Targeting effector memory T cells with the small molecule Kv1.3 blocker PAP-1 suppresses allergic contact dermatitis

Abstract

The voltage-gated potassium channel Kv1.3 has been recently identified as a molecular target that allows for selective pharmacological suppression of effector memory T (T(EM)) cells without affecting the function of naïve and central memory T cells. We here investigated whether PAP-1, a small molecule Kv1.3 blocker (EC50=2 nM), could suppress allergic contact dermatitis (ACD). In a rat model of ACD, we first confirmed that the infiltrating cells in the elicitation phase are indeed CD8+ CD45RC- memory T cells with high Kv1.3 expression. In accordance with its selective effect on T(EM) cells, PAP-1 did not impair sensitization, but potently suppressed oxazolone-induced inflammation by inhibiting the infiltration of CD8+ T cells and reducing the production of the inflammatory cytokines IFN-gamma, IL-2, and IL-17 when administered intraperitoneally or orally during the elicitation phase. PAP-1 was equally effective when applied topically, demonstrating that it effectively penetrates skin. We further show that PAP-1 is not a sensitizer or an irritant and exhibits no toxicity in a 28-day toxicity study. Based on these results we propose that PAP-1 could potentially be developed into a drug for the topical treatment of inflammatory skin diseases such as psoriasis.

Figure 1. Ear infiltrating cells in ACD are CD8⁺, CD45RC⁻, and Kv1.3⁺

(a, c, and e) Consecutive slides from a vehicle-treated rat ear, whereas (b, d, and f) are pictures of consecutive slides from an oxazolone challenged ear. (a and b) Stained for CD8, (c and d) for CD45RC, and (e and f) for Kv1.3. Bar = 200 μm.

Figure 2. Pharmacokinetics of PAP-1

(a) Total PAP-1 plasma concentrations in Lewis rats (n = 3) following intravenous injection at 6mg/kg. The data were fitted as a second-order exponential decay for a two-compartment model with Winnonlin^® software (y = A×exp^−αxt+B ×exp^−βxt where A = 9.58±0.72, B = 2.00±0.16, α = 3.18±0.33 hours⁻¹ and β = 0.23±0.01 hours⁻¹; K10 = 0.99±0.06 hours⁻¹, K12 = 1.68±0.23 hours⁻¹, and K21 = 0.74±0.09 hours⁻¹; steady-state volume of distribution Vss = 1.6±0.06 l/kg. (b) Total PAP-1 plasma concentrations following i.p. injection at 1 (●), 6 (○), or 10 (▴) mg/kg (n = 3 for each concentration). All values are given as the mean±SEM of total PAP-1 concentrations.

Figure 3. PAP-1 inhibits the elicitation phase of ACD when administered i.p., orally, or topically

(a) Rats were treated i.p. with the vehicle Cremophor^®EL/PBS (n = 30), PAP-1 at 1mg/kg (n = 9, 71.5±3.3% of control, P = 0.002), 6mg/kg (n = 20, 43.5±4.8% of control, P = 0.000001), 10 mg/kg (n = 16, 42.0±5.4% of control, P = 0.000001), or with ShK at 16 μg/kg (n = 3, 31.8±14.9% of control, P = 0.00015 vs control and P = 0.45 vs 10 mg/kg PAP-1). (b) Rats were gavaged with the vehicle peanut oil (n = 5) PAP-1 at 5mg/kg (n = 5, 77.2±5.0% of control, P = 0.037), 20 mg/kg (n = 5, 69.5±13.3% of control, P = 0.081) or 50mg/kg (n = 5, 48.4±8.2% of control, P = 0.002). (c) Rats were treated topically on the ears with 50 μl of the vehicle acetone:DMSO (n = 10), PAP-1 at 10 mg/ml (n = 10, 45.1±6.1% of control, P = 0.0002) or AS-85 at 10mg/ml (n = 9, 68.9±8.2% of control, P = 0.03). (d) PAP-1 was applied as a 2% cream in Dermabase^™ (n = 7, 79.8±15.4% of control, P = 0.15) or Eucerin^® (n = 8, 37.2±4.8% of control, P = 0.000001). The controls (n = 14) were treated either with “empty” Dermabase^™ (n = 7) or Eucerin^® (n = 7) and the results (181 μm swelling) averaged as there was no significant difference between the two vehicles. Values are given as the mean±SEM of swelling in percent. The average ear swelling in all controls from all conditions was 168.5 μm.

Figure 4. PAP-1 suppresses oxazolone-induced inflammation by reducing CD8+ T cells infiltration and production of the inflammatory cytokines IFN-γ and IL-17 but not TNF-α

Ears were removed 24 hours after the challenge from (a) vehicle or (b) PAP-1-treated rats and stained for CD8⁺ T cells. Bar = 200 μm. (c and d) mRNA quantities measured for (c) CD8-β, Kv1.3, IL-17, IL-2, and TNF-α or (d) IFN-γ from vehicle (filled black bars) or PAP-1-(10mg/kg, diagonal strips) treated rats. Six unchallenged ears were used as calibrator. Results are expressed as induction factors against the calibrator±SEM (n = 10 ears).

Figure 5. PAP-1 does not prevent sensitization or TPA-induced inflammation

(a) PAP-1 does not prevent sensitization for ACD. Rats were injected i.p. with the vehicle Cremophor^®EL/PBS (n = 8) or with PAP-1 at 10 mg/kg (n = 8) twice daily starting 2 days before sensitization with 1% oxazolone and for 3 days after sensitization. Animals were challenged with 0.2% oxazolone 7 days after sensitization. (b) PAP-1 does not suppress TPA-induced irritation. Rats were treated with 5 μg of TPA in 100 μl of AOO (4:1) on each side of both ears and injected i.p. with the vehicle Cremophor^®EL/PBS (n = 4) or with PAP-1 at 10 mg/kg (n = 4) at 8-hour intervals starting 20 hours before and for 20 hours after TPA treatment. Both ears were measured. The average ear swelling in TPA controls was 110.8 μm. Values are given as the mean±SEM of swelling in percent.

Figure 6. PAP-1 is not a contact sensitizer according to the LLNA IL-2 method

Vehicle (AOO), Triton^®X-100 250 mg/ml, oxazolone 0.2mg/ml, or PAP-1 at 10, 25, or 50mg/ml were applied to mouse ears daily for 3 consecutive days. Two days later, lymph node cells from the draining lymph nodes were collected and cultured for 15 hours in the presence of 10 μg/ml PHA-P and IL-2 concentrations in culture supernatant measured by ELISA. Values are given as stimulation index, which is the ratio of IL-2 released by lymph node cells from compound-treated animals to IL-2 released by lymph node cells from vehicle-treated animals. Values are given as the average of two independent experiments with four mice per group±SEM.

Figure 7. PAP-1 is not a contact sensitizer in vitro

(a) MUTZ-3 and (b) THP-1 myeloid cell lines were cultured for 24 hours in the presence of vehicle (DMSO), the irritant SDS at 200 μM, the sensitizer dinitrochlorobenzene at 3.75 μM or PAP-1 at 10, 20, or 40 μM. CD86 expression was then measured by flow cytometry. Values are given as a stimulation index (SI), which is the ratio of CD86 expression of treated cells over CD86 expression of DMSO-treated cells (SI = (% of cells expressing CD86⁺ ×mean fluorescence intensity of CD86⁺ cells) of treated/(% of cells expressing CD86⁺ ×mean fluorescence intensity of CD86⁺ cells) of control). The values are the average±SEM of two independent experiments.