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Research Article
Attenuation of 1-chloro-2,4-dinitrobenzene-induced inflammation in atopic dermatitis-like skin lesions in rats by a pyrrole containing FELL-NH 2 bioconjugate: Cannabinoid receptor type 1 involvement
expand article infoKonstantinos Papadakis‡, Kadir Bezirci‡, Boryana Borisova§, Stanislava Vladimirova§, Dancho Danalev§, Teodora Handjieva-Darlenska‡, Radka Tafradjiiska-Hadjiolova‡, Hristina Nocheva‡
‡ Medical University, Sofia, Bulgaria
§ University of Chemical Technology and Metallurgy, Sofia, Bulgaria

Corresponding author: Dancho Danalev ( ddanalev@uctm.edu )

Academic editor: Rumiana Simeonova
© 2024 Konstantinos Papadakis, Kadir Bezirci, Boryana Borisova, Stanislava Vladimirova, Dancho Danalev, Teodora Handjieva-Darlenska, Radka Tafradjiiska-Hadjiolova, Hristina Nocheva.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Papadakis K, Bezirci K, Borisova B, Vladimirova S, Danalev D, Handjieva-Darlenska T, Tafradjiiska-Hadjiolova R, Nocheva H (2024) Attenuation of 1-chloro-2,4-dinitrobenzene-induced inflammation in atopic dermatitis-like skin lesions in rats by a pyrrole containing FELL-NH 2 bioconjugate: Cannabinoid receptor type 1 involvement. Pharmacia 71: 1-10. https://doi.org/10.3897/pharmacia.71.e130213
Open Access

Abstract

Atopic dermatitis (AD) is a chronic inflammatory skin condition of significant health and social importance, which justifies the search for new means of treatment. Since the endogenous cannabinoid system appears to be involved in the pathogenesis of AD, the proposed article summarizes the clinical impact on skin inflammation in a rat model of 1-chloro-2,4-dinitrobenzene-induced atopic dermatitis-like condition after exogenous systemic administration of the cannabinoid receptor type 1 (CB1r) agonist anandamide, as well as after local treatment with a newly synthesized pyrrole moiety containing bioconjugate of FELL tetrapeptide with CB1r-dependent analgesic activity. The changes in skin lesions and ear thickness were estimated along with the CB1r expression immunohistochemically determined on skin punch biopsies. The results showed attenuation of skin lesions by anandamide and lack of positive effect after introduction of CB1r antagonist, accompanied by a change in CB1r expression, suggesting the involvement of the cannabinoid system in the defensive functions of the skin. The topically applied newly synthesized bioconjugate also favorably affected skin manifestations of inflammation, but without a change in CB1r expression, suggesting the involvement of other mechanisms in the reported effects.

Keywords

atopic dermatitis, CB1 expression, anandamide, immunohistochemistry, bioconjugates, FELL, peptides, rat model

Introduction

Atopic dermatitis (AD), commonly called atopic eczema, is an inflammatory chronic skin condition characterized by lesions in various parts of the body (Ständer 2021). “Dermatitis” refers to the inflammatory condition of the skin, while “atopic” reflects a genetic predisposition to allergic reactions. AD begins most often in early childhood, affecting two out of ten children (Avena-Woods 2017). The evolution of the pathology includes two possibilities: a chronic recurrent course before disappearing some time before puberty, or persistence into adulthood (Silvestre Salvador et al. 2017). But the onset of AD can occur also in adulthood. Thus, in 2000 Bannister and Freeman introduced the term „adult-onset atopic dermatitis“ (Bannister and Freeman 2000). So regardless of the exact period of the disease occurrence, it occurs in adults. The AD is, in fact, among the leading non-fatal health pathologies, with skin lesions manifesting with erythema, oedema, dryness, fissures, desquamation, itching, and pain, and causing significant psychosocial discomfort to patients and their relatives. The disease is accompanied by a number of additional risks, expressed in atopic comorbidities such as asthma, rhinitis, and food allergy, as well as nonatopic comorbidities like ocular, psychiatric, infectious, endocrine, autoimmune, and cardiovascular diseases, and certain cancers (Thyssen et al. 2023).

The described clinical picture determines the need for effective treatment of AD, and the possibility of local treatment is superior to the systemic treatment (given the danger of systemic reactions). Studying the pathogenesis of the disease would offer promising directions in the search for new approaches. The pathogenesis of AD is multifactorial, being determined by the interaction between genetic factors, impaired immune mechanisms of skin barrier function regulation and neuroimmunological mechanisms of skin protection, features of the microbiome, etc. (Kim et al. 2019b). There are different hypotheses about the pathogenesis of AD, but most authors are united around the impaired skin barrier function and immunopathological processes. A number of experimental models demonstrate the existence of a type 2 inflammation (Gandhi et al. 2016), with an increased number of mainly Th2- and Th22-cells and a weaker presence of Th17-cells, against the background of a typical pro-inflammatory cytokine environment (Suárez-Fariñas et al. 2011). At the same time, the number of reported cases of positive effects on certain skin manifestations (e.g., eczema, psoriasis, acne, pruritus) from experimental application of cannabinoids is increasing (Yoo and Lee 2023). Experimental data are also available on the importance of the endogenous cannabinoid system for the skin barrier—e.g., lack of cannabinoid receptor type 1 (CB1r) correlates with impaired ability to repair the epidermal barrier (Gaffal et al. 2014), suggesting some cannabinoid-mediated anti-inflammatory effect (Bíró et al. 2009).

Experimental modeling of AD follows three main approaches: 1) inbred models, 2) genetically engineered mice, and 3) models induced by topical application of exogenous agents (Kim et al. 2019a). The model used by our team refers to the last group and represents а modification of the one proposed by Chan and co-authors (Chan et al. 2013), in which the pathomorphological manifestations are expressed in thickening of the skin and infiltrate, accompanied by erythema, edema, dryness, and itching, as well as skin lesions (excoriations, erosions) (Feng et al. 2022).

The search for new molecules with a potential anti-inflammatory effect particularly directed to the treatment of AD inflammation led to the development of heterocycles containing one or more heteroatoms, which in principle represent one of the most useful classes of organic compounds in medicinal chemistry (Ivan et al. 2022). The tetrapeptide FELL designed by structure-activity relationship studies by Laurent et al. has shown a promising anti-inflammatory potential (St. Laurent et al. 2015). Further studies of our group revealed a bioconjugate of FELL tetrapeptide containing pyrrole moiety with a general structure of Pyr-Phe-Glu-Leu-Leu-NH2 (named BB19A, Fig. 1), which showed an interesting time-increasing CB1r-dependent analgesic activity, suggesting a possible anti-inflammatory effect (Borisova et al. 2023). Thus, herein we decided to continue our investigation on the molecule of BB19A, taking into account the currently revealed activity.

Figure 1. 

Chemical structure of the target bioconjugate BB19A.

The pathogenesis of AD has not been fully clarified, but there are literature data justifying the search for the involvement of the endogenous cannabinoid system (Shao et al. 2021). As already stated, CB1r-knockout mice showed delayed epidermal barrier recovery (Gaffal et al. 2014); in addition, topical application of some CB1r-specific agonists prevented epidermal thickening in an AD model (Kim et al. 2015). Other experimental data suggested that CB1r agonists may decrease the production of pro-inflammatory cytokines (Gaffal et al. 2014) and mast cell activation (Nam et al. 2016). AEA is widely distributed throughout the body and binds with high affinity to CB1r (Nikan et al. 2016). AM 251 is instead an inverse CB1r agonist (Hodge et al. 2008) used, as in our case, to better elucidate CB1r effects.

Therefore, our study included several goals united by the general idea of revealing elements of the pathogenesis of AD: 1) determining the expression of CB1 receptors in different areas (abdominal and dorsal) of skin and hair follicles after intraperitoneal pretreatment with CB1 agonist (anandamide, 1 mg/kg), resp. CB1 antagonist (AM251, 1.25 mg/kg); 2) determining the expression of CB1 receptors in different areas (abdominal and dorsal) of skin and hair follicles after pre- and post-treatment with the newly synthesized FELL bioconjugate containing pyrrole moiety ВВ19А.

Abbreviations

CB1r Cannabinoid receptor 1

AD Atopic dermatitis

AEA Anandamide

DNCB 1-chloro-2,4-dinitrobenzene

Materials and methods

Chemical substances

Anandamide (AEA), AM251, and 1-chloro-2,4-dinitrobenzene (DNCB) were acquired from Sigma-Aldrich (St. Louis, MO, USA). The latter was administered in acetone/olive oil (3:1) vehiculum. All specifically protected amino acids necessary for the target compound synthesis were purchased from Iris Biotech (Wunsiedel, Germany). Commercially available diethyl 2,4-dimethylpyrrole-3,5-dicarboxylate (CAS Number: 2436-79-5) needed for pyrrole moiety synthesis is from Sigma-Aldrich (Ansbach, Germany). All used solvents are from Valerus (Sofia, Bulgaria), and they are used without any pretreatment. The target substance, as a model one DNCB, was administered in the same acetone/olive oil (3:1) vehiculum.

Synthesis of the targeted bioconjugate BB19A

The synthesis of the targeted bioconjugate Pyr-Phe-Glu-Leu-Leu-NH2 is described in detail by Borisova et al. (2023). Briefly, the pyrrole moiety was obtained by basic catalyzed hydrolysis of commercially available diethyl-2,4-dimethylpyrrole-3,5-dicarboxylate. FELL-NH2 was synthesized on Rink-amide MBHA resin (load 0.63 mmol/g, 200–400 mesh) using a standard solid-phase peptide approach by a Fmoc/Ot-Bu strategy. Both active fragments further were condensed directly on the polymer carrier, and the final bioconjugate was removed from the resin by treatment of the cocktail of 95% TFA, 2.5% TIS, and 2.5% dH2O for 4 h. The final oil was recrystallized in cold diethyl ether, and the purity and the structure were proven by HPLC/MS.

Animals

The experiments were carried out on 68 male Wistar rats weighing 436 ± 18 g. Animals were obtained from the Medical University of Sofia Vivarium (Registration №1431-0001). Rats were subdivided in 11 groups (n = 6–8 per group), described in detail below (and summarized in Table 1), housed under controlled tem­perature (22 °C ± 2 °C) and a 12 h light/dark cycle (light between 08:00 and 20:00). Food (standard laboratory chow) and water were available ad libitum. All experimental protocol procedures were conducted in accordance with the relevant European Community requirements and approved by the BFSA (Permission protocol № 353/30.05.2023).

Table 1.
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Sensibilization (1st, 2nd, and 3rd day), pre-treatments (14th, 17th, 20th, 23rd, 26th, and 29th days), provocation (14th, 17th, 20th, 23th, 26th, and 29th day), and after-treatments (14th, 17th, 20th, 23th, 26th, and 29th day) of animals in each one of the groups.

Group Sensibilization Pretreatment Provocation After-treatment
A D A D A D A D
1 - - - - - - - -
2 200 μl 0.7% DNCB* - - - 200 μl 1% DNCB* - - -
3 - 200 μl 0.7% DNCB* - - - 200 μl 1% DNCB* - -
4 200 μl 0.7% DNCB* - 1 mg/kg AEA, i.p. 200 μl 1% DNCB* - - -
5 - 200 μl 0.7% DNCB* 1 mg/kg AEA, i.p. - 200 μl 1% DNCB* - -
6 200 μl 0.7% DNCB* - 1.25 mg/kg AM251, i.p. 200 μl 1% DNCB* - - -
7 - 200 μl 0.7% DNCB* 1.25 mg/kg AM251, i.p. - 200 μl 1% DNCB* -
8 200 μl 0.7% DNCB* - 50 μl ВВ19А* - 200 μl 1% DNCB* - - -
9 - 200 μl 0.7% DNCB* - 50 μl ВВ19А* - 200 μl 1% DNCB* - -
10 200 μl 0.7% DNCB* - - - 200 μl 1% DNCB* - 50 μl ВВ19А* -
11 - 200 μl 0.7% DNCB* - - - 200 μl 1% DNCB* - 50 μl ВВ19А*

* Topical treatment; A: abdominal skin; D: dorsal skin; i.p.: intraperitoneal administration.

Experimental atopic dermatitis

1-Сhloro-2,4-dinitrobenzene АD has been induced on abdominal/dorsal surfaces, according to a protocol borrowed from Chan et al. (Chan et al. 2013), detailed in the text of Fig. 2.

Figure 2. 

A. Schematic representation of the experimental setup for inducing atopic dermatitis-like lesions. Animals (experimental group AD) were sensitized three times with 200 μl 0.7% DNCB solution on days 1, 2, and 3. Treatment was аpplied to the abdominal/dorsal surface, shaved the day before the start of the experiment. The provocation was carried out 6 times, in 3-day intervals, starting from the 14th day, counting from the first day of sensitization (14th, 17th, 20th, 23rd, 26th, and 29th days) and included: 1) treatment of pre-sensitized abdominal/dorsal surfaces with 200 μl of 1% DNCB solution; 2) treatment of the unsensitized left ear of the animals with 20 μl of 1% DNCB solution. Тhe skin areas that were sensitized are shown in the shaded area B. dorsally and C. abdominally. Densitometric determination of the thickness of the external ear was made in the section pointed by “X” (D); B, C. Schematic representation of the dorsal (B) and abdominal (C) areas treated; D. “X” points to the section in which densitometric determination of the thickness of the external ear was made.

Experimental groups

  1. Negative control (control group) (n = 8): animals that have not been treated;
  2. Positive control-A (atopic dermatitis: AD-А-group) (n = 6): animals in which 1-chloro-2,4-dinitrobenzene АD has been induced on abdominal surfaces (Fig. 2C), detailed in the text of Fig. 2, and schematically represented in Fig. 2A;
  3. Positive control-D (atopic dermatitis: AD-D-group) (n = 6): animals in which 1-chloro-2,4-dinitrobenzene АD has been induced on dorsal surfaces (Fig. 2B), detailed in the text of Fig. 1, and schematically represented in Fig. 2A;
  4. AEA-А-group (n = 6): animals in which abdominal surfaces were sensitized as described in Fig. 2A scheme; 10 min before challenges with 200 μl of 1% DNCB solution on days 14, 17, 20, 23, 26, and 29, animals were intraperitoneally (i.p.) injected with 1 mg/kg anandamide. The left ear of each animal was treated with 20 μl of 1% DNCB solution during challenges on days 14, 17, 20, 23, 26, and 29;
  5. AEA-D-group (n = 6): animals in which dorsal surfaces were sensitized as described in Fig. 2A scheme; 10 min before challenges with 200 μl 1% DNCB solution on days 14, 17, 20, 23, 26, and 29, animals were i.p. injected with 1 mg/kg anandamide. The left ear of each animal was treated with 20 μl of 1% DNCB solution during challenges on days 14, 17, 20, 23, 26, and 29;
  6. AМ251-А-group (n = 6): animals in which abdominal surfaces were sensitized as described in Fig. 2A scheme; 10 min before challenges with 200 μl 1% DNCB solution on days 14, 17, 20, 23, 26, and 29, animals were i.p. injected with 1.25 mg/kg AM251. The left ear of each animal was treated with 20 μl of 1% DNCB solution during challenges on days 14, 17, 20, 23, 26, and 29;
  7. AМ251-D-group (n = 6): animals in which dorsal surfaces were sensitized as described in Fig. 2A scheme; 10 min before challenges with 200 μl 1% DNCB solution on days 14, 17, 20, 23, 26, and 29, animals were i.p. injected with 1.25 mg/kg AM251. The left ear of each animal was treated with 20 μl of 1% DNCB solution during challenges on days 14, 17, 20, 23, 26, and 29;
  8. BB19A-before-А-group (n = 6): animals in which abdominal surfaces were sensitized as described in Fig. 2A scheme; 10 min before challenges with 200 μl of 1% DNCB solution on days 14, 17, 20, 23, 26, and 29, the sensitized abdominal surfaces were treated locally with 200 μl of 2.81 mM BB19A solution. The left ear of each animal was treated with 20 μl of 1% DNCB solution during challenges on days 14, 17, 20, 23, 26, and 29;
  9. BB19A-before-D-group (n = 6): animals in which dorsal surfaces were sensitized as described in Fig. 2A scheme; 10 min before challenges with 200 μl of 1% DNCB solution on days 14, 17, 20, 23, 26, and 29, the sensitized dorsal surfaces were treated locally with 200 μl of 2.81 mM BB19A solution. The left ear of each animal was treated with 20 μl of 1% DNCB solution during challenges on days 14, 17, 20, 23, 26, and 29;
  10. BB19A-after-А-group (n = 6): animals in which abdominal surfaces were sensitized as described in Fig. 2A scheme; 10 min after challenges with 200 μl of 1% DNCB solution on days 14, 17, 20, 23, 26, and 29, the sensitized abdominal surfaces were treated locally with 200 μl of 2.81 mM BB19A solution. The left ear of each animal was treated with 20 μl of 1% DNCB solution during challenges on days 14, 17, 20, 23, 26, and 29;
  11. BB19A-after-D-group (n = 6): animals in which dorsal surfaces were sensitized as described in Fig. 2A scheme; 10 min after challenges with 200 μl of 1% DNCB solution on days 14, 17, 20, 23, 26, and 29, the sensitized dorsal surfaces were treated locally with 200 μl of 2.81 mM BB19A solution. The left ear of each animal was treated with 20 μl of 1% DNCB solution during challenges on days 14, 17, 20, 23, 26, and 29.

Treatment in each experimental group is summarized in Table 1.

Immunohistochemistry

Immunohistochemical evaluation for cannabionid receptor type 1 was carried out by means of rabbit anti-cannabinoid receptor type 1 antibody (clone ab23703, abcam, dilution 1:50), purchased from Sigma-Aldrich. A biotin-free polymer-based detection system (DS9800) was employed on 3 μm sections stained with Leica Microsystems BOND III Autostainer after heat-induced antigen retrieval for 20 minutes in the manufacturer’s alkaline retrieval solution ER2 (AR9640).

Evaluation of dermatitis severity

The severity of dermatitis was measured at the end of the experiment (30th day). Symptoms of erythema (redness), edema (infiltration), dryness (lichenification), and excoriation (scratching marks) were scored according to the severity score from the eczema area and severity index (EASI) (http://www.homeforeczema.org/documents/easi-user-guide-jan-2017-v3.pdf) on a scale of zero (none), one (mild), two (moderate), or three (severe). The intended definitions for zero, mild, moderate, and severe are explained in the text of Table 2. Total dermatitis scores (ranging from 0 to 12) of each one of the experimental groups were the sum of the individual scores, determined by two investigators blind to the experiments; inter-observer error did not vary by more than 3%.

Table 2.
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Clinical evaluation of 1-chloro-2,4-dinitrobenzene-induced AD. Symptoms of erythema (redness), edema (infiltration), dryness (lichenification), and excoriation (scratching marks) were scored on a scale of zero (none), one (mild), two (moderate), or three (severe). Total dermatitis scores (ranging from 0 to 12) of each one of the experimental groups were the sum of the individual scores.

Group Erythema (redness) Еdema (infiltration) Dryness (lichenification) Excoriation (scratching marks) Total
1 0 0 0 0 0
2 3 3 1 2 9
3 1 2 1 3 7
4 1 1 0 1 3
5 1 1 0 1 3
6 3 2 1 2 8
7 1 2 1 2 6
8 2 1 1 1 5
9 1 0 1 0 2
10 2 1 1 2 6
11 1 1 1 1 4
For estimation of erythema, edema, dryness, and excoriation, the following criteria have been adopted:
Sign None Mild Moderate Severe
Erythema No visible redness Slightly visible redness Clearly visible redness Dark red skin
Edema No visible swelling Slight elevation Clearly perceptible elevation Prominent elevation
Dryness No visible thickening or skin lines Barely visible skin lines Clearly exaggerated skin lines Skin thickening with exaggerated skin lines
Excoriation No visible scratching marks Superficial excoriation Deeper excoriation/s Several deep excoriations

The thickness of the left and right ears of the animals of each group was determined by means of an electronic digital calliper (EDC) in the middle of the external ear of the animal, as shown in Fig. 2D. Тhe values ​​of the left (treated) ears were compared both between groups and with the corresponding right (untreated) ears within each group; the right ear was taken as a kind of auto-control.

Results

The negative control group (Table 2, group 1; Fig. 3, Control group) presented no signs of erythema, edema, dryness, or excoriation on both abdominal and dorsal surfaces.

Figure 3. 

Clinical manifestation of experimental 1-chloro-2,4-dinitrobenzene-induced АD.

The induction of AD according to the described methodology led to the development of the characteristic manifestations: erythema (redness), edema (infiltration), dryness (lichenification), and excoriation (scratching marks) (Fig. 2, AD-A- and AD-D-groups). Total scores are presented in Table 2.

The abdominal surfaces of the positive control group (Table 2, group 2) showed dark red skin coloration with prominent elevation, faintly marked skin markings, and several superficial excoriations, while the dorsal ones showed light pink discoloration, perceptible elevation with minimally marked skin markings, but deep excoriations (Table 2, group 3).

After pretreatment with AEA, a weaker appearance of the skin lesions was observed: light pink discoloration, along with barely perceptible elevation, and scant excoriations in the abdominal (Table 2, group 4; Fig. 3, AEA-A-group) and the dorsal (Table 2, group 5; Fig. 3, AEA-D-group) surfaces of the skin. The pretreatment with AM251 instead resulted in lesions comparable with those of the positive controls: the abdominal areas (Table 2, group 6; Fig. 3, AM251-A-group) were more severely affected, presenting with prominent erythema, while the dorsal ones (Table 2, group 7; Fig. 3, AM251-D-group) showed faintly detectable erythema; both abdominal and dorsal areas bore traits of perceptible swelling, slightly marked skin lines, and several excoriations.

Treatment with the newly synthesized substance BB19A also attenuated the clinical manifestations of experimentally induced AD. The pretreatment of abdominal areas had the most prominent effect on the edema (only slight swelling has been observed) and was less prominent on erythema and excoriation; the degree of lichenification remained the same (Table 2, group 8; Fig. 3, BB19A-before-A-group). The pretreatment of dorsal areas had a more prominent effect since the total score decreased from 7 to 2—only barely noticeable redness and skin lines were observed (Table 2, group 9; Fig. 3, BB19A-before-D-group). The post-treatment of abdominal areas attenuated mostly the edema (faintly detectable), with a weaker effect on redness (clearly distinguishable), and no effect on lichenification and excoriation (Table 2, group 10; Fig. 3, BB19A-after-A-group). The post-treatment of dorsal areas affected mostly the excoriation (only superficial lesions were observed) and reduced swelling to a lesser extent, with no effect on erythema and lichenification (Table 2, group 11; Fig. 3, BB19A-after-D-group).

Analysis of left external ear thickness pointed out that in the AD group there was an 87.67% difference for abdominally treated animals and 86.90% for the dorsally treated animals compared to the control group (mean values, in mm, are shown in Table 3; percentage difference is presented in Table 4). AEA-pretreatment reduced this difference to 29.17% for both abdominal and dorsal treatment. After AM251-pretreatment, the difference was comparable to the positive control groups: 82.86% (abdominal treatment) and 82.01% (dorsal treatment). Topical pre- and post-treatment with BB19A showed similar differences: 80.29% (BB19A-before-abdominal treatment), 79.41% (BB19A-before-dorsal treatment), 80.29% (BB19A-after-abdominal treatment), and 77.61% (BB19A-after-dorsal treatment).

Table 3.
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Mean values of the thickness of left/right ears in each one of the groups: control (C); with 1-chloro-2,4-dinitrobenzene-induced AD (AD); with AEA pre-treatment (AEA); with AM251 pre-treatment (AM); with topical BB19A pre-treatment (BB19A before); with topical BB19A after-treatment (BB19A after).

Group of treatment Sensitized area Group number according to Table 2 N Mean of left ears (mm) Mean of right ears (mm) Percentage difference (%)
C 1 8 0.41 0.42 2.41
AD A 2 6 1.05 0.76 32.04
D 3 6 1.04 0.73 34.95
AEA A 4 6 0.55 0.49 11.54
D 5 6 0.55 0.44 22.22
AM A 6 6 0.99 0.62 45.96
D 7 6 0.98 0.61 46.54
BB19A before A 8 6 0.96 0.58 49.35
D 9 6 0.95 0.56 51.66
BB19A after A 10 6 0.96 0.69 32.73
D 11 6 0.93 0.64 36.94

A: abdominal treatment; D: dorsal treatment.

Measurement of the thickness of the outer ear of animals from the negative control group showed no differences between the left and right ears, while in the positive control, a thickening of the left ear by 32.04% compared to the right was reported in the abdominally treated group and 34.95% for the dorsally treated group (results are shown in Table 3). AEA-pretreatment reduced such differences to 11.54% and 22.22% for the abdominally and dorsally treated groups, respectively, whereas in the AM-pretreatment groups the estimated differences were 45.96% and 46.54%, respectively. Topical pretreatment with BB19A also showed left-right ear differences: 49.35% (BB19A-before-abdominal treatment), 51.66% (BB19A-before-dorsal treatment), 32.73% (BB19A-after-abdominal treatment), and 36.94% (BB19A-after-dorsal treatment).

CB1 expression in skin and hair follicles

Results are summarized in Table 4.

Table 4.
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Percentage difference between different experimental groups compared to controls. The indications of the groups are identical to Table 3.

Group of treatment Sensitized area Group number according to Table 2 Comparison to the left ear of the control group (%) Comparison to the right ear of the control group (%)
AD A 2 87.67 57.63
D 3 86.90 53.91
AEA A 4 29.17 15.38
D 5 29.17 4.65
AM A 6 82.86 38.46
D 7 82.01 36.89
BB19A before A 8 80.29 32.00
D 9 79.41 28.57
BB19A after A 10 80.29 48.65
D 11 77.61 41.51

A: abdominal treatment; D: dorsal treatment.

Abdominal punch biopsies from epidermis, basal epidermis, and hair follicle epithelium of negative controls (control group) were positive for CB1r, with CB1r expression most prominent in the epidermis (Table 5, group 1 abd).

Table 5.
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Immunohistochemical determination of CB1 receptor expression in punch biopsies from different areas of the skin.

Group Epidermis Basal epidermis Hair follicle
1 abd* ++ + +
1 dors** - - -
2 - - -
3 - + +
4 + + +
5 - - -
6 - - +
7 - + +
8 - - -
9 - + +
10 - - -
11 - + +

* in punch biopsy from an abdominal region; ** in punch biopsy from a dorsal section; + mild CB1r-expression; ++ moderate CB1r-expression; - lack of CB1r-expression. The description of groups 2–11 is given in the text.

Dorsal punch biopsies from the epidermis, basal epidermis, and hair follicle epithelium of negative controls showed no CB1r-expression (Table 5, group 1 dors).

In the positive controls (AD-A- and AD-D groups), the results showed absence of CB1r expression abdominally (Table 5, group 2) but presence of CB1r expression dorsally in basal epidermis and hair follicle epithelium (Table 5, group 3).

CB1 agonist pretreatment showed CB1r expression similar to controls: positive in abdominal punch biopsies (Table 5, group 4), and negative in dorsal ones (Table 5, group 5).

Pretreatment with CB1r antagonist showed greater similarity in expression to that of positive controls: negative in epidermis and basal epidermis in abdominal punch biopsies (Table 5, group 6) and positive in dorsal ones’ basal epidermis and hair follicle (Table 5, group 7).

Determination of CB1r-expression in the epidermis, basal epidermis, and hair follicle of abdominal and dorsal punch biopsies after pre- and post-treatment with the newly synthesized substance BB19A showed identity with that of the positive controls: absence of CB1r expression abdominally (Table 5, groups 8 and 10) and presence of CB1r expression dorsally in basal epidermis and hair follicles (Table 5, groups 9 and 11).

Discussion

In recent years, the importance of the endocannabinoid system for skin homeostasis has been demonstrated (Sheriff et al. 2020) as well as its involvement in some pathological processes (specifically, the CB1r seems to be involved in the processes of skin fibrosis), which justifies the possibility that its medicinal influence can be used for therapeutic purposes (Bíró et al. 2009; Correia-Sá et al. 2020).

In our experiments, the systemic pre-treatment of experimental animals with a CB1r agonist showed a reduced overall score of clinical manifestations—redness, edema, dryness, excoriation—which is probably due in part to a beneficial effect on itching, confirmed by other literature sources (Schlosburg et al. 2011; Avila et al. 2020), but also improved the skin barrier by modulating the immune response—suppression of Th1-cell immunity and stimulation of Th2-cell immunity, which has also been experimentally confirmed (Gaffal et al. 2014). The possibility that agonists of cannabinoid receptors may favorably affect AD after systemic administration is also suggested by other data, e.g., the use of hempseed oil in the form of nutritional supplements, lessening skin dryness and pruritus (Callaway et al. 2005). We assume that the favorable clinical effect of the skin lesions is due to the decreased levels of pro-inflammatory mediators—TNF, IL-1 β, IL-6, IL-8, and IL-12 (Klein 2005) or others. Our results showed that the indicated beneficial effects were not observed in animals pretreated with a CB1 receptor antagonist, where the total score approached that of the positive controls.

We believe that the thickening of the treated external ears of the animals in the positive control group should be attributed to the systemic manifestations of inflammation due to the sensitization and the subsequent provocation. The reduced thickening after AEA-pretreatment can be explained by the alleviation of such a systemic effect by the CB1 receptor agonist, while the absence of such influence after the systemic introduction of the antagonist AM251 reverses the findings. Local treatment also appears to have a positive effect on systemic manifestations, more significant in pre-treated than in post-treated animals. It is possible that a reduction in itching and local discomfort leads to less scratching and hence to a weaker manifestation of local symptoms (thickening) of the external ear.

Regarding the immunohistochemical findings, we believe that further studies are needed, e.g., to explain differences in abdominal and dorsal cannabinoid receptor expression, as well as the increased expression in basal epidermis and hair follicles in the positive controls. So far, we report that systemic administration of an exogenous agonist of the CB1 receptor changes its expression, and this correlates with clinical manifestations. Notwithstanding that local pretreatment of the sensitized skin areas with the newly synthesized substance BB19A before the provocations showed a decrease in the total score of the appearance of the skin lesions, the immunohistochemical determination of the CB1r expression in the individual skin areas resembled that of the positive controls. The data indicate that the beneficial effect of the newly synthesized pyrrole moiety containing FELL-OH(NH2) tetrapeptide is probably not due to a change in the expression of cannabinoid receptors but rather to an intervention in the pathogenetic mechanisms of the process—e.g., local improvement of the skin barrier function, lowering the effect of pro-inflammatory mediators in the pre-treated areas, or other possible mechanisms, which would be the subject of subsequent studies.

It is likely that topical pretreatment with BB19A reduces the local inflammatory process and attenuates the subsequent systemic manifestations after the provocation, while the topical treatment after the provocation affects the pathological process only locally. Our data support the documented results of successful application of topical agents containing the endocannabinoid palmitoyl-ethanolamide as adjuvants in AD patients with an effect on pruritus, as well as mean time to flare (Eberlein et al. 2008).

Conclusions

Exogenous administration of type 1 cannabinoid receptor agonists reduced local and systemic manifestations of inflammation in a rat model of AD by altering CB1r expression in different skin sites.

The pyrrole containing FELL bioconjugate BB19A showed a positive effect on the local appearance of skin lesions in rats, indirectly affecting the systemic manifestations of inflammation but without affecting the expression of the CB1 receptor.

Acknowledgments

This work was supported by Grant D-147/2023 from the Council of Medical Science, MU-Sofia. In addition, the synthesis and characterization of the targeted molecule as well as Boryana Borisova, Dancho Danalev, Stanislava Vladimirova and Hristina Nochjeva are granted by the project BG-RRP-2.004-0002, “BiOrgaMCT,” financed from the European Un-ion-NextGenerationEU, through the National Recovery and Resilience Plan of the Republic of Bulgaria.

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