Study of Genetic Factors Involved in Pain Perception and Morphine Analgesia in Cancer-related Pain

Pain is one of the most persistent and incapacitating symptoms of cancer. In fact, unsatisfactory treatment of cancer-related pain or absence of analgesic response has an enormous impact on patients’ quality of life. The World Health Organization treatment guidelines include opioid analgesics as the drugs of choice, with morphine as the first line option for moderate to severe pain. However, wide variations in dose requirement, pharmacological efficacy, tolerability and adverse effects have been observed. Age, gender, race/ethnicity, mood states and stress are known influencing factors but have failed to explain the high degree of interindividual variability. In the last decade, pharmacogenetic has been proposed to be an important and influent factor on opioids

response, especially morphine. Polymorphisms in opioid receptors, transporters and metabolizing enzymes are under extensive evaluation, along with genetic variations in modulators/suppressors involved in pain perception and transmission.

The prevalence of cancer-related pain, the unsuccess of the analgesic treatment and the potential of tailored-pain treatment in a foreseeable future prompted us to study important genetic variations in genes involved in opioids and pain mechanisms, along with a more focused study in morphine metabolism. In order to fulfil all the objectives, a method for the quantification of morphine and its major metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), was initially developed. The method revealed to be simple, sensitive, precise and accurate to quantify the three compounds in several antemortem and postmortem matrices, during animal and human studies.

Concerning genetic variations studies, important genes related to opioids action were selected, as µ-opioid receptor (OPRM1); morphine major metabolizing enzyme UDP-Glucuronosyltransferase 2B7 (UGT2B7); transporters ATP binding cassette sub-family B member 1 transporter (ABCB1); and organic anion-transporting polypeptides 1A2 (OATP1A2), along with pain and inflammation modulators, such as catechol-O-methyltransferase (COMT) and several cytokines. The first study of this thesis analyzed the influence of polymorphisms in OPRM1, COMT and ABCB1 genes. The results suggested that COMT Val(108/158)Met polymorphism is associated with opioid requirements, with carriers of Met allele being significantly associated with higher opioid doses. Later, an individual approach was performed and the patients with the higher (Patient 1, 800 mg/day) and lower (Patient 2, 20 mg/day) morphine requirements were analyzed, as Patient 1 reported uncontrolled pain and higher pain intensity. Results of genetic analysis has shown that polymorphisms OPRM1 A118G, COMT Val(108/158)Met

and UGT2B7 C802T and T801A seemed to influence the analgesic effect, with individuals xx GA, Val/Met and T801C802 being related with less morphine efficacy and higher doses. Also, differences in plasma concentrations of metabolites and metabolic ratios were found and correlated with the genetic variances. These observations confirmed the previous result but also highlighted the importance of case series analysis. Polymorphisms in inflammatory mediators were subsequently analyzed (interleukin (IL) 1α, IL-1β, IL-1 receptor antagonist (IL-1Ra), IL-2, IL4 receptor (IL-4R), IL-6, IL-10, tumor necrosis α and interferon γ). In this study, carriers of TT genotype of the C3954T polymorphism in IL-1β were associated with lower levels of IL1-β and lower levels of pain. Also, IL1-β levels were related with cancer onset status and metastatic disease. This result pointed out another non-opioid system that might be involved in pain sensitivity in cancer pain patients.

Finally, a relevant animal model was established to study morphine metabolism and its influence in the analgesic effect. Guinea pig revealed to be an adequate model, with morphine metabolic ratios close to humans. The obtained results showed that morphine metabolism induction leads to higher metabolic ratios (M3G/morphine and M6G/morphine) and faster and better analgesic effect, after a single morphine intraperitoneal administration. On the other hand, opposite results were observed during metabolism inhibition. These results demonstrated the importance of morphine pharmacokinetics in its final analgesic effect and the animal model developed seems promising for future studies concerning morphine metabolism and its implication in clinical practice.

In conclusion, the results of this thesis suggest that genetic variants in opioid and nonopioid systems can affect opioids analgesic effect, especially by influencing opioids requirements and pain perception. Additionally, further studies on the modulation of morphine metabolism might contribute to an improved analgesic effect of morphine, increasing patients’ life quality.