Immunomodulator

Changes of cell-surface thiols induced by chemical treatment may affect the conformations of membrane proteins and intracellular signaling mechanisms. In our previous study, we found that a non-toxic dose of Diphenylcyclopropenone (DPCP), which is a potent skin sensitizer, induced an increase of cell-surface thiols in cells of a human monocytic cell line, THP-1. Here, we examined the influence of DPCP on intracellular signaling. First, we confirmed that DPCP induced an increase of cell-surface thiols not only in THP-1 cells, but also in primary monocytes. The intracellular reduced-form glutathione/oxidized-form glutathione ratio (GSH/GSSG ratio) was not affected by DPCP treatment. By means of labeling with a membrane-impermeable thiol-reactive compound, Alexa Fluor 488 C5 maleimide (AFM), followed by two-dimensional gel electrophoresis and analysis by liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), we identified several proteins whose thiol contents were modified in response to DPCP. These proteins included cell membrane components, such as actin and β-tubulin, molecular chaperones, such as heat shock protein 27A and 70, and endoplasmic reticulum (ER) stress-inducible proteins. Next, we confirmed the expression in DPCP-treated cells of spliced XBP1, a known marker of ER stress. We also detected the phosphorylation of SAPK/JNK and p38 MAPK, which are downstream signaling molecules in the IRE1α-ASK1 pathway, which is activated by ER stress. These data suggested that increase of cell-surface thiols might be associated with activation of ER stress-mediated signaling. [3]

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Topical immunotherapy with diphenylcyclopropenone (DPCP) is considered to be the most effective treatment of severe AA. However, the mechanism is unclear and an early predictor for the efficacy needs to be explored. The TSLP/OX40L/IL-13 pathway is an important pathway to initiate and maintain Th2 immune responses. Our previous work suggests this pathway may play a role in severe AA treated with DPCP. Thus, to further investigate the mechanism of TSLP/OX40L/IL-13 pathway in severe AA treated with DPCP and explore the predictor for the efficacy of DPCP therapy, we conducted a prospective study to compare expression levels of TSLP, OX40L, Th2 cytokines IL-4, IL-5 and IL13, and Th1 cytokine IFN-γ in severe AA patients before and after the treatment. Results showed that 21 AA patients were responsive (responders) to the DPCP therapy and 12 were not responsive (non-responders). Responders had lower levels of TSLP, OX40L and IL-13 than non-responders before the treatment. After the DPCP treatment, TSLP, IL-5 and IL-13 increased and IFN-γ decreased in responders while there were no changes of TSLP, IL-4, IL-13 and IFN-γ in non-responders. Our data suggest that the TSLP/OX40L/IL-13 pathway is down-regulated in some severe AA patients and DPCP might play a therapeutic role by up-regulating the pathway in these severe AA patients. The TSLP/OX40L/IL-13 pathway could be a predictor of response to the DPCP therapy for severe AA patients[1].

Diphenylcyclopropenone enhanced antigen-specific IgG2a antibody responses as well as IL-10 cytokine production after epicutaneous immunization with ovalbumin (OVA). Epicutaneous allergen-specific immunotherapy (EPIT) with OVA and DCP also protected sensitized mice from anaphylaxis and asthma. The protective effect was more robust than that of conventional SCIT, which did not significantly alleviate the symptoms of allergy in the murine models of anaphylaxis and asthma.

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p-Phenylenediamine (PPD) and Diphenylcyclopropenone (DPCP) are two potent haptens. Both haptens are known to cause delayed-type hypersensitivity, involving a cytokine response and local infiltration of T-cell subpopulations, resulting in contact dermatitis. We investigated the systemic immune effects of PPD and DPCP, two relatively unexplored skin allergens. The dorsal sides of the ears of BALB/c mice were exposed to PPD or DPCP (0.1 % w/v or 0.01 % w/v), or vehicle alone. Mice were treated once daily for 3 days (induction period) and subsequently twice per week for 8 weeks. Local and systemic immune responses in the auricular and pancreatic lymph nodes, spleen, liver, serum, and ears were analyzed with cytokine profiling MSD, flow cytometry, and qPCR. Ear swelling increased significantly in mice treated with 1 % PPD, 0.01 % DPCP or 0.1 % DPCP, compared with vehicle treatment, indicating that the mice were sensitized and that there was a local inflammation. Auricular lymph nodes, pancreatic lymph nodes, spleen, and liver showed changes in regulatory T-cell, B-cell, and NKT-cell frequencies, and increased activation of CD8+ T cells and B cells. Intracellular cytokine profiling revealed an increase in the IFN-γ- and IL-4-positive NKT cells present in the liver following treatment with both haptens. Moreover, we saw a tendency toward a systemic increase in IL-17A. We observed systemic immunological effects of PPD and DPCP. Furthermore, concentrations too low to increase ear thickness and cause clinical symptoms may still prime the immune system. These systemic immunological effects may potentially predispose individuals to certain diseases[4].

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Background/aim: Alopecia areata (AA) is an inflammatory disease with a genetic and autoimmune basis. Herein, it was aimed to study the efficacy and safety of an immunomodulatory therapeutic agent, Diphenylcyclopropenone, while manifesting its association with histopathological features, prognostic factors, and side effects. Materials and methods: In this retrospective study, 98 patients (60 males, 38 females) with alopecia, who were referred to the Hair Disease Polyclinic at the Department of Dermatology, between 2011 and 2015, were included. Together with medical histories and dermatological examinations, a skin biopsy for histopathological examination was conducted for all of the patients prior to therapy. Therapeutic success was evaluated on the basis of the hair regrowth percentage. Results: Regarding the overall treatment success, 33 (34%) patients had complete response, 16 (16%) had partial response (between 50% and 99%), 27 (28%) had minimal response (between 1% and 49%), and 22 (22%) were nonresponders. Both sexs were equally represented in the outcome. Conclusions: There was a significant relation between the severity of alopecia and the treatment outcome (P = 0.038). Patients with AA had significantly better response when compared to those with alopecia totalis and universalis. There was no statistically significant relation with other parameters, such as disease duration, age, sex, atopy history, age of onset, and histopathological features [2].

Changes of cell-surface thiols induced by chemical treatment may affect the conformations of membrane proteins and intracellular signaling mechanisms. In our previous study, we found that a non-toxic dose of diphenylcyclopropene (DPCP), which is a potent skin sensitizer, induced an increase of cell-surface thiols in cells of a human monocytic cell line, THP-1. Here, we examined the influence of DPCP on intracellular signaling. First, we confirmed that DPCP induced an increase of cell-surface thiols not only in THP-1 cells, but also in primary monocytes. The intracellular reduced-form glutathione/oxidized-form glutathione ratio (GSH/GSSG ratio) was not affected by DPCP treatment. By means of labeling with a membrane-impermeable thiol-reactive compound, Alexa Fluor 488 C5 maleimide (AFM), followed by two-dimensional gel electrophoresis and analysis by liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), we identified several proteins whose thiol contents were modified in response to DPCP. These proteins included cell membrane components, such as actin and β-tubulin, molecular chaperones, such as heat shock protein 27A and 70, and endoplasmic reticulum (ER) stress-inducible proteins. Next, we confirmed the expression in DPCP-treated cells of spliced XBP1, a known marker of ER stress. We also detected the phosphorylation of SAPK/JNK and p38 MAPK, which are downstream signaling molecules in the IRE1α-ASK1 pathway, which is activated by ER stress. These data suggested that increase of cell-surface thiols might be associated with activation of ER stress-mediated signaling[3].

In a mouse model of allergy, we tested the adjuvant potential of diphenylcyclopropenone (DCP), a strong contact sensitizer, which is currently used for the treatment of a T cell-mediated hair loss disease (alopezia areata).[3]

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Female CBA mice were used at the age of 6–8 weeks. Before epicutaneous immunization, mice were shaved on their belly (2 × 2 cm). After 4 h, the mice were anaesthetized with xylazin 8 mg/kg and ketamine 50 mg/kg intraperitoneally (i.p.) 13. The skin was tape stripped 10 times with a scotch tape. Thereafter, the mice were immunized with 25-μg ovalbumin (OVA; Grade V) dissolved in water/acetone/dibutylphthalate (1 : 1 : 2) ± 1% DCP. The vaccination was repeated after 7, 14, and 28 days (Fig. 1A). Sham controls were treated with the vehicle with 1% DCP. For reference, some mice were immunized four times subcutaneously with 25-μg OVA in PBS/aluminum hydroxide (2 : 1).

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Hapten treatment for mice and measurement of ear thickness [4]
The mice were sedated with hypnome/midazolam (0.01 μl per gram), and weight and ear thickness were measured prior to the first application of hapten. The haptens were dissolved in the vehicle acetone/olive oil 4:1 (AOO), and the mice were exposed on the dorsal sides of both ears to 25 μl of the respective hapten, p-Phenylenediamine, Diphenylcyclopropenone, or vehicle. For the PPD groups, the concentrations were 1.0 or 0.1 % (w/v). For the Diphenylcyclopropenone/DPCP groups, the concentrations were 0.1 or 0.01 %. Sensitization was achieved by treatment once daily for 3 days followed by challenge twice per week for 8 weeks. The mice were euthanized by cervical dislocation 24 h after the last treatment, and the draining auricular lymph nodes (ALN), pancreatic lymph nodes (PLN), spleen, liver, ears, and pancreas were removed and used for flow cytometry and quantitative PCR. Serum samples were collected by jaw puncture prior to euthanization.

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Treatment method with Diphenylcyclopropenone/DPCP [2]
Topical immunotherapy with Diphenylcyclopropenone/DPCP was performed following a standard protocol of sensitization. This protocol comprised the application of a 2% concentration of DPCP solution diluted in acetone, over an area of 2 × 2 cm on the occipital region of the scalp. The patients were told to avoid water contact of the sensitized area for 2 days and avoid sun exposure by using a wig or protective hat. After 2 days, the patients were checked to detect whether or not sensitization to DPCP had occurred. There was a lag period of 2 weeks after the sensitization, and then the treatment began. The initial DPCP concentration was 0.001%. DPCP solution was applied to all of the affected areas on the scalp, together with the eyebrows, once every week. Again after each session, the patients were told to avoid water contact for 2 days, including excessive sweating and sun exposure. DPCP solution was left on the scalp for 48 h and then washed off with a mild shampoo. The concentration of the DPCP solutions were increased weekly, unless there were serious side effects, including irritant contact dermatitis and photoallergic reactions, and the final concentration of 2% was reached at the end of week 6. Concentrations of 0.001%, 0.01%, 0.1%, 0.2%, 0.5%, 1%, and 2% were applied sequentially.

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Follow-up and assessment of efficacy [2]
During follow-up visits, the side effects of the patients were recorded, as well as the grade of hair regrowth. Once complete or cosmetically acceptable hair regrowth (amount of growth that eliminated the need for using a wig or hat) was achieved, the intervals of the Diphenylcyclopropenone/DPCP application were prolonged to 2 weeks, 3 weeks, and monthly. By this method, the DPCP immunotherapy was discontinued gradually. In the case of hair loss during this tapering-off period, therapy was restored at weekly intervals. If there was no obvious response at the end of 6 months, immunotherapy was considered as noneffective and discontinued.

Many adverse events have been reported due to the use of Diphenylcyclopropenone/DPCP, including eczematous reactions, urticaria, vitiligo, lymphadenopathy, hyperpigmentation, or erythema multiforme-like reactions. In the current study, during the sensitization process, almost all of the patients experienced minimal erythema, itching, and burning sensation. Overall, the most commonly encountered side effect was erythema and itching, followed by the formation of papules, vesicles, bullae, and flu-like symptoms. Other less frequent side effects were lymph node enlargement, fever, general malaise, irreversible hyperpigmentation of the head and neck area, and vitiligo macules.
The use of DPCP in children is still a controversial area, although some studies have shown good results with acceptable side effect profiles. In the current study, children above 5 years of age were also included and the treatment results or safety parameters were similar to those of the adult patients.[2]

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A total of 33 severe AA patients and 20 healthy volunteers (normal controls) were enrolled. There were no statistical differences between AA patients and normal controls for age and gender. All the patients were treated with Diphenylcyclopropenone/DPCP, 21 (63.6%) had satisfactory hair regrowth (responders), and 12 (36.4%) were not responsive to the treatment (non-responders). Information of patients is presented (Table 1). No significant differences in gender, age, onset age, disease duration and SALT scores were found between responders and non-responders (Table 1). No significant associations were found between responses to the treatment and the final concentration of DPCP/Diphenylcyclopropenone applied, treatment reaction, side effects, nail involvement and other clinical features (data not shown). A total of 30 patients (90.9%) experienced one or more side effects during the treatment. Dermatitis in the Diphenylcyclopropenone/DPCP contact area was the most common finding (27/30, 90%). Lymphadenopathy in the draining area was another common side effect (14/30, 46.7%). Other side effects included scalp hyperpigmentation (12/30, 40.0%), systemic contact dermatitis (5/30, 16.7%), scalp hypopigmentation (2/30, 6.7%) and hyperpyrexia (1/30, 3.3%). Side effects resolved after application of topical corticosteroids, use of systemic antihistamines or treatment suspension for one week, but they did not lead to discontinuation of therapy.[1]

[1]. Diphenylcyclopropenone plays an effective therapeutic role by up-regulating the TSLP/OX40L/IL-13 pathway in severe alopecia areata. Exp Dermatol. 2021 Feb;30(2):278-283.
[2]. ssessment of treatment efficacy of diphenylcyclopropenone (DPCP) for alopecia areata. Turk J Med Sci. 2020 Dec 17;50(8):1817-1824.
[3]. Changes of cell-surface thiols and intracellular signaling in human monocytic cell line THP-1 treated with diphenylcyclopropenone. J Toxicol Sci. 2010 Dec;35(6):871-9.
[4]. The contact sensitizer diphenylcyclopropenone has adjuvant properties in mice and potential application in epicutaneous immunotherapy. Allergy. 2012 May;67(5):638-46.

Diphenylcyclopropenone is a cyclopropenone compound having phenyl substituents at the 2- and 3-positions. It has a role as a photosensitizing agent, a hapten and a drug allergen.
Diphencyprone has been used in trials studying the treatment and basic science of Melanoma, Ultraviolet Rays, Immunosuppression, Neoplasm Metastasis, and Hypersensitivity, Delayed, among others.
Diphencyprone is a synthetic, potent allergic contact sensitizer with potential immunostimulatory activity. After sensitization process by repeated topical application of diphencyprone to a specific area, further application of this agent to the affected area may stimulate an immune response and may potentially be useful to clear the affected area from infection or cancer.

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To our knowledge, this is the first study investigating the expression of the TSLP/OX40L/IL13 pathway in severe AA patients and immunotherapy with DPCP. Results showed that expression levels of Th2 cytokines IL-5 and TSLP were significantly lower and Th1 cytokine IFN-γ was higher in serum of severe AA patients than normal. It was similar in the scalp skin. TSLP, OX40L, IL-4 and IL-13 were found to be much lower, and IFN-γ was higher than the normal control, which is consistent with the previous report detecting mRNA levels of Th1-type cytokines in scalp skin. Results of both serum and scalp skin suggest that the TSLP/OX40L/IL13 pathway is down-regulated, Th2 responses are inhibited and Th1 immune is dominant in severe AA patients.
Down-regulation of the TSLP/OX40L/IL13 pathway reflects a lower Th2 activity in severe AA patients. Therefore, as an effective treatment for severe AA, DPCP might play its role in the process by up-regulating the TSLP/OX40L/IL13 pathway, promoting Th2 immune activity and then restoring equilibrium between Th1 and Th2 responses. We compared expression levels of related cytokines before and after the DPCP treatment. It was shown that TSLP, IL-13 and another Th2 cytokine IL-5 significantly increased whereas Th1 cytokine IFN-γ decreased to a normal level in severe AA patients after being treated with DPCP. The result proves that DPCP plays a therapeutic role in severe AA by up-regulating the TSLP/OX40L/IL13 pathway and reconstructing Th1/Th2 immune balance.
Topical immunotherapy with DPCP is considered to be an effective and safe therapeutic method for AA, but not all patients respond to it. In this study, 12 out of 33 (36.36%) AA patients did not respond to the therapy. Was there any difference between responders and non-responders which leads to the different responses to the DPCP therapy? Comparing the expression levels of Th2- and Th1-related cytokines between the responder and non-responder groups, we found that before the DPCP treatment, the expression levels of TSLP, OX40L and IL-13 in responders were significantly lower than non-responders, while non-responders had similar expression levels of these cytokines with normal controls. After being treated with DPCP, TSLP and IL-13 in serum greatly increased in responders while there were no changes of these cytokines in non-responders. There were no significant differences of Th1 cytokine IFN-γ and other Th2 cytokines IL-5 and IL-4 between the responder and non-responder groups, no matter before or after the DPCP treatment. These results indicate that the difference between the responders and the non-responders is in the TSLP/OX40L/IL-13 pathway. Patients with down-regulation of the TSLP/OX40L/IL-13 pathway were responsive to the DPCP treatment while those who without down-regulation of the pathway were not responsive. The results further demonstrate that DPCP plays its therapeutic role by up-regulating TSLP/OX40L/IL-13 pathway in some severe AA patients.
In conclusion, this study shows that the TSLP/OX40L/IL-13 pathway is down-regulated and Th2 immune is inhibited in some severe AA patients. Topical treatment with DPCP is only effective in patients with down-regulation of the TSLP/OX40L/IL-13 pathway. The TSLP/OX40L/IL-13 pathway could be a predictor of response to DPCP therapy for severe AA patients.[1]

C15H10O

206.244

206.073

C, 87.36; H, 4.89; O, 7.76

886-38-4

886-38-4;

65057

Typically exists as Off-white to light yellow solids at room temperature

1.2±0.1 g/cm3

407.2±45.0 °C at 760 mmHg

118-122 °C(lit.)

182.7±23.7 °C

0.0±0.9 mmHg at 25°C

1.669

3.78

0

1

2

16

284

0

O=C1C(C2C=CC=CC=2)=C1C1C=CC=CC=1

HCIBTBXNLVOFER-UHFFFAOYSA-N

InChI=1S/C15H10O/c16-15-13(11-7-3-1-4-8-11)14(15)12-9-5-2-6-10-12/h1-10H SMILES

2,3-Diphenylcycloprop-2-en-1-one

Diphencyprone; DPCP; Diphenylcyclopropenone; Diphencyprone; 2,3-Diphenylcycloprop-2-en-1-one; 2,3-Diphenylcycloprop-2-enone; 2,3-Diphenylcyclopropenone; 1,2-Diphenylcyclopropen-3-one; Cyclopropenone, diphenyl-;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~484.87 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (12.12 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (12.12 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (12.12 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)