ABC Transporters and P-glycoprotein

• ABC transporters are membrane proteins that use ATP hydrolysis to transport substrates across membranes, functioning as either importers or exporters, but not both.
• Exporters include P-gp (MDR1), while importers are mainly found in prokaryotes, such as maltose or methionine uptake transporters.
• These transporters are typically expressed on either the apical or basolateral side of polarized cells, not both.
• ABC transporters protect the body from xenobiotics and have physiological roles, like bile transport in the liver and insulin regulation.
• They can interfere with drug circulation by pumping compounds back into the bloodstream.
• BCRP, MDR3, and MRP1 exhibit multi-drug resistance.

P-glycoprotein (P-gp) -- ABCB1

• P-gp, encoded by the ABCB1 gene, is a major contributor to multi-drug resistance in cancer cells.
• The ABCB1 gene was discovered in 2000, with subsequent studies focusing on the functional consequences of its polymorphisms.
• It functions as an ATP-dependent pump with broad specificity, also known as MRP1.
• P-gp is found in animals, fungi, and bacteria, and has evolved as a defense against toxins.
• It is present in renal tubule brush border membranes, bile canaliculi, astrocyte foot processes in brain micro-vessels, and the GI tract.
• Polymorphisms in ABCB1 and SLCs contribute to individual genetic variation in drug responsiveness and can lead to drug-drug interactions.
• Collie dogs lacking MRP1 (involved in toxin transport from CSF across the BBB) are susceptible to ivermectin-induced neurotoxicity.
• The ABCB1 gene, located on chromosome 7 in humans, encodes this transporter.
• P-gp's primary functions include removing xenobiotics from enterocytes, transporting xenobiotics from bile across the canalicular membrane, and preventing xenobiotic transport in the brain by transporting them to the kidney lumen via brush-border membranes.
• Cancer cells can exhibit high levels of P-gp, causing drug resistance.
• Rarer forms of ABCB4 and ABCB11 may contribute to inherited cholestasis.

Polymorphisms in ABCB1 Gene

• The ABCB1 gene contains 28 introns and exons, spanning approximately 12kb, with an mRNA of 4.7kb.
• Over 50 single nucleotide polymorphisms (SNPs) have been identified.
• The three most common and studied polymorphisms are:
  • Exon 12: Silent C1236T (nucleotide 1236)
  • Exon 21: Ala393Ser (G2677T/A), resulting in an Ala to Ser change.
  • Exon 26: Silent C3435T, which can result in a non-functional protein.
• The 3435G>T polymorphism affects RNA stability.
• The three common haplotypes are TTC, CGT, and CGC, with allele frequencies varying by ethnicity.

Proposed Effects of ABCB1 Polymorphisms

• Studies suggest ABCB1 polymorphisms can affect mRNA and protein expression and stability of the MRP1 transporter.
• Synonymous SNP 3435C>T is associated with decreased mRNA and protein levels through an unknown mechanism.
• SNP 3435C>T is associated with Parkinson's Disease (PD), inflammatory bowel disease, and other conditions.
• Common SNPs in the coding region of ABCB1 are 1236C>T, 2677G>T/A, and 3435C>T.
• 1236 and 3435 are synonymous mutations, while 2677 causes an amino acid substitution (889 Ala>Ser/Thr).
• 1236 and 2677 are functional SNPs.
• The C1236T variant lowers p-gp expression by 11%, while G2677T/A and C3435T variants lower it by 16%.
• Homozygotes of TT are associated with 42% and 47% increases in p-gp transport activity.
• TT SNPs in MDR1 are associated with increased P-gp protein efflux activity, while C3435T and G2677T/A are related to decreased placental P-gp protein expression.
• Majority variants of MDR1 accumulated Rh123 intracellularly and lowered efflux of R123 compared to the wt MDR1.

ABCB1 Haplotypes and Drug Availability/Efficacy

• Simvastatin and Atorvastatin Pharmacokinetics:
  • In a study with Finnish volunteers, the frequencies of occurrence of haplotypes were 42.7% and 34.4%.
  • Simvastatin AUC was 60% larger and atorvastatin half-life was 24% longer in patients with the ABCB1 TTT/TTT genotype compared to those with GGC/GGC.
  • No differences were observed between the two genotypes regarding the pharmacokinetics of lactones of these drugs.

Simvastatin, Haplotypes, and Cholesterol Reductions

• Common polymorphisms in ABCB1, CYP3A4, and CYP3A5 genes and lipid-lowering efficacy and safety (using Simvastatin) were investigated.
• The CYP3A subfamily metabolizes lactone and hydroxy acid forms of simvastatin.
• CYP3A4 and CYP3A5 catalyze the formation of the main simvastatin metabolites.
• Functional polymorphisms of CYP3A41B and CYP3A53 were found.
• Simvastatin hydroxy acid is a P-gp substrate, while the lactone form has more efficacy than the acid form.

More Evidence on ABCB1 and Statins

• Patients with the TT genotype had a higher risk of developing Statin-Associated Muscle Symptoms (SAMS) compared to wild-type and heterozygous carriers.
• The presence of the TT genotype in all three ABCB1 polymorphisms nearly doubled the risk of SAMS.

Contradictory Findings

• No significant effect of ABCB1 haplotypes on the pharmacokinetics of fluvastatin, pravastatin, lovastatin, and rosuvastatin was observed (Keskitalo et al, 2009).
• Human MDR1 polymorphisms G2677T/A and C3435T did not affect MDR1 transport activities (Maria N et al, 2003).
• No significant observations were made between the 5 polymorphisms of MDR1 and the wild type MDR1 gene, so SNPs had no effect on transport activities of MDR1 proteins expressed in LLC-PK1 cells in vitro.
• Subjects possessing the 3435T allele had a significantly higher AUC of 3'-phydroxypaclitaxel compared to those possessing the 3435C allele
• An allelic variant of MDR1 may functionally affect PKs of its metabolite although genotyping of CYP2C8, CYP3A4 and MDR1 genes might not be essential to predict adverse effects of paclitaxel in Japanese patients
• The ABCB1 (C1236T) polymorphism affects P-gp-mediated transport of osteosarcoma drugs in a drug-specific way.

Importance of the ABCB1 (C1236T) SNP for P-gp Activity

• In vivo studies show a close relationship between ABCB1 (C1236T) SNPs and the efficacy of osteosarcoma drugs.
• Two Cao-cell lines were generated by transfection with either wild-type 1236C or 1236T variant allele, and compared in terms of drug resistance in osteocarcinoma cells
• Actinomycin D accumulated to similar extents in cells overexpressing wild-type or variant P-gp.
• Methotrexate and etoposide were transported to a greater extent by variant P-gp than wild-type protein, but doxorubicin was transported to a greater extent by wild-type P-gp.

Summary of ABCB1 Effects

• Despite extensive studies, there is no consensus on overall effects and relevance to drug efficacy, possibly due to transporter complexity and links between transport and metabolism.

ABCG2 - Breast Cancer Resistance Protein

• ABCG2 has a similar location to P-gp and high levels in lactating breasts.
• It secretes xenobiotics into milk, which has implications for breast-fed infants.
• BCRP evidence suggests imatinib is a substrate for BCRP, CML stem cells appear to have higher levels of ABCG2.
• Genetic polymorphism in an amino acid change in BCRP is a risk factor for gout (accumulation of uric acid crystals in joints occurs resulting in pain ability to excrete this compound).
• Uric acid is a substrate for BCRP, and an unstable form of the protein results in a poor ability to excrete this compound.
• Uric acid accumulation in the body can cause issues in the joints (high levels in synovial fluid) and kidneys (uric acid kidney stones).
• ABCG2 makes an unstable protein by the second mutation H155A.

ABCG2 Polymorphisms

• C421A is one of the most common ABCG2 polymorphisms with high allele frequency.
• Allele frequency varies with ethnicity, being higher in Asians (29-36%) than Europeans (4.5-12%) and Africans (0-2.3%).
• C421A is a significant risk factor for developing gout.
• This polymorphism results in the amino acid change Gln141Lys.

Effects of ABCG2 Polymorphisms

• Impaired protein folding and cellular processing occurs
• Protein expression in the cell membrane is reduced by 30-40%.
• Transport activity is reduced (by >20% in vivo), leading to reduced efflux and increased drug absorption.

ABCG2 Genotype and Rosuvastatin Pharmacokinetics

• BCRP normally pumps a large proportion of rosuvastatin back into the lumen of the small intestine.
• Individuals with the variant C421A may have lower BCRP protein concentration.
• Individuals homozygous for the A/A allele have a high concentration of this mutant transporter in the GI tract lumen compared to the C/C genotype
• This can lead to muscle toxicity if drugs are given in higher concentrations.

SLOC1B1

• SLOC1B1 is part of the OATP family (organic anion transporting polypeptide).
• Transporters are encoded by SLOC1 to 6 genes
• OATP1B1, OATP1B3 and OATP2B1 - most important in the liver
• Families 1 and 2 are most important in relevance to drug disposition (SLOC1 and SLOC2 also called OATP1 and OATP2)
• These transport drugs into the sinusoidal membrane of the liver.
• Drug substrates are anionic, have a high MW, and are generally plasma protein bound.
• Examples of substrates for OATP1B1 are statins, rifampicin, and benzylpenicillin.
• Non-synonymous polymorphisms in SLOC1B1 appear to affect the pharmacokinetics of several widely used drugs, including statins.

SLOC1B1 Polymorphisms

• This gene encodes for OATP1B1; also called OATC, SLC21A6.
• OATP1B1 transports anionic drugs into cells, especially hydrophilic compounds, into the liver, reducing systemic availability.
• Located at the sinusoidal membrane.
• Many polymorphisms give rise to amino acid substitutions in the SLOC1B1 coding sequence.
• Some lead to decreased transport. However, increased transport activity is seen with some rarer amino acid substitutions.
• Haplotype frequency varies with ethnicity.

Val174Ala Polymorphism and Pravastatin

• Japanese subjects with the SLCO1B1*1B/*15 genotype had a significantly reduced oral clearance of pravastatin compared to subjects with the *1B/*1B genotype.
• White German subjects with the *1A/*5 genotype had a significantly greater area under the plasma concentration-time curve (AUC) of pravastatin.
• White Finnish subjects with the SNPs g.−11187G>A and c.521T>C and the haplotypes *17 (g.−11187A, c.388G, c.521C) and *15 were associated with an AUC of pravastatin that was increased by about 2-fold, and the short-term effect of pravastatin on hepatic cholesterol synthesis was significantly reduced in carriers of the *17 haplotype.
• No studies on the effects of SLCO1B1 polymorphism on the pharmacokinetics of fluvastatin.

SLOC1B1 Genotypes and Rosuvastatin Pharmacokinetics

• The SLOC1B1 genotype can predict OATPC function.
• Homozygous for *14 (AA) or *20 (CC) have highly increased OATPC Funtion.
• Heterozygous for *14 (CA) or *20 (AC) have increased OATPC Funtion.
• Normal TT, decreased = heterozygous for *5 (TC) funtion, and poor = Homozygous for *5 (CC).

SLOC1B1 Genotypes and Simvastatin

• Nguyen et al, 2023 found The most well-known variant is rs4149056 [c.521T>C (Val174Ala)], which contributes to various haplotypes including the allele SLCO1B1*5.
• The frequency of this function-decreased allele is about 2% in European and less than 1% in other populations.
• Another extensively studied variant of the SLCO1B1 gene is rs2306283 [c.388A>G (Asn130Asp)], which contributes to several alleles including SLCO1B1 *14,*15,*20,*31,*37,*46,*47.
• The rs2306283 frequency is higher in Asian and African people than in European people.
• Besides, some others such as rs11045819 [c.463C>A (Pro155Thr)] which uniquely belongs to the allele SLCO1B1*14 and rs4363657 [g.89595T>C (intron)] are available in the dbSNP database.

SLOC22A1

• It is a solute carrier and encodes cationic transporters OCT1 & OCT2.
• These transporters play an important role in transport of cationic drugs into hepatocytes and renal tubule cells, respectively.
• There is increasing evidence that coding region polymorphisms in these genes are relevant to metformin response and to cisplatin-induced nephrotoxicity.

Glucose-6-phosphate dehydrogenase (G6PD)

• Required to produce the antioxidant GSH (Glutathione).
• GSH limiting due to low G6PDH and lack of NADPH, haemolytic anaemia can result due to low levels of GSH in erythrocytes.

G6PDH Deficiency

• G6PD is a genetic defect.
• G6PDH is a 14 exon gene located on the X chromosome.
• G6PDH deficiency is therefore more common in males.
• Quite common in individuals of African or Mediterranean origin - protective against malaria.
• Exposure to certain drugs or pro-oxidant xenobiotics (e.g., fava beans) may trigger haemolytic anaemia.
• Over 230 mutations with reduced protein function have been identified.
• Clinical severity varies from class 1 (most severe) to 5 (mild) based on enzyme residual activity.
• Affect catalytic properties, structural parameters and 3D structure

Some G6PDH Variants

• Some G6PDH variants include: Asp142Asn and Val68Met
• Mahidol: Gly163Ser
• Mediterranean: Ser188Phe
• Vanua Lava: Leu128Pro
• Viangchan: Val291Met
• Affect catalytic properties, structural parameters and 3D structure
• Clinical severity varies - class 1 (most severe) - 5 (mild) based on enzyme residual activity

Compounds Associated with Drug-Induced Haemolysis

• Nitrofurantoin
• Primaquine
• Sulphamethoxazole
• Acetanilide
• Naphthalene

ABC transporters use ATP to move substrates across membranes, functioning as importers or exporters. P-glycoprotein (P-gp), encoded by ABCB1, contributes to multi-drug resistance in cancer. ABCB1 was discovered in 2000, with studies focusing on its polymorphisms and ATP-dependent functions.