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In response to adverse outcomes from prescription opioids, the Centers for Disease Control and Prevention (CDC) released the Guideline for Prescribing Opioids for Chronic Pain in March 2016.To test the hypothesis that the CDC guideline release corresponded to declines in specific opioid prescribing practices.Interrupted time series analysis of monthly prescribing measures from the IQVIA transactional data warehouse and Real-World Data Longitudinal Prescriptions population-level estimates based on retail pharmacy data. Population size was determined by U.S. Census monthly estimates.United States, 2012 to 2017.Persons prescribed opioid analgesics.Outcomes included opioid dosage, days supplied, overlapping benzodiazepine prescriptions, and the overall rate of prescribing.The rate of high-dosage prescriptions (≥90 morphine equivalent milligrams per day) was 683 per 100 000 persons in January 2012 and declined by 3.56 (95% CI, −3.79 to −3.32) per month before March 2016 and by 8.00 (CI, −8.69 to −7.31) afterward. Likewise, the percentage of patients with overlapping opioid and benzodiazepine prescriptions was 21.04% in January 2012 and declined by 0.02% (CI, −0.04% to −0.01%) per month before the CDC guideline release and by 0.08% (CI, −0.08% to −0.07%) per month afterward. The overall opioid prescribing rate was 6577 per 100 000 persons in January 2012 and declined by 23.48 (CI, −26.18 to −20.78) each month before the guideline release and by 56.74 (CI, −65.96 to −47.53) per month afterward.No control population; inability to determine the appropriateness of opioid prescribing.Several opioid prescribing practices were decreasing before the CDC guideline, but the time of its release was associated with a greater decline. Guidelines may be effective in changing prescribing practices.CDC.