Mu-Opioid Receptor Genetic Polymorphism and Intrathecal Analgesia
Condition: Labor Pain; Post-cesarean Delivery
MeSH terms: Labor Pain
Study type: Observational

Summary: 
      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.
    

Detailed description: 
      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.
    

Intervention name(s): Blood Draw
Intervention type(s): Procedure
Intervention description(s): Blood for OPRM1 analysis
Observational model: Cohort
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Pregnant women
      
Biospecimen description: 
      Blood Samples
    
Keywords: µ-opioid receptor; neuraxial labor analgesia; pharmacogenetics