P-glycoprotein modulates morphine uptake into the CNS: a role for the non-steroidal anti-inflammatory drug diclofenac.

Our laboratory has previously demonstrated that peripheral inflammatory pain (PIP), induced by subcutaneous plantar injection of λ-carrageenan, results in increased expression and activity of the ATP-dependent efflux transporter P-glycoprotein (P-gp) that is endogenously expressed at the blood-brain barrier (BBB). The result of increased P-gp functional expression was a significant reduction in CNS uptake of morphine and, subsequently, reduced morphine analgesic efficacy. A major concern in the treatment of acute pain/inflammation is the potential for drug-drug interactions resulting from P-gp induction by therapeutic agents co-administered with opioids. Such effects on P-gp activity can profoundly modulate CNS distribution of opioid analgesics and alter analgesic efficacy. In this study, we examined the ability of diclofenac, a non-steroidal anti-inflammatory drug (NSAID) that is commonly administered in conjunction with the opioids during pain therapy, to alter BBB transport of morphine via P-gp and whether such changes in P-gp morphine transport could alter morphine analgesic efficacy. Administration of diclofenac reduced paw edema and thermal hyperalgesia in rats subjected to PIP, which is consistent with the known mechanism of action of this NSAID. Western blot analysis demonstrated an increase in P-gp expression in rat brain microvessels not only following PIP induction but also after diclofenac treatment alone. Additionally, in situ brain perfusion studies showed that both PIP and diclofenac treatment alone increased P-gp efflux activity resulting in decreased morphine brain uptake. Critically, morphine analgesia was significantly reduced in animals pretreated with diclofenac (3 h), as compared to animals administered diclofenac and morphine concurrently. These novel findings suggest that administration of diclofenac and P-gp substrate opioids during pain pharmacotherapy may result in a clinically significant drug-drug interaction

Diclofenac treatment increases P-gp expression in rat brain microvessels.

<p>Animals received subcutaneous injections of saline (Sal) or λ-carrageenan (Carr) into the plantar surface of the right hind paw. 15 min after paw treatment animals received saline (Sal, 1 mL/kg, i.p.) or diclofenac (Diclo, 30 mg/kg, i.p.). 3 h after paw treatment, animals were sacrificed and brain microvessels were isolated and prepared for Western blot analysis. Whole microvessels (10 µg) were resolved on a 10% SDS-polyacrylamide gel and transferred to a PVDF membrane. Samples were analyzed for expression of P-gp. <b>A</b>) Representative blot illustrating increased P-gp expression in animals treated with λ-carrageenan, diclofenac or both λ-carrageenan and diclofenac. <b>B</b>) Relative levels of P-gp expression in samples from <b>A</b> were determined by densitometric analysis. Results are expressed as mean ± SEM of three separate experiments (9 animals per group). Asterisks represent data points that are significantly different from saline control (*p<0.05).</p

Diclofenac increases P-gp-mediated transport of morphine at the BBB.

<p>The effect of λ-carrageenan treatment on brain uptake [³H]morphine (36.7 µg/rat) was determined by <i>in situ</i> brain perfusion. Animals received subcutaneous injections of saline or λ-carrageenan into the plantar surface of the right hind paw. 15 min after paw treatment animals received saline (1 mL/kg, i.p.) or diclofenac (30 mg/kg, i.p.). 3 h post paw treatment animals were perfused with [³H]morphine (0.5 mCi/mL) for 10 min. Morphine brain accumulation was significantly decreased in animals that received saline paw injections with diclofenac i.p. injections (Sal/Diclo) and in animals that received λ-carrageenan paw injections with saline i.p. injections (Carr/Sal) as compared to saline treated (Sal/Sal) animals. Results are expressed as mean ± SEM of at least 6 individual animals per treatment group. Asterisks represent data points that are significantly different (*p<0.05).</p

Diclofenac increases P-gp immunofluorescence.

<p>Animals received subcutaneous injections of saline (Sal) or λ-carrageenan (Carr) into the plantar surface of the right hind paw. 15 min after paw treatment animals received saline (Sal, 1 mL/kg, i.p.) or diclofenac (Diclo, 30 mg/kg, i.p.). 3 h after paw treatment animals were sacrificed and brain microvessels were isolated, plated on glass slides and fixed. Vessels were probed with mouse monoclonal anti-P-glycoprotein antibody and rabbit polyclonal antibody directed against von Willebrand factor (VWF). All slides were then incubated with appropriate Alexa Fluor-conjugated secondary antibodies. <b>A</b>. Representative immunofluorescent images from animals treated with saline paw and saline i.p. (Sal/Sal), saline paw and diclofenac i.p. (Sal/Diclo), λ-carrageenan paw and saline i.p. (Carr/Sal) or λ-carrageenan paw and diclofenac i.p. (Carr/Diclo). <b>B</b>. Semiquantitative analysis of the mean fluorescent intensity of P-gp. Microvessels isolated from treated and control animals were randomly selected for image analysis. Each bar represents the mean ± SEM of 75 microvessels from 3 separate experiments. Asterisks represent data points that are significantly different from Sal/Sal (*p<0.05).</p

Diclofenac administration following λ-carrageenan injection attenuates PIP-induced paw edema and hyperalgesia.

<p>Animals received subcutaneous injections of saline (Sal) or λ-carrageenan (Carr) into the plantar surface of the right hind paw (injected). 15 min after paw treatment animals received saline (Sal, 1 mL/kg, i.p.) or diclofenac (Diclo, 30 mg/kg, i.p.) <b>A</b>) Administration of diclofenac resulted in a significant decrease in paw edema in λ-carrageenan treated animals 3 h post paw treatment. Edema of the untreated left hind paw (contralateral) was comparable in all treatment groups. <b>B</b>) Administration of diclofenac attenuated λ-carrageenan -induced hyperalgesia 3 h post paw treatment. Data are reported as mean ± SEM from at least nine animals per treatment group. Asterisks represent data points that are statically different as indicated (*p<0.05).</p

PIP increases P-gp-mediated transport of morphine at the BBB.

<p>The effect of λ-carrageenan treatment on brain uptake [³H]morphine (36.7 µg/rat) was determined by <i>in situ</i> brain perfusion. Animals received subcutaneous injections of saline or λ-carrageenan into the plantar surface of the right hind paw. 3 h post paw treatment animals were perfused with [³H]morphine (0.5 mCi/mL) for 10 min. As previously shown, λ-carrageenan treatment induced an increase in P-gp function as exemplified by decreased brain morphine uptake. P-gp specific efflux of morphine was confirmed using the P-gp selective inhibitory peptide reversin 205. Animals were perfused with reversin 205 for 10 min prior to perfusion with [³H]morphine. Animals were then perfused with [³H]morphine (.5 mCi/mL) for 10 min in the presence of reversin 205. Results are expressed as mean ± SEM of at least 6 individual animals per treatment group. Asterisks represent data points that are significantly different (*p<0.05).</p

Diclofenac pretreatment alters morphine antinociception in warm-water (52°C) tail flick assay.

<p>Effect of diclofenac (Diclo, 30 mg/kg) administration on morphine (10 mg/kg) antinociception was assessed by warm-water tail flick. <b>A</b>) Time-effect curves showing an increase in tail flick latency that peaks 60 min post morphine dosing and is declining by 120 min. Two-way ANOVA indicates a decrease in morphine-induced analgesia in animals that received morphine 3 h post diclofenac treatment as compared to animals that received both morphine and diclofenac concurrently at the 45 and 60 min time points (*p<0.05). Animals receiving only saline (Sal) or diclofenac showed no change in antinociception over the 120 min time course. Results are expressed as mean ± SEM of 10 animals per treatment group. <b>B</b>) Histogram bars represent areas under the time-effect curves illustrated in <b>A</b>. Asterisks represent data points that are significantly different (*p<0.05).</p