Mu-Opioid Receptor Genetic Polymorphism and Intrathecal Analgesia

Pharmacogenetics has allowed clinicians to identify associations between an individual's genetic profile and his/her response to drugs. The A118G (c.188A\>G)is a single nucleotide polymorphism (SNP) of the mu-opioid receptor (OPRM1). The mutated protein, N40D, appears to increase the binding affinity and potency of beta-endorphin approximately 3-fold. Individuals carrying the variant receptor gene (A118G) may show differences in some of the functions mediated by beta-endorphin action at the altered OPRM1. Combined spinal-epidural (CSE) analgesia is a commonly utilized technique for labor analgesia. Analgesia is initiated with the intrathecal administration of a lipid-soluble opioid (e.g. fentanyl), sometimes combined with a local anesthetic. The mean (± SD) duration of analgesia after intrathecal fentanyl 25 microgram was 89 ± 43 min. The ED50 of intrathecal fentanyl for labor analgesia varies between 14 microgram to 18.2 microgram. The wide variability in the duration of analgesia, as was well the differences in ED50 may result from differences known to affect labor pain (e.g., ethnicity, parity, stage of labor). Another possible explanation for the differences in opioid requirements and duration, as well as incidence of side effects such as itching and nausea/vomiting, is that opioid responsiveness is determined by genetic variability of the µ-opioid receptor. The ED50 for intrathecal fentanyl labor analgesia was significantly lower for parturients carrying the A118G variant of the mu-opioid receptor, compared to parturients with the A118 wild type receptor. The purpose of this study is to determine whether polymorphism at nucleotide 118 of OPRM1 influences the duration of intrathecal opioid (fentanyl) labor analgesia, and intrathecal opioid (morphine) postoperative analgesia.

Study 1 (intrathecal fentanyl): The primary outcome variable is duration of intrathecal fentanyl analgesia. A two-sided log rank test with an overall sample size of 152 subjects (wild type OPRM1 = 106, variant OPRM1 = 46) achieves 80% power at α = 0.05 to detect a difference of 0.2 between 0.5 and 0.3 (the proportion of subjects with continuing intrathecal fentanyl analgesia after 70 min). This assumes that 70% of subjects will have the wild-type MUOR1 phenotype, and 30% the variant phenotype. To account for anticipated subject dropout, 175 subjects will be enrolled in the study.

Study 2 (intrathecal morphine): The primary outcome variable is amount of rescue analgesia (morphine equivalents) necessary for 24 h after the intrathecal morphine injection. An overall sample size of 71 subjects (wild type OPRM1 = 50, variant OPRM1 = 21) achieves 81% power to detect a difference of 15 mg morphine equivalents between the null hypothesis that both group means are 40 mg morphine equivalents and the alternative hypothesis that the mean of one group is 25 mg morphine equivalents. The estimated group standard deviations are 20 mg morphine equivalents with alpha = 0.05 using a two-side Mann-Whitney test assuming that the actual distribution is uniform. This assumes that 70% of subjects will have the wild-type OPRM1 phenotype, and 30% the variant phenotype. To account for anticipated subject dropout, 90 subjects will be enrolled in the study.

Protocol specific methods:

Study 1: Eligible parturients admitted to the Labor and Delivery Unit of Prentice Women's Hospital will be approached for study participation immediately after the routine preanesthetic evaluation. This occurs shortly after admission to the Labor and Delivery Unit. Women who agree to participate will give written, informed consent at this time.

Venous blood will be obtained for genetic analysis of the 118 position of the µ-opioid receptor gene shortly after the subject consents to study participation, either through an intravenous catheter placed for routine intravenous access for labor and delivery, or through a fresh venipuncture. A total of 10 mL blood will be collected into two 5 mL EDTA tubes. The tubes will be batched, coded and stored in a 40C refrigerator until they will be send (1x/month) to the laboratory of Dr. J. L. Blouin, care of Dr. Landau, at the Hopitaux Universitaires de Geneve. Genetic analysis will be performed as described below. When the subject first requests analgesia, her cervix will be examined (this is routine procedure prior to initiating analgesia). If the cervix is dilated between 2 and 5 cm, the parturient will be included in the study. Visual analogue score (VAS) for pain (100 mm line where 0 mm = no pain and 100 mm = worst possible pain) will be determined immediately before initiation of analgesia. Combined spinal-epidural analgesia will be initiated in the sitting position per routine with intrathecal fentanyl 25 microgram. An epidural catheter will be sited. No drug will be injected through the epidural catheter until the parturient requests analgesia again. The parturient will be placed in the lateral position after the epidural catheter is secured. A VAS will be determined 10 min after the intrathecal injection.

The primary outcome variable is duration of intrathecal fentanyl analgesia.At the time the parturient requests additional analgesia, the cervix will be examined. A VAS will be determined. In addition, the parturient will be asked about the presence of pruritus since the initiation of analgesia (none, mild, moderate, severe), nausea (none, mild, moderate or severe), and vomiting (yes, no). An epidural test dose will be administered (lidocaine 1.5% with epinephrine 1:200,000). Assuming a negative test dose, bupivacaine 0.125% will be injected incrementally to a T10 sensory level. Epidural analgesia will be maintained with patient controlled epidural analgesia (PCEA) (bupivacaine 0.0625% with fentanyl 1.95 micro grams/mL: background infusion 15 mL/h, PCEA bolus 5 mL, lockout 10 min, maximum 30 mL/h) as per routine.

The study ends after the parturient delivers and the epidural infusion is discontinued. At this time the subject will be asked about her satisfaction with labor analgesia (100 mm scale, 0 mm = not satisfied at all, 100 mm = very satisfied).

The following data will be collected: maternal age, height, weight, race (self-described), cervical dilation at initiation of analgesia, time and cervical dilation at 2nd request for analgesia, maximum oxytocin dose, time to complete cervical dilation (10 cm), time to delivery, mode of delivery, neonatal weight, Apgar scores and umbilical blood gas values (obtained as part of routine care), total dose of epidural bupivacaine and other local anesthetics, total dose of epidural fentanyl, number of PCEA boluses and number of manual boluses (by the anesthesiologist).

Cases will be excluded from data analysis if CSE analgesia is not performed, if there is no analgesia (VAS \> 10 mm) 10 minutes after the intrathecal injection (failure of CSE technique), if cervical dilation is 8 cm within 60 minutes of intrathecal injection, or if the patient has a cesarean delivery. These cases will be reported.

Study 2: Eligible women admitted to the Labor and Delivery Unit of Prentice Women's Hospital for planned Cesarean delivery will be approached for study participation immediately after the routine preanesthetic evaluation. This occurs shortly after admission to the Labor and Delivery Unit. Women who agree to participate will give written, informed consent at this time.

Venous blood will be obtained for genetic analysis of the 118 position of the µ-opioid receptor gene shortly after the subject consents to study participation, either through an intravenous catheter placed for routine intravenous access for labor and delivery, or through a fresh venipuncture. A total of 10 mL blood will be collected into two 5 mL EDTA tubes. The tubes will be batched, coded and stored in a 4oC refrigerator until they are sent (1time/month) to the laboratory of Dr. J. L. Blouin, care of Dr. Landau, at the Hospitaux Universitaires de Geneve. Genetic analysis will be performed as described below Routine aspiration prophylaxis will be administered (intravenous ranitidine and metoclopramide, and oral antacid). Spinal anesthesia will be initiated in the sitting position in the routine manner with bupivacaine 12 mg (1.6 mL 0.75% hyperbaric bupivacaine), fentanyl 15 µg, and morphine 150 micrograms. Subjects will be placed in the supine left uterine displacement position immediately after the intrathecal injection. The level of cephalad sensory blockade will be determined 30 min after the intrathecal injection using von Frye hairs. Subjects with a sensory level below T6, or those that require intraoperative systemic opioid supplementation, will be excluded from further study participation.

In the PACU and for 24 hours after surgery, patients will receive ibuprofen 600 mg po q6h as per standard protocol. Patients may request rescue analgesia if they are experiencing discomfort. Rescue medication will consist of hydrocodone 10 mg plus acetaminophen 325 mg per os. An additional dose of hydrocodone 10 mg plus acetaminophen 325 mg will be provided after 1 hour if the pain is not relieved. These are routine oral analgesic medications for postoperative Cesarean delivery analgesia. Standard orders will be written for monitoring sedation and respiratory rate, and treatment of side effects (nausea, vomiting, pruritus and respiratory depression).

The primary outcome variable is amount of rescue morphine equivalent analgesia required for the 24 hours after the intrathecal morphine injection.18 The time of first rescue analgesia request will be noted, and the VAS will be determined at the time of request for rescue analgesia. In addition, the parturient will be asked to provide a VAS for pain at 4, 8, 12, 18, and 24 hours after the intrathecal injection. The presence of pruritus (none, mild, moderate, severe), nausea (none, mild, moderate or severe), and vomiting (yes, no) will be determined at 4 and 24 h after the intrathecal injection.

The study ends 24 h after the intrathecal injection. At this time the subject will be asked about her satisfaction with postoperative analgesia (100 mm scale, 0 mm = not satisfied at all, 100 mm = very satisfied). Further analgesia will not be dictated by study protocol.

The following data will be collected: maternal age, height, weight, race (self-described), requirement for supplemental intraoperative sedation, neonatal weight, intra- or postoperative treatment for pruritus, nausea or vomiting. In addition to the time to first request for supplemental oral analgesia, the following will be recorded: supplement analgesia dose requirements (number of acetaminophen/hydrocodone tablets) at 4, 8, 12, 18, and 24 h after the intrathecal injection.

DNA collection: Peripheral blood will be collected in 2 5ml EDTA tubes (total 10ml). DNA will be prepared by non-phenolic methods using Puregene Blood Extraction Kit (Gentra, Minneapolis, MN) and tested for molecular weight on gel electrophoresis and purity quality by optical densitometry measure (ratio 260/280 nm).

SNP genotyping: For identification of allelic distribution of the A118G SNP, 20-60 ng of DNA from individuals will be first amplified by PCR (on thermocycler apparatuses equipped with a 96 well-microtiter plate block) using primers designed in the vicinity of the SNPs. The SNP will be then genotyped in amplified products by minisequencing (Pyrosequencing).

Pyrosequencing: PCR using cDNA specific primers (spanning an intron in genomic DNA), in which the forward primer is labeled with 5' biotin is performed under standard conditions, and the product is analyzed by the Pyrosequencing method. Briefly, an internal primer is designed two nucleotides before the mutation site, so that the two mRNA populations could be assayed by quantifying the relative amounts of each allele present in the PCR product. DNA from normal and affected subjects are used as controls.

PCR products are immobilized with Dynabeads (Dynal, Oslo, Norway) by a 15 min, 65 oC incubation in a buffer containing 10mM Tris-HCl, 2M NaCl, 1mM EDTA and 0.1% Tween 20. PCR products are then removed from solution using magnetic separation, denatured with NaOH 0.5 M and washed with 200mM Tris-Acetate, 50mM MgAc2. The remaining single stranded DNA is then hybridized with the internal 'sequencing' primer, by heating the mix to 80oC, and slowly cooling it to room temperature. Next enzyme and substrate mixes are automatically added to each well, and the reactions proceed at 28oC, by the sequential addition of single nucleotides at a predetermined order. Luciferase peak heights are proportional to the number of nucleotide incorporations, which has been shown to be very quantitative (5% error rate) in a number of experimental settings.

Coded DNA samples will be stored in Dr. Blouin's laboratory. No further sequencing will be done unless subjects signed the consent form for further future studies.

Eligibility Criteria:
Inclusion Criteria:

Study 1: Laboring Women

* Nulliparous women in spontaneous labor or with spontaneous rupture of membranes
* Term pregnancy (≥ 37 weeks gestation)
* Vertex presentation
* Healthy, ASA PS 1-2
* Desire neuraxial labor analgesia.

Study 2: Cesarean Delivery

* Nulliparous women undergoing elective primary Cesarean delivery (e.g., for breech presentation, macrosomia)
* Term pregnancy (≥ 37 weeks gestation)
* Healthy, ASA PS 1-2
* Desired spinal anesthesia.

Exclusion Criteria:

Study 1: Laboring Women

* Chronic or pregnancy induced disease
* Chronic opioid use
* History of substance abuse
* Systemic opioid analgesia before initiation of neuraxial labor analgesia
* Cervical dilation \< 2 cm or \> 5 cm of time of request for neuraxial analgesia
* Allergy to fentanyl

Study 2: Cesarean delivery

* Chronic or pregnancy induced disease
* Chronic opioid use
* Previous abdominal or pelvic surgery
* Allergy to fentanyl, morphine, or bupivacaine
* BMI ≥ 40 kg/m2
* History of substance abuse
* Failed spinal anesthesia
* Requirement for systemic opioid supplementation during Cesarean delivery.