Pharmacology & Therapeutics

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1 Pharmacology & Therapeutics 124 (2009) Contents lists available at ScienceDirect Pharmacology & Therapeutics journal homepage: Associate editor: P. Holzer Genetic modulation of the pharmacological treatment of pain Jörn Lötsch, Gerd Geisslinger, Irmgard Tegeder pharmazentrum frankfurt/zafes, Institute of Clinical Pharmacology, Johann Wolfgang Goethe-University, Theodor-Stern-Kai 7, D Frankfurt am Main, Germany article info abstract Keywords: Pharmacogenetics Pain Personalized therapy Opioids Non-opioid analgesics Inadequately treated acute and chronic pain remains a major cause of suffering and dissatisfaction in pain therapy. A cause for the variable success of pharmacologic pain therapy is the different genetic disposition of patients to develop pain or to respond to analgesics. The patient's phenotype may be regarded as the result of synergistic or antagonistic effects of several genetic variants concomitantly present in an individual. Variants modulate the risk of developing painful disease or its clinical course (e.g., migraine, fibromyalgia, low back pain). Other variants modulate the perception of pain (e.g., OPRM1 or GCH1 variants conferring modest pain protection by increasing the tone of the endogenous opioid system or decreasing nitric oxide formation). Other polymorphisms alter pharmacokinetic mechanisms controlling the local availability of active analgesic molecules at their effector sites (e.g., decreased CYP2D6 related prodrug activation of codeine to morphine). In addition, genetic variants may alter pharmacodynamic mechanisms controlling the interaction of the analgesic molecules with their target structures (e.g., opioid receptor mutations). Finally, opioid dosage may be increased depending on the risk of drug addiction (e.g., DRD2 polymorphisms decreasing the functioning of the dopaminergic reward system). With the complex nature of pain involving various mechanisms of nociception, drug action, drug pharmacology, pain disease and possibly substance addiction, a multigenic or even genome wide approach to genetics could be required to base individualized pain therapy on the patient's genotype Elsevier Inc. All rights reserved. Contents 1. Introduction Genetics of painful diseases Genetic modulation of nociception Genetic modulation of the effects of drugs used in pain treatment Pharmacogenetics of drug interactions in pain therapy Genetics of drug addiction Special populations, age, sex and ethnic background Conclusions and perspectives Acknowledgments References Abbreviations: ABCB1, P-glycoprotein gene (ATP-binding cassette, subfamily B, member 1 gene); ADRB2, β 2 adrenoceptor gene; BH4, tetrahydrobiopterin; COMT, catechol-omethyltransferase; COX, cyclooxygenase; CYP2C9, cytochrome P450 2C9; CYP2C19, cytochrome P450 2C19; CYP2D6, cytochrome P450 2D6; CYP3A5, cytochrome P450 3A5; CNS, central nervous system; DRD2, dopamine receptor 2 gene; EM, CYP2D6 extensive metabolizer phenotype; FAAH, fatty acid amide hydrolase; GCH1, guanosin triphosphate cyclohydrolase 1; GIRK, G protein-coupled inwardly-rectifying potassium channel (=potassium inwardly-rectifying channel); GRK, G protein-coupled receptor kinase; HSAN, hereditary sensory and autonomic neuropathy; IL, interleukin; KCNJ, potassium inwardly-rectifying channel; MC1R, melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) gene; OPRD1, δ-opioid receptor gene; OPRM1, µ-opioid receptor gene; P-gp, P-glycoprotein; PTGS2, cyclooxygenase 2 gene (prostaglandin-endoperoxide synthase 2 gene); PM, CYP2D6 poor metabolizer phenotype; SNP, single nucleotide polymorphism; TRPA1, transient receptor potential cation channel, subfamily A, member 1; TRPV1, transient receptor potential cation channel, subfamily V, member 1; UGT2B7, uridine diphosphate glucuronosyltransferase 2 family, polypeptide B7; UM, CYP2D6 ultrafast metabolizer phenotype. Corresponding author. Tel.: ; fax: address: j.loetsch@em.uni-frankfurt.de (J. Lötsch) /$ see front matter 2009 Elsevier Inc. All rights reserved. doi: /j.pharmthera

2 J. Lötsch et al. / Pharmacology & Therapeutics 124 (2009) Introduction The genetic control of pain has been demonstrated impressively by quantitative sensory trait approaches in mice (Mogil et al., 1996b; Mogil, 1999). Mice of 11 inbred strains displayed different nociceptive responses in 12 different pain models (Mogil et al., 1999). There was no uniform high-pain/low-pain strain distribution across models, but the strains were comparatively sensitive to pain in some models and comparatively insensitive in others. The differences in nociceptive sensitivity varied between 1.2 and 54-fold. Moreover, mouse strains differed with respect to the antinociceptive effects of morphine in different pain models (Mogil et al., 1996a). For example, in HA/LA and HAR/LAR mice, an inverse relationship was seen with respect to the antinociceptive sensitivity to morphine. By using quantitative sensory trait association techniques, several candidate genes were identified in mice and rats that probably underlie such hereditary transmittable differences in nociception and opioid antinociception (Devor & Raber, 1990; Chesler et al., 2001; Abiola et al., 2003; Kest et al., 2004; Mogil et al., 2006). Animal models of pain research (Mogil, 2009) serve as a major source of candidates for genetic association studies of pain in humans. This is facilitated by the public availability of a pain-genes database (Lacroix-Fralish et al., 2007) that is updated and maintained to the actual level of knowledge at enter.html (accessed May 16, 2009). In humans, the risk of developing pain diseases varies with certain genotypes, which is known for example in migraine (Colson et al., 2007) or low back pain (Tegeder & Lötsch, 2009). Furthermore, the severity of pain is controlled by several genetic variants affecting the expression or function of components of the nociceptive sensory system (Kim & Dionne, 2005; Lötsch & Geisslinger, 2007; Lacroix-Fralish & Mogil, 2009). These factors indicate that the individual pain variability is to a high degree genetically controlled, alongside genetically determined differences in the efficacy or kinetics of analgesics. Substances administered for pain treatment are subject to distributional processes or metabolic modifications that involve genetic modulations at all stages of the interactions of the drug with the body (Evans & Relling, 1999), including the interaction with the drug's targets. Associations of single nucleotide polymorphisms (SNP) with increased or decreased pain, or modified effects of pain killers, have demonstrated that pain therapy is subject to pharmacogenetics. The effects of genetics are exerted via (i) factors modulating the risk of developing a pain disease or its clinical course and severity, (ii) factors modulating the individual perception of pain, (iii) factors altering pharmacokinetic mechanisms controlling the local availability of analgesic molecules at their site of action, (iv) factors altering pharmacodynamic mechanisms controlling the interaction of the analgesic molecules with their target structures influencing the intensity of pain, or (v) factors modulating opioid dosage by conferring a risk of drug addiction and side effects. In complex settings such as pain therapy, several genetic factors contribute to the patient's phenotype in pain treatment. This is the result of synergistically or antagonistically working genetic variants, modifying the perception and emotional processing of pain and the response to its treatment in one or other direction relative to the population average (Fig. 1). The approach to the pharmacogenetics of pain includes increasingly more than one gene, and the first successes of this approach, such as the possibility to predict the response to morphine in cancer patients by genotyping for the µ-opioid receptor variant gene OPRM1, variant 118ANG, and the P-glycoprotein (P-gp) gene ABCB1, variant 3435CNT (Campa et al., 2008), are encouraging to incorporate the genetics of pain mechanisms and pain therapy into the development of personalized treatment approaches. Since a fifth of adults in Europe suffers from moderate or severe chronic pain (Breivik et al., 2006) and successful analgesia is still one of the main healthcare issues, the identification of functional genetic polymorphisms modulating the individual response to nociceptive input, or to analgesic therapy, has received a high level of interest. It is expected that the pharmacogenetics of pain treatment will (i) explain poor drug responses and (ii) provide advice on personalized therapy at optimal efficacy. Therefore, the aim of this review is to summarize the human genetic variants that have been identified so far to modulate the pharmacotherapy of pain by any of the above mentioned mechanisms. Importantly, the focus is on the evidence for a modulation of pain pharmacotherapy rather than the underlying disease, with a particular emphasis on the clinical effects of analgesics. Candidate polymorphisms that might be expected to modulate pain because of biologically plausible associations, but with regard to pain are not yet backed by clinical evidence, have been mostly excluded. 2. Genetics of painful diseases Migraine was amongst the first painful diseases associated with genetics (Godinova, 1967), and today several variants have been identified that underlie its hereditary features (Colson et al., 2007). Other pain syndromes are also under genetic control, and genetic risk factors have been summarized for fibromyalgia (Ablin et al., 2006), low back pain (Tegeder & Lötsch, 2009) and complex regional pain syndrome (Mailis & Wade, 1994). For example, interleukin related genetic polymorphisms, namely in the IL-1 receptor antagonist gene (IL1RN), the IL-1α (IL1A) and IL-1β (IL1B) genes, have been found to be associated with a higher risk of low back pain and a higher number of sick days (Solovieva et al., 2004), probably due to an enhanced IL-1 production. Major haplotypes of the β 2 adrenoceptor gene (ADRB2) have been found to define the risk of developing a musculoskeletal pain disorder (Diatchenko et al., 2006a). However, these genetic associations only relate to disease risk or disease subtypes, but not to pain pharmacotherapy. 3. Genetic modulation of nociception A higher or lower intensity of pain is very likely to require higher or lower doses of analgesics for efficacious therapy. The genetic control of human pain perception and processing is therefore likely to modulate analgesic therapy. However, clinical evidence for this relationship is sparse. Genetic control of pain is most pronounced in people carrying mutations that confer congenital insensitivity to pain. Several syndromes have been described, all being characterized by an interruption of transmission or processing at key points of the nociceptive system (for full details, see the Online Mendelian Inheritance in Man database (OMIM): db=omim) (Oertel & Lötsch, 2008). Examples include (i) the channelopathy associated insensitivity to pain, which is based on loss-of-function mutations of the alpha-subunit of the voltage-gated sodium channel, Na v 1.7 (Cox et al., 2006; Goldberg et al., 2007), (ii) the hereditary sensory and autonomic neuropathy type I (HSAN-I), caused by mutations in the serine palmitoyltransferase, long chain base subunit 1 gene (Bejaoui et al., 2001; Dawkins et al., 2001), (iii) the HSAN-II, based on mutations in the hereditary sensory neuropathy, type II gene (Lafreniere et al., 2004; Riviere et al., 2004; Roddier et al., 2005; Cho et al., 2006), (iv) the HSAN-III due to mutations in the inhibitor of kappa light polypeptide gene enhancer in B-cells kinase complex-associated protein gene (Anderson et al., 2001; Slaugenhaupt & Gusella 2002; Leyne et al., 2003), (v) the HSAN-IV, also called congenital insensitivity to pain with anhidrosis (CIPA) and based on mutations in the neurotrophic tyrosine kinase receptor type 1 gene (Indo et al., 1996; Miura et al., 2000), and (vi) the HSAN-V caused by mutations in the nerve growth factor beta polypeptide gene (Einarsdottir et al., 2004). These syndromes probably have no importance on the pharmacotherapy of pain as they are very rare and the affected people probably do not require

3 170 J. Lötsch et al. / Pharmacology & Therapeutics 124 (2009) Fig. 1. Gene abbreviations and gene products: CYP2D6 cytochrome P450 2D6 (UM: ultrafast metabolizer phenotype, PM: poor metabolizer phenotype), CYP3A5 cytochrome P450 3A5, CYP2C9 cytochrome P450 2C9, ABCB1 P-glycoprotein, UGT2B7 uridine diphosphate glucuronosyltransferase 2 family, polypeptide B7, COMT catechol-omethyltransferase, MC1R melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor), OPRM1 µ-opioid receptor, OPRD1 δ-opioid receptor, PTGS2 cyclooxygenase 2, GCH1 bguanosin triphosphate cyclohydrolase 1, FAAH fatty acid amide hydrolase, TRPA1 transient receptor potential cation channel, subfamily A, member 1, TRPV1 transient receptor potential cation channel, subfamily V, member 1, DRD2 dopamine receptor 2; HSAN = Hereditary Sensory and Autonomic Neuropathy, i.e, a group of disease caused by mutations in several genes. Pharmacogenetic scale of the consequences of functional polymorphisms for analgesic. The patient's phenotype, with respect to pain therapy, is the result of several synergistic and concurring genetic variants. Genes affecting pain and pharmacotherapy at several levels are grouped in the boxes at the bottom. In the average population (green) variants increasing analgesic are in equilibrium with variants decreasing analgesic. In some patients genetic variants are imbalanced in either direction (red). Known genes with variants with a shown function inpain therapy are given on the respective sides of the scale, for details, see Tables 1 and 2. Variants with evidence of a modulation of pain or of the pharmacology of drugs used as analgesics, but without evidence for a modulation of the effects of analgesic or dosage, are indicated with a question mark. pain therapy. However, defining the molecular causes for hereditary insensitivity to pain may serve as an important source of information to find new targets for analgesic drugs. Pain in the average population is controlled by fairly frequent genetic variants (allelic frequencies 10 50%, Tables 1 and 2). Each of them, however, modifies the pain phenotype to only a modest degree, and most information so far has only been found with experimental pain models. A modulating influence on analgesic efficacy and dosage (Fig. 1) has been shown for only a few of the variants seen to modulate pain.

4 J. Lötsch et al. / Pharmacology & Therapeutics 124 (2009) Table 1 Evidence for a statistically significant genetic modulation of pain in the average population. Gene Variant Minor allele frequency [%] GCH1 (guanosine triphosphate cyclohydrolase 1) COMT (catechol-o-methyl transferase) MC1R (melanocortin-1 receptor) TRPV1 (transient receptor potential cation channel, subfamily V, member 1) TRPA1 (transient receptor potential cation channel, subfamily A, member 1) Type of pain rs gna 17 Chronic low back pain with radicular pain; heat, ischemic and pressure pain rs ant 19 Decreased rs tnc 28 Decreased rs ang 19 Decreased rs752688gna 19 Decreased Haplotype from 15 GCH1 SNPs Increased or Reference decreased pain Decreased Tegeder et al., Chronic low back pain with Decreased radicular pain; heat, ischemic and pressure pain, 14.7 Capsaicin induced hyperalgesia Decreased Tegeder et al., 2008 Capsaicin induced hyperalgesia Decreased Campbell et al., Pain of various reasons in tertiary care Shorter need for pain therapy Doehring et al., in press rs tnc 33 Cold pain intensity Decreased Kim et al., 2006 rs6269ang 44 Cold pain intensity Decreased or increased rs4633tnc 49 Pressure pain, thermal pain Decreased Diatchenko et al., 2005 rs4680 GNA Muscle pain due to hypertonic Increased Zubieta et al., 2003 saline Heat pain temporal summation Increased Diatchenko et al., 2006b rs6269g/rs4633c/ 36.5 Pressure pain, thermal pain Low Diatchenko et al., 2005 rs4818g/rs4680g rs6269a/rs4633t/ 48.7 Average rs4818c/rs4680a rs6269a/rs4633c/ 10.7 High rs4818c/rs4680g Haplotypes from 6 COMT SNPs Cold pain intensity Changed Kim et al., 2006 rs cnt, 2 Electrical pain tolerance Decreased Mogil et al., 2005 rs cnt, rs gnc and other rs ang 36.8 Cold pain withdrawal time Decreased Kim et al., 2004 rs gna 41 Cold pain withdrawal time Increased Kim et al., 2006 Haplotypes from rs tna and rs gna Cold pain withdrawal time, heat pain intensity Changed (direction not given) OPRM1 (µ-opioid receptor) rs ANG 11.2 Pressure pain threshold Decreased Fillingim et al., Cortical responses to Decreased Lötsch et al., 2006c trigeminal pain stimuli IVS2+31GNA 2.8 Pressure pain threshold Decreased Huang et al., 2008 OPRD1 (δ-opioid receptor) rs tng 10.9 Heat pain intensity Decreased or Kim et al., 2004 unchanged rs tnc 35.6 Heat pain intensity Increased FAAH (fatty acid amide hydrolase) rs932816gna 19 Cold pain intensity Increased Kim et al., 2006 rs tnc 49 Cold pain intensity Increased Kim et al., 2006 Cold pain withdrawal time Increased rs gna 43 Cold pain intensity Increased Kim et al., µ-opioid receptors Opioid receptors are the natural targets of the endogenous pain defense opioid system with its transmitters endorphin, enkephalin and dynorphin. A variant in exon 1 of the µ-opioid receptor gene (OPRM1 118ANG; dbsnp rs according to the NCBI SNP database at has been shown to be associated with a 0.8-fold lower pressure pain intensity, especially in males (Fillingim et al., 2005) and with a 0.5-fold smaller amplitude of pain-related cortical potentials after a specific stimulation of nasal nociceptors with gaseous carbon dioxide (Lötsch et al., 2006c). The only clinical study suggesting that pain protection conferred by this variant translates to a reduced analgesic requirement is a report that women carrying the variant required lower intrathecal fentanyl doses for labor analgesia than non-carriers (Landau et al., 2008). All other studies available thus far suggest the opposite effect on opioid dosing (see below). In addition, an intronic variant appears to be associated with increased pressure pain thresholds (Huang et al., 2008) Melanocortin-1 receptors Subjects carrying non-functional melanocortin-1 receptors (MC1R) have a red hair and fair skin phenotype. One study holds that they have a 1.3 times higher tolerance to electrical pain stimuli than controls with functional MC1Rs (Mogil et al., 2005). However, this association was not found in another study (Liem et al., 2005). In addition, another cohort of redheads was reported to be more sensitive to thermal pain stimuli than controls (Liem et al., 2005). In contrast, mice (C57BL/6- Mc1r(e/e)) with non-functional MC1Rs exhibit a consistently decreased sensitivity to pain across a broad range of nociceptive modalities (Mogil et al., 2005). The MC1R variants modulate opioid efficacy in a sexspecific manner. Women with red hair and white skin, carrying two

5 172 J. Lötsch et al. / Pharmacology & Therapeutics 124 (2009) non-functional MC1R alleles, benefit from a greater analgesic effect of the k-opioid agonist pentazocine than women carrying only one or none of the MC1R variants, or men with the same MCR1 genotype (Mogil et al., 2003). The impact of MC1R variants on analgesia was related to the binding of the endogenous k-opioid receptor ligand dynorphin, which binds only to non-mutated MC1Rs (Quillan & Sadee, 1997). In hypothesizing that MC1R activation by endogenous neuromodulators exerts anti-opioid actions (Mogil et al., 2003), the greater effect of pentazocine in carriers of those mutations may be explained by an omission of this anti-opioid effect. The greater analgesic efficacy of k- opioids in female carriers of MC1R non-functional variants suggests that this neurochemical pain modulation has a sex-specific regulation when the k-opioid system is involved (Mogil et al., 2003). MC1R inactivation also enhances the µ-opioid receptor-mediated analgesic effect of morphine-6-glucuronide, but this change appears to be gender-independent (Mogil et al., 2005) Guanosine triphosphate cyclohydrolase 1 Guanosine triphosphate cyclohydrolase 1 (GCH1) is the rate limiting enzyme catalyzing the production of tetrahydrobiopterin (BH4) (Auerbach & Nar, 1997). BH4 is an essential co-factor for the three isoforms of the nitric oxide synthase. Excessive BH4 in peripheral sensory neurons following axonal injury contributes to the manifestation of neuropathic pain (Tegeder et al., 2006). This is mediated in part by increasing calcium influx and nitric oxide production (Tegeder et al., 2006). A decrease in pain associated with reduced-function GCH1 variants, in particular with a haplotype composed of 15 GCH1 polymorphisms (Fig. 2), has been reported in well defined cohorts of patients after lumbar disk surgery and in healthy young volunteers (Tegeder et al., 2006; Campbell et al., 2009; Tegeder et al., 2008). Two studies have disputed such an effect in chronic pancreatic pain (Lazarev et al., 2008) and in patients after dental surgery (Kim & Dionne, 2007). With respect to analgesic therapy, carriers of the pain protective GCH1 haplotype have recently been observed in a cross-sectional assessment to have been on tertiary pain care for a shorter time before the day of study inclusion than non-carriers (Doehring et al., in press). This was interpreted as a prophylactic role of decreased GCH1 upregulation delaying the need for pain therapy. The hypothesis was subsequently verified (Lötsch et al., 2009b) in a cohort of 251 non-related patients with cancer pain. The interval between cancer diagnosis and opioid therapy initiation was longer in homozygous carriers of this genetic variant (78 ± 65.2 months) than in heterozygous subjects (37 ±46.5 months) and non-carriers (30.4±43.8 months) Other variants modulating pain in the general population The δ-opioid receptor is the main target of enkephalin within the endogenous opioid system. A variant of the δ-opioid receptor gene, OPRD1, has been associated with lower heat pain intensity (Kim et al., 2004). Other functional variants (Table 1) include the catechol-o-methyl transferase (COMT), which degrades catecholamine neurotransmitters such as norepinephrine and dopamine. Since COMT inhibition may lead to increased pain sensitivity via β-adrenoceptor dependent mechanisms (Nackley et al., 2007), genetic variants associated with high COMT enzymatic activity could potentially decrease pain. However, COMT haplotypes associated with higher COMT activity were also associated with a lower pain sensitivity (Diatchenko et al., 2005; Diatchenko et al., 2006b), although this association was not reproduced in another study (Kim et al., 2006). The frequent COMT major 472G variant (wild type of dbsnp rs4680) was associated with lower temporal summation of heat stimuli (Diatchenko et al., 2006b). The transient receptor potential cation channel, subfamily V, member 1 (TRPV1), mediates pain induced by noxious heat or capsaicin. A subject insensitive to capsaicin, with 50% reduced mrna and protein expression levels of TRPV1, carried seven intronic TRPV1 single nucleotide polymorphisms (SNPs) (Park et al., 2007a). Women carrying a coding TRPV1 variant were found to be less sensitive to cold (Kim et al., 2004), a finding that was not reproduced in another study (Kim et al., 2006). Thus far, these polymorphisms have not been seen to concern pain therapy. Low temperatures and menthol stimulate mainly the cold- and menthol-sensitive transient receptor potential channel, subfamily M (melastin), member 8 (TRPM8). This channel plays a role in the development of cold allodynia following peripheral nerve injury (Colburn et al., 2007). The other cold-sensitive receptor, TRPA1 (transient receptor potential cation channel, subfamily A, member 1), is mainly activated by noxious cold, chemical and endogenous irritants (Bandell et al., 2004). A decrease in cold pain withdrawal time was associated with TRPA1 variants in a study of healthy subjects (Kim et al., 2006). Endogenous cannabinoids bind to cannabinoid receptors, comprising neuronal CB 1 and mostly non-neuronal CB 2 receptors (Pertwee, 1999), and participate in physiological functions related to cognition and memory, nociception, motor coordination, temperature and vascular homeostasis, and inflammatory processes (Smita et al., 2007). The endogenous cannabinoid anandamide is degraded by fatty acid amide hydrolase (FAAH). Decreased FAAH expression or net activity may therefore be expected to cause higher synaptic availability of anandamide and thereby be more efficient as an endogenous pain defense mechanism. Conversely, a rise of FAAH activity might increase pain. A non-coding FAAH variant has been associated with increased cold pain perception (Kim et al., 2006), which suggests that the enzymatic activity of FAAH is enhanced, but the biochemical consequences of these SNPs have not yet been analyzed. The relevance of these variants to analgesic treatment is also unknown. 4. Genetic modulation of the effects of drugs used in pain treatment 4.1. Genetic modulation of the effects of traditional analgesics The major groups of classical analgesics comprise (i) opioids and (ii) antipyretic non-opioid analgesics, which include traditional nonselective cyclooxygenase (COX) inhibitors (e.g., ibuprofen, diclofenac, acetylsalicylic acid) and selective COX-2 inhibitors (e.g., celecoxib, parecoxib, etoricoxib). Metamizole (dipyrone) and acetaminophen also act by COX inhibition, although they are weaker and less specific (Muth-Selbach et al., 1999; Pierre et al., 2007; Hinz et al., 2008; Schildknecht et al., 2008). In a broader sense, (iii) triptans acting on serotonin receptors and (iv) anticonvulsants and antidepressants used in the treatment of migraine and neuropathic pain can also be considered as analgesic agents. The effects of all analgesic compounds are based on their local availability at the site of their target. When the drug molecules have reached their site of action, their effects may be modulated by an altered interaction with their targets or by altered consequences of this interaction Pharmacokinetics Drug metabolism and drug transport mainly govern the time course of the analgesic's concentration at the site of action. Clinically, this is most often associated with detectable alterations in plasma levels because modified drug metabolism or transport leads to changes in drug clearance or distribution. However, due to the therapeutic range of analgesics, not all measurable changes in the drug's plasma concentrations have consequences for the drug's effect. We have therefore focused on variants shown to alter the clinical efficacy of analgesics Drug metabolizing enzymes. Analgesics are subject to metabolic clearance by several enzymes. In the case of the administered compound being the active principle, decreased metabolism would lead to increased effects due to slower systemic elimination. The

6 J. Lötsch et al. / Pharmacology & Therapeutics 124 (2009) Table 2 Evidence for a pharmacogenetic modulation of the pharmacodynamic effects of analgesic drugs in humans. Gene Variant Minor allele frequency a Single genes OPRM1 118ANG (µ-opioid receptor) (rs ) COMT (Cathecol-Omethyl transferase) MC1R (Melanocortin-1 receptor) ABCB1 (P-glycoprotein) CYP2D6 (cytochrome P450 2D6) UGT2B7 (UDP glucuronosyl transferase 2B7) PTGS2 (Cyclooxygenase 2) Affected analgesic 13.5% for minor allele Morphine-6- glucuronide Clinical effect Study population n Reference Decreased miotic effects Healthy volunteers 12 Lötsch et al., 2002a Decreased miotic effects Healthy volunteers 12 Skarke et al., 2003a Decreased side effects Single case 1 Lötsch et al., 2002b Decreased analgesia Healthy volunteers 20 Romberg et al., 2004 Decreased analgesia Healthy volunteers 16 Romberg et al., 2005 Morphine Decreased miotic effects Healthy volunteers 12 Skarke et al., 2003 a Increased opioid Single case 1 Hirota et al., 2003 Increased opioid Cancer pain patients 207 Klepstad et al., 2004; Reyes-Gibby et al., 2007 Increased opioid Postoperative 80 Chou et al., 2006a patients Increased opioid Postoperative 147 Chou et al., 2006b patients Increased opioid Postoperative 588 Sia et al., 2008 patients Alfentanil Decreased analgesia and Healthy volunteers 20 Oertel et al., 2006 respiratory depression Decreased analgesia Healthy volunteers 25 Oertel et al., 2008 Fentanyl Decreased opioid Labouring women 223 Landau et al., 2008 Levomethadone Decreased miotic effects Healthy volunteers 51 Lötsch et al., 2006b Increased opioid Postoperative 138 Hayashida et al., 2008 patients Several Slightly increased pain Chronic pain patients 352 Lötsch et al., 2009d 472 GNA (rs4680) 48.1% for minor allele Morphine Decreased morphine Cancer pain patients 207 Rakvag et al., insA, 451CNT 0 8.5%; 2.2% with two M6G Increased analgesia Healthy volunteers 47 Mogil et al., 2005 (rs ), 478CNT variant alleles Pentazocine Increased analgesia Healthy volunteers 42 Mogil et al., 2003 (rs ), 880GNC in women (rs ), other 3435 CNT (rs ) 34.5% for minor a allele Fentanyl Respiratory depression Postoperative 126 Park et al., 2007 b 1236TT/2677TT/3435TT 35.7% for minor allele patients 3435 CNT (rs ) 50.4% for minor a allele Several Decreased opioid Chronic pain patients 352 Lötsch et al., 2009d Only non-functional 5.3% of the patients Codeine Decreased analgesia Healthy volunteers 24 Sindrup et al., 1990 alleles Decreased analgesia Healthy volunteers 18 Eckhardt et al., 1998) Decreased analgesia Single case 1 Foster et al., 2007) Decreased analgesia Women after 11 Persson et al., 1995 hysterectomy Decreased respiratory, Healthy volunteers 16 Caraco et al., 1996 psychomotor and miotic effects Oxycodone Insufficient or inadequate Pain patients Each Susce et al., 2006; clinical responses 1 Foster et al., 2007 Tramadol Decreased analgesia Healthy volunteers 27 Poulsen et al., 1996 Decreased analgesia Postoperative 300 Stamer et al., 2003 patients More than two copies of functional alleles 1.7% of the patients Codeine Increased effects up to toxicity Tramadol Increased effects up to toxicity Increased analgesia and miosis and more nausea 2 allele b Codeine Increased effects up to toxicity Single cases 765CNG (rs20417) 17 Rofecoxib Decreased analgesia Post molar surgery pain Each 1 Dalen et al., 1997; Gasche et al., 2004; Koren et al., 2006; Madadi et al., 2007; Voronov et al., 2007 Single case 1 Stamer et al., 2008 Healthy volunteers 22 Kirchheiner et al., 2008 Single cases 2 Madadi et al., Lee et al., 2006 Combinations of genes OPRM1 and COMT Group 1: OPRM1 118 AA and COMT 472 AA Group 2: OPRM1 118 AA and COMT 472 G Group 3: OPRM1 118 G and COMT 472 AA Group 4: OPRM1 118 G and COMT 472 G 15.9% of the patients Morphine Morphine increasing from group % of the patients to group 4 7.1% of the patients 18.2% of the patients Cancer pain patients 207 Reyes-Gibby et al., 2007 (continued on next page)

7 174 J. Lötsch et al. / Pharmacology & Therapeutics 124 (2009) Table 2 (continued) Gene Variant Minor allele frequency a Combinations of genes OPRM1 and ABCB1 Group 1: OPRM1 118 AA and ABCB TT Group 2: OPRM1 118 AA 59.6% of the patients and ABCB C or OPRM1 118 G and ABCB TT Group 3: OPRM1 118 G 20.5% of the patients and ABCB C Affected analgesic Clinical effect Study population n Reference 19.9% of the patients Morphine Opioid analgesia decreasing Cancer pain patients 145 Campa et al., 2008 from group 1 to group 3 a Minor refers to the allele reported to be minor in gene databases. When its reported allelic frequency is close to 50%, it can happen that the minor allele has a frequency N50% in the actual cohort. We nevertheless preserved the denomination minor to be consistent with the literature. b The distribution of homozygous, heterozygous and non-carriers of the minor alleles of the selected variants agreed with the expectations from the Hardy Weinberg law (χ 2 goodness-of-fit tests: P 0.17). opposite applies for a prodrug where decreased effects due to reduced production of the active metabolite are the consequence Prodrug activation. Prodrugs are substances administered in an almost inactive form that need to be activated to obtain a clinical effect. Typical prodrugs among analgesics are codeine, tilidine, parecoxib and metamizole (dipyrone). Less typical is tramadol, which is metabolically activated but also has a complex clinical pharmacodynamic activity, including analgesia on its own (Gillen et al., 2000). In contrast, the existence of an active metabolite, such as morphine-6-glucuronide, does not necessarily render the parent compound a prodrug, because morphine itself produces the main clinical effect, unless the metabolite accumulates in special populations (Lötsch & Geisslinger, 2001). Codeine is extensively metabolized by the cytochrome P450 (CYP) isoenzyme 2D6. After a single oral dose of 30 mg, 81% is transformed to codeine-6-glucuronide, 2.2% to norcodeine, 0.6% to free morphine, 2.1% to morphine-3-glucuronide, 0.8% to morphine-6-glucuronide, and 2.4% to normorphine (Vree & Verwey-van Wissen, 1992). Adding morphine and its glucuronides, 6% of codeine is thus metabolized to morphine. The clinical analgesic effect of codeine is mainly attributed to its conversion to morphine, which has a 200-times higher affinity and a 50-times higher intrinsic activity at µ-opioid receptors than codeine itself (Mignat et al.,1995; Schmidt et al., 2003). Morphine is therefore considered to be the active principle of codeine despite some evidence that codeine, or codeine-6-glucuronide, contributes to the pharmacodynamic effects (Srinivasan et al., 1996; Srinivasan et al., 1997; Eckhardt et al., 1998; Vree et al., 2000; Lötsch et al., 2006a). Since CYP2D6 (Dayer et al., 1988) is genetically highly polymorph (Cascorbi, 2003), the effects of codeine are under pharmacogenetic control (Fagerlund & Braaten, 2001). Genetically altered codeine effects may occur in subjects with either decreased, absent or highly increased CYP2D6 activity when compared with the population average. Decreased codeine effects may occur in approximately 7 11% of the Caucasian population in whom CYP2D6 is inactive for genetic reasons (Lovlie et al., 1996; Bertilsson et al., 2002; Tamminga et al., 2003). In this CYP2D6 poor metabolizer phenotype (PM), with interethnic differences (Cascorbi, 2003), very low levels of morphine or no morphine at all are formed after administration of codeine. On the other hand, increased codeine effects may occur in up to 7% of the Caucasian population in whom CYP2D6 is extremely active (Steijns & Van Der Weide, 1998). In this CYP2D6 ultrafast metabolizer phenotype (UM) codeine yields very high levels of morphine. Methadone, which is also a CYP2D6 substrate, has no relevant active metabolite. Because it is cleared by CYP3A, CYP2B6 and other CYPs (Wang & DeVane, 2003; Gerber et al., 2004), CYP2D6 genetics play a minor role in its pharmacokinetics (Coller et al., 2007) and pharmacodynamics (Lötsch et al., 2006b). Only a tendency towards a greater responder fraction among PMs than among UMs has been reported (Eap et al., 2001). Thus, roughly one out of seven Caucasians (Zanger et al., 2004) is at risk of either failure or toxicity of codeine therapy due to extremely low or high morphine formation, respectively. Nevertheless, the fraction of 7 11% CYP2D6 variants is too low to explain the numbers needed to treat of 50 or 9.1 for dental or postsurgical pain, respectively, to achieve 50% pain relief with 60 mg codeine (Moore & McQuay, 1997). Non-genetic factors, such as pain characteristics or simply the low amount of 4 5 mg morphine, which in the CYP2D6 extensive metabolizer phenotype (EM) results from 60 mg of oral codeine, is likely to play a role. In addition, codeine analgesia is probably modulated by additional genetic factors that modify the effects of morphine, such as variants altering drug transport (Campa et al., 2008), opioid receptor expression (Zhang et al., 2005) or signaling (Oertel et al., 2009), nociception or pain (for review, see Kim & Dionne, 2005; Lötsch & Geisslinger, 2007) and genetic variants in other enzymes, such as CYP3A (Lalovic et al., 2004) or UGT2B7 (Madadi et al., 2009), accounting for approximately 70 80% of the metabolism of codeine (Chen et al., 1991; Vree & Verwey-van Wissen, 1992). Similarly, high morphine formation is probably a prerequisite, but not a sufficient single cause, to produce codeine toxicity. This is documented in only a few case reports (Table 3) and therefore rarer than would be expected from the proportion (up to 7%) of Caucasian phenotypic UMs or carriers (1 3%) of CYP2D6 gene amplification (Johansson et al., 1993). However, mild toxicity might be overlooked or misdiagnosed, but can still jeopardize effective pain therapy and the quality of life in chronic pain patients. Tramadol is a µ-opioid receptor agonist but has a lower affinity at µ-opioid receptors than its metabolite O-desmethyltramadol. Tramadol is not without analgesic effects when CYP2D6 is blocked (Collart et al., 1993). It possesses opioid activity and also acts through nonopioid dependent mechanisms which involve serotonin or noradrenaline mediated pain inhibition originating in the brainstem (Enggaard et al., 2006). However, its antinociceptive effects are modulated by CYP2D6 activity (Garrido et al., 2003). The analgesic efficacy on experimental pain is reduced in CYP2D6 PMs (Poulsen et al., 1996), a finding later confirmed in pain patients (Stamer et al., 2003). As regards other CYP2D6 substrates with active metabolites, the evidence for altered analgesic effects with altered CYP2D6 function is either negative, such as for dihydrocodeine (Platten et al., 1998; Webb et al., 2001; Schmidt et al., 2003), or explained at a non-genetic level, such as for oxycodone (Heiskanen et al., 1998). In other cases, the evidence is based only on animal studies, such as for hydrocodone (Lelas et al., 1999) or restricted to single case reports giving a reasonable mechanism-based interpretation for the lack of effect (Susce et al., 2006) or inadequate activity of oxycodone (Foster et al., 2007). Tilidine is activated to nortilidine, and parecoxib is activated into valdecoxib via CYP3A. This enzyme is phenotypically highly variable, but only a minor part of this variability can be attributed to genetics. Individuals with at least one CYP3A5 1 allele copy (adenine in position 6986) produce high levels of full-length CYP3A5 mrna and express active CYP3A5 (Kuehl et al., 2001). However, the majority (95%) of Caucasians have no active CYP3A5 due to a premature stop codon (Hustert et al., 2001). Positive associations in prodrug activation and analgesic action have so far not been reported.

8 J. Lötsch et al. / Pharmacology & Therapeutics 124 (2009) Fig. 2. Molecular and systemic consequences of decreased GCH1. A: Upregulation of GTP cyclohydrolase (GCH1)mRNA in L4/5 dorsal root ganglion neurons (DRGs) in the spared nerve injury (SNI) model of peripheral neuropathic pain as detected by in situ hybridization 7 d after SNI (scale bar 100 μm). GCH1 catalyzes the rate limiting first step in the synthesis cascade of the coenzyme tetrahydrobiopterin (BH4). B: Tetrahydrobiopterin (BH4) levels analyzed as total biopterin (ng per mg tissue) and BH4-dependent nitric oxide (NO) levels in the DRGs 7 days after nerve injury in the SNI model. BH4 and NO also increase at the site of nerve injury and in the dorsal horn of the spinal cord. Inhibition of GCH1 catalytic enzyme activity with diaminohydroxypyrimidine (DAHP) normalizes both BH4 and NO levels in the DRGs. C: Reduction of mechanical allodynia (i.e. increase of von Frey threshold) with the GCH1 inhibitor DAHP in the SNI model. Daily treatments with DAHP started 17 days after SNI. The withdrawal threshold to von Frey Hair stimulation of the injured hind paw was recorded. The antinociceptive efficacy of DAHP extended to the post-treatment period. Oppositely, intrathecal (i.t.) spinal delivery of BH4 in naïve rats caused hyperalgesia, i.e., enhanced nociceptive excitability assessed by recording the paw withdrawal latency to radiant heat in the Hargreaves test. D: Locations of 15 GCH1 single nucleotide polymorphisms in non-coding regions of GCH1 including 5 and 3 UTR regions (Ensemble database v.38 Apr2006), those significantly associated with low pain scores are coded in red ( P b0.05). Genotype phenotype associations of eight haplotypes with frequencyn1% and accounting for 94% of chromosomes studied, were analyzed. Letters in each haplotype are alleles for the 15 GCH1 SNPs. Pain scores for each haplotype are the mean z-score for leg pain calculated from four questions assessing frequency of pain at rest, after walking, and their improvement after surgery. Lower scores correspond to less pain. The upper haplotype that included all five SNPs significantly was associated with low pain scores, and further 3 rare alleles (highlighted in blue) were associated with lower leg pain scores compared with the seven other haplotypes; P = E: Effect of number of copies of the pain protective haplotype on frequency of leg pain at rest. Wild, HET (heterozygous) and HOM (homozygous) denote patients with 0, 1 and 2 copies of the haplotype. Numbers on y-axis correspond to the mean pain frequency scores at rest over a follow-up period of 24 months after surgery. F: GCH1 mrna (QRT-PCR) and protein in Epstein Barr Virus immortalized white blood cells (WBCs) of wild type (n=7), heterozygous (n=5) and homozygous (n=4) lumbar root pain patients, stimulated with forskolin (10 μm, 12 h), relative to unstimulated levels of wild type individuals (100%). Colored bars show unstimulated levels of the mrna; white bars show mrna after stimulation. Biopterin in supernatants of forskolin stimulated immortalized WBCs relative to baseline in lumbar root pain patients. Results represent means with SEM. Linear regression analysis revealed significant effects of the number of copies of the pain protective haplotype for forskolin induced changes in GCH1 mrna (Pb0.001), protein (P=0.037) and biopterin (P=0.001) (Tegeder et al., 2006, 2008) Drug inactivation. The glucuronidation of morphine, codeine, buprenorphine, flurbiprofen and other analgesics is mainly mediated by the UDP glucuronosyl transferase (UGT) 2B7 (Coffman et al., 1997; de Wildt et al.,1999; Mano et al., 2007), for which a couple of genetic polymorphisms have been described (Bhasker et al., 2000; Innocenti et al., 2001; Holthe et al., 2003). The main proportion of morphine (approximately 70%) is metabolized to morphine-3-glucuronide and to a lesser degree to morphine-6-glucuronide (10%) (Hasselström & Säwe, 1993). Both metabolites are active, with effects opposite to each other, consisting in excitation and anti-analgesia for morphine-3-glucuronide, and in typical opioid agonist effects for morphine-6-glucuronide (Lötsch & Geisslinger, 2001; Lötsch, 2005; Skarke et al., 2005). Thus, morphine glucuronidation does not lead to complete inactivation. However, morphine is not a prodrug, except in long term therapy. Especially in the presence of renal failure, glucuronide metabolites play a minor role when compared with morphine itself (Lötsch et al., 1997).

9 176 J. Lötsch et al. / Pharmacology & Therapeutics 124 (2009) Table 3 Reported opioid toxicity after codeine administration in which CYP2D6 gene amplification was a cause. Clinical case n Oral codeine dose Clinical effect Reference 33-year old dental pain 1 60 mg Euphoria, dizziness, blurred vision, epigastric pain Dalen et al., 1997 patient 62-year old pneumonia patient with leukemia history 1 25 mg 3 times a day for 3 days Arterial P O2 =56 mm Hg, score of 6 on the Glasgow coma scale (no eye opening, 1no verbal response, and limb withdrawal after pain stimulation) Gasche et al., 2004 Breastfed newborn 1 Mother: 30 mg 2 times a day for 2 days, 15 mg 2 times a day subsequently Child: intermittent periods of difficulty in breastfeeding and lethargy starting on day 7, regained his birth weight at day 11, grey skin and his milk intake fallen at day 12, dead at day 13. Koren et al., 2006; Madadi et al., months old child mg/kg Unresponsiveness, pinpoint pupils, the patient became apnoeic, complete Voronov et al., 2007 after tonsillectomy recovery Breastfed newborns 2 of 17 Mother: 120 mg/day Mother: sedation, nausea, dizziness, weakness Madadi et al., 2009 Child: extreme drowsiness, poor feeding; complete reversal of the symptoms after switch to formula feeding Polymorphisms of the UGT2B7 gene are functional (Innocenti et al., 2008) and have been associated with altered plasma concentration ratios of opioids and their glucuronide metabolites. For example, the Tyr268 UGT2B7 glucuronidates buprenorphine at a 10-fold higher rate (expressed as pmol/min/mg protein) in vitro than the His268 isoform (Coffman et al., 1998). In vivo, different variants in the 5 untranslated region of UGT2B7 are associated with reduced morphine-6-glucuronide/morphine ratios in patients (Court et al., 2003; Sawyer et al., 2003; Duguay et al., 2004; Darbari et al., 2008). In addition, two mothers whose breast-fed children exhibited severe neonatal toxicity, carried an UGT2B7 2/ 2 genotype and were CYP2D6 UMs (Madadi et al., 2009). This combination may have decreased the main clearance pathway of codeine to codeine-6-glucuronide and thus contributed to the intensive formation of morphine. However, the consequences of UGT variants were restricted to alterations of plasma concentrations, while none of the UGT variants alone have been associated with the altered efficacy of analgesics. The increased enzyme activity associated with the CYP3A5 1 allele (Hustert et al., 2001; Kuehl et al., 2001) may cause accelerated elimination of CYP3A substrates, such as alfentanil (Klees et al., 2005a,b), fentanyl (Jin et al., 2005) or sufentanil (Guitton et al., 1997). However, positive associations of CYP3A polymorphisms with analgesic actions have not been reported so far. The CYP3A5 genotype did not affect the systemic or apparent oral clearance as well as the pharmacodynamics of alfentanil (Kharasch et al., 2007) and levomethadone (Lötsch et al., 2006b) Transmembrane transporters. P-glycoprotein (P-gp) coded by the ABCB1 gene is mainly located in organs with excretory functions, such as liver, kidney and the gastrointestinal tract (Fromm et al., 1999). It is expressed at the blood brain barrier where it forms an outward transporter (Cordon-Cardo et al., 1989). Therefore, functional impairment of P-gp mediated drug transport may be expected to result in increased bioavailability of orally administered drugs, reduced renal clearance or an increased brain concentration of its substrates. These mechanisms give rise to the expectation of decreased dosage or increased clinical effects of analgesics that are substrates of P-gp. The ABCB1 3435CNT variant is associated with decreased dosage in opioids that are P-gp substrates, as assessed in an outpatient pain therapy setting (Lötsch et al., 2009d). Moreover, a diplotype consisting of three polymorphic positions in the ABCB1 gene (1236TT, 2677TT, and 3435TT) is associated with increased susceptibility to the effect of fentanyl to cause respiratory depression (Park et al., 2007b). Also, the opioid loperamide, clinically prescribed as antidiarrheic which does not produce effective CNS concentrations due to its low absorption and rapid excretion from the CNS by P-gp (Wandel et al., 2002), produces central nervous opioid effects associated with an ABCB1 3435TT genotype (Skarke et al., 2003b). With the OPRM1118ANG variant, the ABCB1 3435CNT predicted the response to morphine in cancer patients with a sensitivity close to 100% and a specificity of more than 70% (Campa et al., 2008). Finally, methadone analgesia may be subject to P-gp pharmacogenetic modulation. The pupillary effects of perorally administered methadone are increased following the pharmacological blockade of P-gp by quinidine (Kharasch et al., 2004), and the methadone dosing for heroin substitution can be decreased in carriers of ABCB1 variants associated with decreased transporter expression, e.g., ABCB1 2435CNT and others (Coller et al., 2006) Pharmacodynamics Decreased effects of analgesics may result from pharmacodynamic interferences, consisting of insufficient receptor binding, activation or signaling, or of a decreased expression of the drug's target, such as opioid receptors or cyclooxygenases. Genetic factors have been found to act via any of these mechanisms Opioid receptors. The µ-opioid receptor is the clinically most relevant target of opioid analgesics (Matthes et al., 1996). The OPRM1 gene is highly polymorphic (Thony & Blau, 2006; Shabalina et al., 2009), with 1799 human SNPs currently listed in the NCBI SNP data base ( accessed on January 18, 2009). Coding mutations affecting the third intracellular loop of the µ-opioid receptor (e.g., 779GNA, 794GNA, 802TNC) result in reduced G-protein coupling, receptor signaling and desensitization (Koch et al., 2000; Befort et al., 2001; Wang et al., 2001) leading to an expectation that opioids should be almost ineffective in patients carrying those polymorphisms. However, this has yet to be shown due to their extremely low allele frequency ( 1%) and is restricted to rare single cases. Evidence for a function of OPRM1 variants with allelic frequencies N5% is sparse (Hirota et al., 2003) or negative (Lötsch & Geisslinger, 2006), except for the 118ANG SNP (Lötsch & Geisslinger, 2006). The latter causes an amino acid exchange of the aspartate with an asparagine at position 40 of the receptor protein deleting a putative extracellular glycosylation site. This may cause decreased µ-opioid receptor expression (Zhang et al., 2005) or signaling (Oertel et al., 2009). These molecular changes translate to decreased clinical effects of various opioids in experimental settings (Skarke et al., 2003a; Romberg et al., 2004, 2005; Oertel et al., 2006) and clinical studies (Klepstad et al., 2004; Chou et al., 2006a; Campa et al., 2008; Sia et al., 2008), except for a single report with opposite observations (Landau et al., 2008). The consequences of the 118ANG SNP have consistently been observed to be a decrease in opioid potency for pupil constriction, which results in a right-shift of the pupil size versus the opioid concentration curve. Evidence for this is available for various opioids, such as morphine (Skarke et al., 2003a), morphine-6-glucuronide (Lötsch et al., 2002a; Skarke et al., 2003a) and methadone (Lötsch et al., 2006b). For analgesia, the SNP decreases the concentration-dependent effects of alfentanil on experimental pain (Oertel et al., 2006). Specifically, the variant decreases the effects of opioids on pain-related activation mainly in those regions of the brain that are processing the sensory dimension of pain including the primary and secondary somatosensory cortices and

10 J. Lötsch et al. / Pharmacology & Therapeutics 124 (2009) the posterior insular cortex (Oertel et al., 2008) (Fig. 3). In clinical settings, greater postoperative of alfentanil (Caraco et al., 2001) and morphine (Klepstad et al., 2004; Chou et al., 2006a,b) have been reported for carriers of the variant, and higher concentrations of alfentanil (Oertel et al., 2008; Oertel et al., 2006) or morphine- 6-glucuronide (Romberg et al., 2004; Romberg et al., 2005) were needed to produce analgesia in experimental pain models. In addition, a single case of a patient heterozygously carrying the variant 118G allele was reported in whom a daily oral dose of 2 g morphine was necessary for satisfactory pain relief (Hirota et al., 2003). The OPRM1 118ANG SNP results in a broadened therapeutic range of alfentanil in healthy homozygous carriers. It decreases opioid induced respiratory depression more than opioid induced analgesia (Oertel et al., 2006). This was not present in heterozygous carriers (Romberg et al., 2005; Oertel et al., 2006). Finally, the variant is involved in modulating opioid effects as a joint genetic consequence with COMT (Reyes-Gibby et al., 2007) and ABCB1 (Campa et al., 2008) variants. The clinical consequences agree with the expectations from the single SNPs (see below). Newer insights into the OPRM1 architecture might reveal further functional variants (Shabalina et al., 2009). The intracellular opioid receptor signaling cascade involves G protein-coupled receptor kinases (GRK) and G protein-coupled inwardly-rectifying potassium channels (GIRK). They are first-line candidates for a genetic modulation of pain and opioid effects, although direct evidence is not yet available. GRK and subsequent arrestin binding to phosphorylated receptors is a key mechanism of opioid receptor desensitization (Ferguson et al., 1996). GIRKs are primary post-synaptic effectors of opioids in the CNS. Opioid tolerance is modulated by the functioning of GRK2 (Saland et al., 2008), GRK3 (Terman et al., 2004), and GRK2/6 plus β-arrestin 2 (Ferrer-Alcon et al., 2004). Moreover, GRK2 and 6 play a role in inflammation and allodynia (Kleibeuker et al., 2007; Eijkelkamp et al., 2009). GIRK1 (KCNJ3) and GIRK2 (KCNJ6) are involved in opioid induced analgesia (Ikeda et al., 2002; Marker et al., 2004). Other potassium inwardly-rectifying channels, such as GIRK3 (KCNJ9), are modulators of pain sensitivity (Smith et al., 2008). So far, functional GRK variants are known only in the cardiovascular context (Liggett et al., 2008) Catechol-O-methyl transferase. COMT degrades catecholamine neurotransmitters such as norepinephrine and dopamine. Increased dopamine concentrations suppress the production of endogenous opioid peptides (George & Kertesz, 1987). Opioid receptor expression is compensatorily upregulated (Zubieta et al., 2003), which has been shown with the V158M variant of the COMT, coded by the COMT 472GNA SNP, in human post-mortem brain tissue (Berthele et al., 2005), and in vivo by assessing radiolabeled 11 C-carfentanil µ-opioid receptor binding (Zubieta et al., 2003). This variant leads to a low-function COMT enzyme that fails to degrade dopamine, which may cause a depletion of enkephalin. Patients with cancer pain carrying the V158M variant needed less morphine for pain relief than patients not carrying this variant (Rakvag et al., 2005). Finally, the variant exerts its opioid enforcing effects also in cross-relation with the OPRM1118ANG variant (Reyes-Gibby et al., 2007) Cyclooxygenases. Polymorphisms in the prostaglandin-endoperoxide synthase 2 gene (PTGS2) coding for cyclooxygenase 2 may modulate the development of inflammation and its response to treatment with inhibitors of cyclooxygenases, especially those with a COX-2 preference (Esser et al., 2005). This has been proposed for the PTGS2-765GNC SNP, which was reported to be associated with more than a two-fold decrease in COX-2 expression (Cipollone & Patrono, 2002). By altering a putative Sp1 binding site (Hernandez-Avila et al., 2004), this PTGS2 gene variant was found to decrease the promoter activity by 30% (Papafili et al., 2002). However, a report pointing to an association of this with a net decrease in COX-2 function, quantified by prostaglandin E2 production from peripheral blood monocytes after stimulation with bacterial LPS (Cipollone & Patrono, 2002), has been withdrawn (Fazia et al., 2007). Interestingly, a year before withdrawal neither the 765GNC SNP-associated decrease in COX-2 expression nor the reduction of COX-2 inhibitor effects were reproduced in a study with healthy volunteers receiving celecoxib (Skarke et al., 2006). Nevertheless, rofecoxib lacked analgesic effects in carriers of the 765C variant allele (Lee et al., 2006) Co-analgesics The major types of co-analgesic drugs used for the treatment of chronic pain, in addition to the classical analgesics, comprise of (i) tricyclic antidepressants, (ii) modulators of the α 2 δ subunit of L-type voltage-gated calcium channels (i.e., gabapentin and pregabalin) and (iii) other anti-epileptic agents targeting mainly sodium channels. These classes of drugs have been developed for indications other than pain, namely as psychopharmaceuticals or anticonvulsants, respectively, but have gained a role in chronic pain therapy Pharmacokinetics Several antidepressants administered during therapy of chronic neuropathic pain are substrates of polymorphic CYP2D6 or CYP2C19 (Kirchheiner et al., 2004),which has led to specific genotype dependent dosage recommendations (Kirchheiner et al., 2001). Pharmacogenetic associations in pain therapy have not yet been reported. P-gp also plays a role in the brain concentrations of antidepressants (Mihaljevic-Peles et al., 2008). ABCB1 variants have been reported to modulate responses to several antidepressants (Gex-Fabry et al., 2008; Kato et al., 2008; Uhr et al., 2008). However, this is information about CYP or ABCB1 related genetic modulation of antidepressant responses from a psychiatric rather than a pain perspective Pharmacodynamics The target structures of antidepressants, namely the norepinephrine or serotonin transporters SLC6A2 and SLC6A4, respectively, have been reported to display functional genetic variants that may modulate the response to antidepressants (Rausch, 2005; Brown & Harris, 2008; Xu et al., 2008). Again, this information is primarily from psychiatric indications. Similarly, the relevance of variants in the α 2 δ subunit of L-type voltage-gated calcium channels has not yet been reported in human pain treatment. Nevertheless, a genotype dependence of gabapentin and pregabalin sensitivity depending on the type of pain has been shown in laboratory animals (Chesler et al., 2003). 5. Pharmacogenetics of drug interactions in pain therapy Small functional alterations associated with genetic variants may play an important role when the pharmacological target system is addressed by an additional drug, because due to the genetic variation its compensation capacity may be decreased. For example, the effects of loperamide on the central nervous system are significantly enhanced by the co-administration of the P-gp blocker quinidine in carriers of the P-gp gene variant ABCB CNT or in a haplotype that additionally contains the common allele of ABCB GNT(A) (Lötsch et al., 2003). Thus, drug interactions may affect only a part of the population who have a susceptible genotype (Samer et al., 2005). By inhibiting CYP2D6, paroxetine has been found to increase the steady state plasma concentrations of (R)-methadone in EMs but not PMs (Begre et al., 2002). Conversely, the effect of pharmacogenetic variants may be compensated as long as a second factor does not additionally challenge the affected system. CYP2C9 non-functional variants may increase the plasma concentrations of COX inhibitors, such as ibuprofen, diclofenac or celecoxib (Kirchheiner et al., 2002; Kirchheiner, 2003a,b). However, due to their broad therapeutic range, this might not translate into altered clinical effects unless warfarin, also a CYP2C9

11 178 J. Lötsch et al. / Pharmacology & Therapeutics 124 (2009) Fig. 3. Opioid effects on pain related brain activation (Oertel et al., 2008) assessed by pharmacological functional magnetic resonance imaging (pharm-fmri). Top: Glass brain representation of pain-associated brain activation at different alfentanil concentrations (successively targeted at 0, 25, 50 and 75 ng/ml) in non-carriers of µ-opioid receptor variant N40D. The opioid decreased the pain-stimulus associated brain activation (top and bottom, white columns) in a concentration-dependent manner. Bottom: Brain regions where the alfentanil effects on pain related activation differed between carriers and non-carriers of the OPRM1 118 ANG SNP. While in wild type subjects, the concentration versus effect relationship was linear (bottom, white columns, it was almost flat in carriers of N40D variant µ-opioid receptors (bottom, yellow columns). The pharmacogenetic differences occurred in brain regions of the pain matrix that are known to be involved in the processing of the sensory component of pain and thus of pain intensity, i.e., the primary and secondary somatosensory areas, S I and S II, the anterior cingulate cortex, ACC, and the anterior part of the insula.