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Chair of Pharmacology, Department of Experimental and Clinical Medicine, Faculty of Medicine and Surgery, University Magna Græcia of Catanzaro; Clinical Pharmacology and Pharmacovigilance Unit, Mater Domini University Hospital, Catanzaro, Italy
Researcher, Chair of Pharmacology, Department of Experimental and Clinical Medicine, Faculty of Medicine and Surgery, University Magna Græcia of Catanzaro; Clinical Pharmacology and Pharmacovigilance Unit, Mater Domini University Hospital
Toxicology Unit, ASL 8, Lamezia Terme, Italy
Full Professor, Chair of Pharmacology, Department of Experimental and Clinical Medicine, Faculty of Medicine and Surgery, University Magna Græcia of Catanzaro; Clinical Pharmacology and Pharmacovigilance Unit, Mater Domini University Hospital
Clinical Specialist, Department of Medical Sciences, Clinical Pharmacology, Uppsala University, Uppsala, Sweden
Reprints: Dr. Gallelli, Department of Experimental and Clinical Medicine, School of Medicine, University Magna Græcia of Catanzaro, Clinical Pharmacology Unit, Mater Domini University Hospital, via Tommaso Campanella, 115, 88100 Catanzaro, Italy, fax 39-0961-774424, luca_gallelli{at}hotmail.com
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CASE SUMMARY: A 25-year-old man (weight 55 kg, height 165 cm) with a 12-year history of polydrug abuse was admitted to an opiates withdrawal methadone program. At the time of our observation, he was using both cannabinoids and heroin; no other medical conditions were discovered. Within the opiates withdrawal methadone program, under medical supervision, the patient started methadone therapy (20 mg/day). Two weeks later, an Abuscreen assay for methadone screening in the urine was negative and, to prevent the development of withdrawal symptoms, the dose of methadone was increased to 60 mg/day. One day later, the patient was asked to collect another urine sample in the presence of a nurse. The Abuscreen for methadone in urine remained negative. Evaluation of urinary samples collected over 24 hours documented low concentrations of methadone and high levels of 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (the primary metabolite of methadone). Evaluation for the presence of the most common polymorphisms in the cytochrome P450 and P-glycoprotein genes showed that the patient was heterozygous for the CYP3A5*1 allele and for 2 single nucleotide polymorphisms in the P-glycoprotein gene (1236C/T and 3435C/T).
DISCUSSION: In this patient, poor methadone adherence was ruled out because of the presence of physicians and nurses during both methadone maintenance treatment and Abuscreen screening. Moreover, because the patient reported only heroin and cannabis at the time of evaluation, drug interactions were ruled out as possible causes for the rapid clearance of methadone.
CONCLUSIONS: In this case, CYP3A5 polymorphism may have played a role in the rapid methadone metabolism.
Key Words: methadone, CYP3A5, CYP2C19, P-glycoprotein gene, polydrug abuser
Published Online, May 6, 2008. www.theannals.com, DOI 10.1345/aph.1K539
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Blood chemical tests confirmed the presence of microcythemia (hemoglobin
11.8 g/dL; mean corpuscular volume 64.7 mm3; mean
corpuscular hemoglobin 20.7 µg, mean corpuscular hemoglobin content 32%).
Tests for hepatitis A, B, and C viruses; Epstein-Barr virus; HIV; and
cytomegalovirus showed the presence of hepatitis C infection with no signs of
liver failure (aspartate aminotransferase 18 U/L, alanine aminotransferase 27
U/L, 
-glutamyl-transpeptidase 120 U/L).
On December 2, under medical supervision, the patient started methadone therapy (20 mg/day). Two weeks later, a radioimmunoassay screening test (Abuscreen, Roche Diagnostics) for methadone in urine was negative, and to prevent the development of withdrawal symptoms, the methadone dose was increased to 60 mg/day. One day later, the patient was asked to collect a urine sample in the presence of a nurse; the Abuscreen test for methadone in urine remained negative. Poor adherence was ruled out because of the presence of physicians and nurses during both methadone treatment and urine screening test. At the time of this observation, the patient had reported use of only heroin and cannabis. Therefore, drug interactions were ruled out as a possible cause for the rapid clearance of methadone.
To evaluate the methadone kinetics during maintenance treatment (60 mg/day), plasma samples were analyzed using liquid chromatography–mass spectrometry. The tests documented a low methadone concentration (200 ng/mL)7 with an increase in EDDP levels (51.5 ng/mL).8 Evaluation of urine samples collected at different times (6, 12, 18, and 24 h) and analyzed for methadone and EDDP concentrations by a validated high-performance liquid chromatography (HPLC) assay9 documented low concentrations of methadone and high levels of EDDP (Table 1).
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The intra- and interassay precision for both HPLC and liquid chromatography–mass spectrometry methods was validated at the cut-off concentration used for the analysis (300 ng/mL).
To evaluate the role of genetic factors in methadone kinetics in this patient, genotyping was performed for the main allelic variants of the cytochrome P450 isoforms 2C19, 2D6, and 3A4/5, as well as for the P-glycoprotein coding gene (MDR1) polymorphisms. The CYP2C19 and MDR1 polymorphisms were analyzed with real-time polymerase chain reaction (PCR; TaqMan kits, Applied Biosystems), while the presence of allelic variants of CYP2D6, CYP3A4, and CYP3A5 were identified by PCR-restriction fragment length polymorphism, as previously described.10-18
The patient did not carry any CYP2D6, CYP2C19, or CYP3A4 detrimental alleles. Furthermore, he did not carry extra copies of a functional CYP2D6 allele or the CYP2C19*17 allele; therefore, he was not genetically classifiable as an ultra-rapid metabolizer for these 2 enzymes. Conversely, he was found to be heterozygous for CYP3A5*1 (genotype CYP3A5*1/*3), therefore expressing a functional CYP3A5 protein. In addition, he was heterozygous for 2 SNPs in the P-glycoprotein gene (MDR-1): ABCB1 1236C/T (rs1128503) and ABCB1 3435C/T (rs1045642).
Urinary and plasma concentrations of methadone and EDDP were measured at the Mater Domini Hospital of Catanzaro; genetic tests were performed at Uppsala University.
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To evaluate the role of genetic factors in the drug's kinetics, genetic tests were performed. Our patient did not carry any CYP2C19, CYP2D6, or CYP3A4 detrimental allele. Conversely, he was heterozygous for CYP3A5*1 (genotype CYP3A5*1/*3).
Methadone metabolism is predominantly mediated by cytochrome P450, in particular by CYP3A4,3,19,20 CYP2B6,20-22 CYP2D6,23 and, to some extent, by CYP2C19.21 The primary metabolic pathway is N-demethylation to EDDP, which is further N-demethylated to 2-ethyl-5-methyl-3,3-diphenyl-1-pyrroline.20-24 Other minor metabolic pathways include formation of methadol and normethadol. Human intestinal microsomes also N-demethylate methadone, and CYP3A4 is considered to play a pivotal role in this metabolic pathway.2,25 The predicted in vivo first-pass methadone extraction, based on in vitro kinetics, is 21%.26 These in vitro results suggest that both the intestine and liver act as sites of methadone first-pass metabolism and clearance, and that both intestinal and hepatic CYP3A4 may be responsible for methadone oral and systemic clearance, thereby playing a pivotal role in interindividual metabolic variability. CYP3A4 expression and activity vary greatly among individuals.27 Some polymorphic sites in the regulatory region of the CYP3A4 gene have been described, but their functional importance remains unclear.
Unlike CYP3A4, the hepatic expression of CYP3A5 is bimodally distributed, indicating the existence of polymorphisms.27 Several genetic variants have been described for CYP3A5, and the most common, the CYP3A5*3 allele, causes the loss of CYP3A5 activity. Thus, only individuals carrying at least one CYP3A5*1 allele express large amounts of CYP3A5.28,29 This polymorphism has been reported to influence total CYP3A activity and shows racial differences in its frequency.30
Thus, a substantial change in CYP3A5 activity might influence the pharmacokinetics of CYP3A substrates.31 In fact, it has been shown that patients with CYP3A5*1/*1 and *1/*3 genotypes require a significantly higher sirolimus daily dose to achieve the same blood concentration at steady-state as *3/*3 patients.32 In addition, Utecht et al.33 reported that carriers of CYP3A5*1 have higher clearance rates of tacrolimus than CYP3A5*3 homozygotes. Furthermore, in most cases, subjects expressing CYP3A5 express very high levels of CYP3A4. Therefore, even if CYP3A5 was not shown to play an active role in methadone metabolism in vitro,20,25 since it may represent up to 50% of the total hepatic CYP3A content in subjects expressing it,28 and in view of the fact that subjects expressing it also have very high levels of CYP3A4 activity, it might be an important contributor to the interindividual variability in methadone metabolism.
Conversely, previous results suggest that, although CYP2B6 influences (S)-methadone plasma levels, since only (R)-methadone contributes to the opioid effect of this drug, a major influence of CYP2B6 genotype on response to treatment is unlikely and has not been shown.34
It has been reported that methadone is a P-glycoprotein substrate, a transmembrane efflux transporter belonging to the adenosine triphosphate–binding cassette (ABCB1) family, encoded by the multidrug resistance 1 gene (MDR1).35,36 P-glycoprotein is expressed in various human tissues, such as gut, liver, kidneys, testes, lymphocytes, and blood–brain barrier.37 P-glycoprotein actively transports xenobiotics from the intracellular to the extracellular domain, resulting in a protective role against their potentially toxic accumulation by enhancement of their elimination and limitation of their distribution in the body.38 Several drugs are able to modulate plasma concentrations of methadone through P-glycoprotein interaction. However, in our patient, we excluded a drug interaction. Our patient was heterozygous for 2 SNPs: ABCB1 1236C/T (rs1128503) and ABCB1 3435C/T (rs1045642). Recently, it has been documented that MDR1 genetic variability may influence daily methadone dose requirements.39 Crettol et al.40 showed, however, that MDR1 genotypes have little association with the pharmacokinetics of most commercially available drugs after oral administration, as a result of a probable sufficient concentration in the gut to saturate the efflux transporters. Thus, intestinal absorption is not restricted. Moreover, it has been documented that the SNP of the ABCB1 gene 3435C>T, which is associated with lower P-glycoprotein expression, plays a role on trough but not peak methadone plasma concentrations.41,42 Furthermore, it has been documented that 3435C>T is common (24.3%) in the white population.43 Therefore, we assume that even if our patient carried such polymorphisms, absorption was not limited, resulting in normal disposition of methadone. Such polymorphisms are unlikely to have played a role in the rapid methadone metabolism documented in our patient. We assume that, in our patient, CYP3A5 polymorphism contributed to the methadone clearance augmentation by CYP3A4 enhancement.
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