BCRP, MRPs) in cancer cells
• Transporter inhibitors can be used to overcome MDR by blocking the efflux of chemotherapeutic agents, increasing their intracellular accumulation and efficacy (verapamil, a P-gp inhibitor, in combination with chemotherapy)
• Designing drugs that are poor substrates for efflux transporters or using nanoparticle drug delivery systems can also help overcome MDR and improve therapeutic outcomes

In vitro and in vivo transporter assays

• In vitro and in vivo transporter assays are essential tools for studying transporter function, identifying substrates and inhibitors, and predicting drug-transporter interactions
• These assays provide valuable information for drug discovery, development, and regulatory decision-making, and help optimize drug therapy and safety

Cell-based transporter assays

• Cell-based assays use cell lines overexpressing a specific transporter to study its function and interaction with drugs
• Bidirectional transport assays measure the permeability of drugs across cell monolayers, comparing the apical-to-basolateral (A-B) and basolateral-to-apical (B-A) transport to assess transporter-mediated efflux (Caco-2, MDCK-MDR1 cells)
• Uptake assays measure the accumulation of drugs in cells expressing uptake transporters, often using radiolabeled or fluorescent substrates (HEK-OATP1B1, CHO-OCT2 cells)
• Inhibition assays assess the ability of compounds to inhibit transporter function, using known substrates and measuring changes in their transport (calcein-AM assay for P-gp inhibition)

Vesicle-based transporter assays

• Vesicle-based assays use membrane vesicles prepared from cells expressing a specific transporter to study its function and interaction with drugs
• Inside-out membrane vesicles allow the direct measurement of transporter-mediated uptake or efflux, without the influence