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. 2023 Dec 1;164(12):2653-2664.
doi: 10.1097/j.pain.0000000000002993. Epub 2023 Jul 18.

Topical coapplication of hyaluronan with transdermal drug delivery enhancers attenuates inflammatory and neuropathic pain

Ivan J M Bonet  1 , Dionéia Araldi  1 , Paul G Green  1   2 , Jon D Levine  1   3
Affiliations

Topical coapplication of hyaluronan with transdermal drug delivery enhancers attenuates inflammatory and neuropathic pain

Ivan J M Bonet et al. Pain. .

Abstract

We have previously shown that intradermal injection of high-molecular-weight hyaluronan (500-1200 kDa) produces localized antihyperalgesia in preclinical models of inflammatory and neuropathic pain. In the present experiments, we studied the therapeutic effect of topical hyaluronan, when combined with each of 3 transdermal drug delivery enhancers (dimethyl sulfoxide [DMSO], protamine or terpene), in preclinical models of inflammatory and neuropathic pain. Topical application of 500 to 1200 kDa hyaluronan (the molecular weight range used in our previous studies employing intradermal administration), dissolved in 75% DMSO in saline, markedly reduced prostaglandin E 2 (PGE 2 ) hyperalgesia, in male and female rats. Although topical 500- to 1200-kDa hyaluronan in DMSO vehicle dose dependently, also markedly, attenuated oxaliplatin chemotherapy-and paclitaxel chemotherapy-induced painful peripheral neuropathy (CIPN) in male rats, it lacked efficacy in female rats. However, following ovariectomy or intrathecal administration of an oligodeoxynucleotide antisense to G-protein-coupled estrogen receptor (GPR30) mRNA, CIPN in female rats was now attenuated by topical hyaluronan. Although topical coadministration of 150 to 300, 300 to 500, or 1500 to 1750 kDa hyaluronan with DMSO also attenuated CIPN, a slightly lower-molecular-weight hyaluronan (70-120 kDa) did not. The topical administration of a combination of hyaluronan with 2 other transdermal drug delivery enhancers, protamine and terpene, also attenuated CIPN hyperalgesia, an effect that was more prolonged than with DMSO vehicle. Repeated administration of topical hyaluronan prolonged the duration of antihyperalgesia. Our results support the use of topical hyaluronan, combined with chemically diverse nontoxic skin penetration enhancers, to induce marked antihyperalgesia in preclinical models of inflammatory and neuropathic pain.

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Conflict of interest statement

Conflict of Interest: The authors declare no competing financial interests.

Figures

Figure 1.
Figure 1.. Topical co-administration of 500–1200 kDa hyaluronan (HA) with DMSO inhibits prostaglandin E2 (PGE2) hyperalgesia in male and female rats
PGE2 was injected intradermally (100 ng/5 μL, i.d.) at the site of nociceptive threshold testing on the dorsum of one hind paw of male and female rats followed, 5min later, by 500–1200 kDa hyaluronan (2 μg/μL in a volume of 30 μL), or its DMSO vehicle (30 μL), was applied topically at the same site. Mechanical nociceptive threshold was measured before and again 30 and 60 min after PGE2 administration. A. Topical administration of high molecular weight hyaluronan 500–1200 kDa hyaluronan in DMSO vehicle attenuated PGE2-induced hyperalgesia in male rats (repeated measures one-way ANOVA, 30 min: Treatment F(1.35,6.75)=40.11, P=0.0003, Tukey’s multiple comparison test PGE2 alone vs. PGE2 + hyaluronan P=0.0003, PGE2 + hyaluronan vs PGE2 + DMSO vehicle P=0.0014; 60 min: Treatment F(1.84,9.2)=40.15, P<0.0001, Tukey’s multiple comparison test PGE2 alone vs. PGE2 + hyaluronan P=0.0009, PGE2 + hyaluronan vs PGE2 + DMSO vehicle P=0.0039). B. Topical administration of 500–1200 kDa hyaluronan in DMSO vehicle also attenuated PGE2-induced hyperalgesia in female rats (repeated measures one-way ANOVA, 30 min: Treatment F(1.85,9.26)=60.23, P<0.0001, Tukey’s multiple comparison test PGE2 alone vs. PGE2 + hyaluronan P=0.0005, PGE2 + hyaluronan vs PGE2 + vehicle P=0.0011; 60 min: Treatment F(1.39,6.93)=29.87, P=0.0006, Tukey’s multiple comparison test PGE2 alone vs. PGE2 + hyaluronan P=0.004, PGE2 + hyaluronan vs PGE2 + DMSO vehicle P=0.0004). n=6 per group.
Figure 2.
Figure 2.. Dose-response relationship for topical hyaluronan-induced anti-hyperalgesia in rats with chemotherapy-induced peripheral neuropathy (CIPN)
A. Male rats received oxaliplatin (2 mg/kg, i.v.) on day 0. On day 7, at which time oxaliplatin-induced hyperalgesia was established, three different doses of 500–1200 kDa hyaluronan, at (1, 2 or 4 μg/μL) or DMSO vehicle, each in a volume of 30 μL, was administered topically on the dorsum of one hind paw, at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before and 7 days after oxaliplatin administration, and then again 10, 30, 60 and 120 min after 500–1200 kDa topical hyaluronan (in DMSO vehicle). Oxaliplatin decreased mechanical nociceptive threshold (i.e., produced hyperalgesia) (paired Student’s t-test, t(23)=15.62, P<0.0001), and topical administration of 500–1200 kDa hyaluronan in DMSO vehicle dose-dependently attenuated oxaliplatin-induced hyperalgesia (Two-way repeated measures ANOVA, dose F(3,20=10.08), P=0.0003). n=6 per group. B. Male rats received oxaliplatin (2 mg/kg, i.v.) on day 0. On day 7 after oxaliplatin administration, 500–1200 kDa hyaluronan (2 μg/μL in a volume of 30 μL) dissolved in 0.9% saline (without DMSO), or vehicle (0.9% saline, 30 μL) alone, was administered topically on the dorsum of one hind paw, at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before oxaliplatin and 7 days after oxaliplatin administration, and then again 30, 60 and 120 min after topical administration of hyaluronan. Oxaliplatin decreased mechanical nociceptive threshold (i.e., produced hyperalgesia) (paired Student’s t-test, t(11)=14.65, P<0.0001). Topical administration of 500–1200 kDa hyaluronan dissolved in saline, without DMSO, did not attenuate oxaliplatin-induced hyperalgesia (Two-way repeated measures ANOVA, dose F(1,10)=0.11, P=0.75). n=6 per group.
Figure 3.
Figure 3.. Topical hyaluronan-induced anti-hyperalgesia for CIPN is sexually dimorphic
Male and female rats received oxaliplatin (2 mg/kg, i.v.) on day 0. On day 7, 500–1200 kDa hyaluronan (2 μg/μL in a volume of 30 μL) or its DMSO vehicle alone (30 μL) was topically applied to the dorsum of the hind paw, at the site of nociceptive threshold testing. Additional groups of male and female rats received paclitaxel (1 mg/kg, i.p.), every other day for a total of 4 doses (days 0, 2, 4 and 6). On day 7, approximately 24 h after the last dose of paclitaxel, hyaluronan (2 μg/μL in a volume of 30 μL) or its DMSO vehicle (30 μL) was applied topically on the dorsum of one hind paw, at the site of nociceptive threshold testing. Finally, an additional group of rats received bortezomib (0.2 mg/kg, i.v., every other day for a total of 4 doses). On day 7, approximately 24 h after the last dose of bortezomib, 500–1200 kDa hyaluronan (2 μg/μL in a volume of 30 μL) or its DMSO vehicle (30 μL) was applied topically on the dorsum of the hind paw, at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before administration of oxaliplatin, paclitaxel or bortezomib and 7 days after, and then again 30, 60 and 120 min after topical administration of hyaluronan. A. Oxaliplatin decreased mechanical nociceptive threshold (i.e., produced hyperalgesia) in male rats. Topical administration of 500–1200 kDa hyaluronan attenuated oxaliplatin-induced hyperalgesia (Two-way repeated measures ANOVA, dose F(1,10)=24.98, P=0.0005) (results in this figure are reproduced from FIG. 2A, for comparison). B. Paclitaxel decreased mechanical nociceptive threshold (i.e., produced hyperalgesia) in male rats. Topical administration of 500–1200 kDa hyaluronan attenuated the hyperalgesia induced by paclitaxel (Two-way repeated measures ANOVA, dose F(1,10)=78.74, P<0.0001). C. Bortezomib decreased mechanical nociceptive threshold (i.e., produced hyperalgesia) in male rats. Topical administration of hyaluronan attenuated bortezomib-induced hyperalgesia (Two-way repeated measures ANOVA, dose F(1,10)=39.39, P<0.0001). D. In female rats, oxaliplatin-induced hyperalgesia was not attenuated by topical administration of 500–1200 kDa hyaluronan (Two-way repeated measures ANOVA, dose F(1,10)=0.039, P=0.8464). n=6 per group. E. In female rats, paclitaxel-induced hyperalgesia was not attenuated by topical administration of 500–1200 kDa hyaluronan (Two-way repeated measures ANOVA, dose F(1,10)=0.004, P=0.9503). n=6 per group. F. In female rats, bortezomib-induced hyperalgesia was not attenuated by topical administration of 500–1200 kDa hyaluronan (Two-way repeated measures ANOVA, dose F(1,10)=1.41, P=0.2471). n=6 per group.
Figure 4.
Figure 4.. In female rats hyaluronan-induced anti-hyperalgesia is nociceptor GPER dependent
A. A group of female rats underwent surgical ovariectomy 3 weeks prior to receiving oxaliplatin (2 mg/kg, i.v.), administered on day 0. On day 7 they received 500–1200 kDa hyaluronan (2 μg/μL in a volume of 30 μL) or DMSO vehicle (30 μL), administered topically on the dorsum of the hind paw, at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before and 7 days after oxaliplatin, and then again 30, 60 and 120 min after topical hyaluronan. Oxaliplatin decreased mechanical nociceptive threshold, measured 7 days after its administration. On day 7, topical administration of 500–1200 kDa hyaluronan attenuated the hyperalgesia induced by oxaliplatin in ovariectomized (Two-way repeated measures ANOVA, dose F(1,10)=37.75, P=0.0001), but not gonad intact, female rats (Fig. 3C). n= 6 per group. B. Another group of female rats underwent ovariectomy 3 weeks prior to receiving a first dose of paclitaxel (1 mg/kg, i.p.). Paclitaxel was administered every other day for a total of 4 doses (days 0, 2, 4 and 6). Seven days after the first dose of paclitaxel, rats were treated with 500–1200 kDa hyaluronan (2 μg/μL in a volume of 30 μL) or its DMSO vehicle (30 μL), applied topically on the dorsum of the hind paw, at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before and 7 days after the first dose of paclitaxel, and then again 30, 60 and 120 min after topical hyaluronan. Paclitaxel decreased mechanical nociceptive threshold, measured 7 days after its first dose, in ovariectomized and gonad-intact female rats. Topical 500–1200 kDa hyaluronan attenuated the hyperalgesia induced by paclitaxel in gonadectomized (Two-way repeated measures ANOVA, dose F(1,10)=66.27, P<0.0001) but not gonad intact female rats (Fig. 3D). n= 6 per group. C. Female rats received oxaliplatin (2 mg/kg, i.v.) on day 0. Four days later, they were treated i.t. with ODN antisense or mismatch (120 μg/20 μL, i.t.) for GPR30 mRNA, daily for 3 consecutive days. On day 7, approximately 24 h after the last dose of ODN, 500–1200 kDa hyaluronan (2 μg/μL in a volume of 30 μL) or its DMSO vehicle (30 μL) was administered topically on the dorsum of the hind paw, at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before oxaliplatin, 3 and 7 days after its administration, and then again 30, 60 and 120 min after topical hyaluronan. Oxaliplatin decreased mechanical nociceptive threshold in both GPR30 antisense- and mismatch-ODN treated rats. However, 500–1200 kDa hyaluronan attenuated the hyperalgesia induced by oxaliplatin only in the GPR30 antisense-treated group (Two-way repeated measures ANOVA, dose F(1,10)=24.83, P=0.0006). n= 6 per group. D. Another group of female rats received paclitaxel (1 mg/kg, i.p. every other day for a total of 4 doses). Starting four days after the 1st dose of paclitaxel, rats were treated with antisense or mismatch ODN for GPR30 mRNA (120 μg/20 μL, i.t.), daily for 3 consecutive days. On day 7, approximately 24 h after the last dose of ODN and the last dose of paclitaxel, 500–1200 kDa hyaluronan (2 μg/μL in a volume of 30 μL) or its DMSO vehicle (30 μL) was administered topically on the dorsum of the hind paw, at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before paclitaxel, 3 and 7 days after the first administration of paclitaxel, and then again 30, 60 and 120 min after topical 500–1200 kDa hyaluronan. Paclitaxel decreased mechanical nociceptive threshold in both GPR30 antisense- (one-way repeated measures ANOVA F(1.53,7.63) P<0.0001) and mismatch-treated groups (one-way repeated measures ANOVA F(1.13,5.65) P=0.0002). However, 500–1200 kDa hyaluronan only attenuated the paclitaxel-induced hyperalgesia in the GPR30 antisense-treated group (Two-way repeated measures ANOVA, dose F(1,10)=15.51, P=0.0028). n= 6 per group.
Figure 5.
Figure 5.. Steep DMSO concentration dependence for hyaluronan-induced anti-hyperalgesia
A. Male rats received oxaliplatin (2 mg/kg, i.v.) on day 0. On day 7, 500–1200 kDa hyaluronan (2 μg/μL in a volume of 30 μL) in six different percentages of DMSO (75%, 50%, 25%, 20%, 15% and 5% DMSO) or DMSO vehicle (30 μL) was administered topically on the dorsum of the hind paw at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before oxaliplatin and 7 days after its administration, and then again 30, 60 and 120 min after topical hyaluronan. Results are presented as mechanical nociceptive threshold in grams. Oxaliplatin decreased mechanical nociceptive threshold (i.e., produced hyperalgesia; paired Student’s t-test, baseline versus post-oxaliplatin before hyaluronan, for each group: Vehicle P=0.003956). Topical administration of hyaluronan in 75%, 50%, 25% and 20% produced anti-hyperalgesia (75% of DMSO P<0.0001, 50% of DMSO P<0.0001, 25% of DMSO P<0.0001, 20% of DMSO P<0.0001). However, topical administration of hyaluronan in 15% or 5% of DMSO did not produce significant anti-hyperalgesia (15% of DMSO P>0.99, 5% of DMSO P=0.99). Two-way repeated measures ANOVA, F(24.140)=13.01, P<0.0001. Dunnet’s multiple comparison post-hoc test. n=6 per group. B. Area Under Curve (AUC) representation of 30 and 60 min data in FIG. 5A.
Figure 6.
Figure 6.. Repeated administration of high molecular weight hyaluronan prolongs anti-hyperalgesia
A. Male rats received oxaliplatin (2 mg/kg, i.v.) on day 0. On day 7, 500–1200 kDa hyaluronan (2 μg/μL in a volume of 30 μL) or DMSO vehicle (30 μL) was administered topically on the dorsum of the hind paw, at the site of nociceptive threshold testing. Once the anti-hyperalgesic effect of hyaluronan wore off, hyaluronan was again administered, at the same site. Mechanical nociceptive threshold was evaluated before oxaliplatin, 7 days after its administration, 30, 60 and 120 min after the first topical administration of 500–1200 kDa hyaluronan, and then again 30, 60, 120, 180 and 240 min after the second topical administration of hyaluronan. Rats treated with oxaliplatin showed a prolongation of hyaluronan-induced anti-hyperalgesia after a second administration (two-way repeated measures ANOVA, F(1,5)=138.0, P<0.0001). n= 6 per group. B. Male rats received paclitaxel (1 mg/kg, i.p. every other day for a total of 4 doses). On day 7 hyaluronan (2 μg/μL in a volume of 30 μL) or DMSO vehicle (30 μL) was administered topically on the dorsum of the hind paw, at the site of nociceptive threshold testing. Once the anti-hyperalgesic effect of hyaluronan wore off, another dose of hyaluronan was administered at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before paclitaxel, 7 days after its first administration, 30, 60 and 120 min after the first topical administration of hyaluronan, and then again 30, 60, 120, 180 and 240 min after the second topical administration of hyaluronan. Paclitaxel-treated rats showed a prolongation of 500–1200 kDa hyaluronan-induced anti-hyperalgesia after the second administration, (two-way repeated measures ANOVA, F(1,5)=29.0, P=0.003). n= 6 per group.
Figure 7.
Figure 7.. Effect of hyaluronan molecular weight, and use of other transdermal drug delivery enhancers on topical hyaluronan-induced anti-hyperalgesia
A. Male rats received oxaliplatin (2 mg/kg, i.v.) on day 0. On day 7, hyaluronan of 5 different molecular weight ranges (75–120, 150–300, 300–500, 500–1200, and 1500–1750 kDa), was administered, on the dorsum of the hind paw, at the site of nociceptive threshold testing, in separate groups of rats. Mechanical nociceptive threshold was evaluated before, 7 days after administration of oxaliplatin, and then again 10, 30, 60 and 120 min after topical hyaluronan or DMSO vehicle. Oxaliplatin decreased mechanical nociceptive threshold (i.e., produced hyperalgesia, paired Student’s t-test, baseline versus post-oxaliplatin, before hyaluronan, for each group: Vehicle P=0.003956). Topical administration of all molecular weight ranges of hyaluronan, except 70–120 kDa which did not produce anti-hyperalgesia, produced robust anti-hyperalgesia, of similar magnitude (70–120 kDa P=0.000526, 150–300 kDa P=0.000122, 300–500 kDa P=0.000009, 500–1200 kDa P=0.000169, 1500–1750 kDa P=0.000026). The anti-hyperalgesia induced by all molecular weight ranges of hyaluronan was significant 60 min post-administration (two-way repeated measures ANOVA, F(5,30)=20.60, P<0.0001). n= 6 per group. B. Male rats received oxaliplatin (2 mg/kg, i.v.) on day 0. On day 7 after oxaliplatin hyaluronan combined with protamine (2 μg/μL in a volume of 30 μL) or protamine alone as a control (30 μL) was administered topically on the dorsum of the hind paw, at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before, 7 days after administration of oxaliplatin, and then again 10, 30, 60 and 120 min after topical application of hyaluronan. Compared with protamine alone, topical administration of hyaluronan combined with protamine produced robust anti-hyperalgesia lasting 120 min post-administration (two-way repeated measures ANOVA, F(4,40)=24.50, P<0.0001). n= 6 per group. C. Male rats received oxaliplatin (2 mg/kg, i.v.) on day 0. On day 7 after oxaliplatin hyaluronan combined with terpene (2 μg/μL in a volume of 30 μL) or terpene alone as a control (30 μL) was administered topically on the dorsum of the hind paw, at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before, 7 days after administration of oxaliplatin, and then again 10, 30, 60 and 120 min after topical application of hyaluronan. Compared with terpene alone, topical administration of hyaluronan combined with terpene produced anti-hyperalgesia (two-way repeated measures ANOVA, F(4,40)=41.02, P<0.0001). n= 6 per group. D. Male rats received paclitaxel (1 mg/kg, i.p. every other day for a total of 4 doses). On day 7 hyaluronan combined with protamine (2 μg/μL in a volume of 30 μL) or protamine alone as a control (30 μL) was administered topically on the dorsum of the hind paw, at the site of nociceptive threshold testing in the end of the day. On day 8, another dose of hyaluronan was administered at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before paclitaxel, 7 and 8 days after its administration, and then again 30, 60, 120, 180, 240, 300 and 360 min after the second topical administration of hyaluronan. Paclitaxel-treated rats showed a prolongation of 500–1200 kDa hyaluronan-induced anti-hyperalgesia after the second administration, (two-way repeated measures ANOVA, F(9,90)=21.12, P<0.0001). n= 6 per group.
Figure 8.
Figure 8.. CD44 dependence of topical hyaluronan-induced anti-hyperalgesia
A. Male rats received oxaliplatin (2 mg/kg, i.v.) on day 0. Starting four days later they were treated intrathecally (i.t.) with ODN antisense or mismatch for CD44 mRNA (120 μg/20 μL, i.t.), daily for 3 days. On day 7, approximately 24 h after the last dose of ODN, 500–1200 kDa hyaluronan (2 μg/μL in a volume of 30 μL) or its DMSO vehicle (30 μL) was administered topically on the dorsum of the hind paw, at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before oxaliplatin and 7 days after its administration, and then again 30, 60 and 120 min after topical hyaluronan. Results are presented as mechanical nociceptive threshold in grams. Oxaliplatin decreased mechanical nociceptive threshold, measured 4 days after its administration, in both CD44 antisense- (one-way repeated measures ANOVA F(1.00,5.01)=41.13, P=0.0014) and mismatch- (one-way repeated measures ANOVA F(1.30,6.51)=48.95, P=0.0002) treated groups. However, 500–1200 kDa hyaluronan only attenuated the hyperalgesia induced by oxaliplatin in the CD44 mismatch-treated group (two-way repeated measures ANOVA F(1,10)=40.72, P<0.0001). n= 6 per group. B. Male rats received paclitaxel (1 mg/kg, i.p., every other day for a total of 4 doses). Four days after the first paclitaxel injection, rats were treated with ODN antisense or mismatch (120 μg/20 μL, i.t.) for CD44 mRNA, daily for 3 consecutive days. On day 7, approximately 24 h after the last ODN dose, and the last administration of paclitaxel, hyaluronan (2 μg/μL in a volume of 30 μL) or DMSO vehicle (30 μL) was applied topically on the dorsum of the hind paw, at the site of nociceptive threshold testing. Mechanical nociceptive threshold was evaluated before paclitaxel, 7 days after its administration, and then again 30, 60 and 120 min after topical 500–1200 kDa hyaluronan. Hyaluronan attenuated the hyperalgesia induced by paclitaxel only in the CD44 mismatch-treated group (two-way repeated measures ANOVA F(1,10)=54.02, P<0.0001). n= 6 per group.
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