WO2014182655A1 - Assay for screening a compound for the ability to modulate proinflammatory cytokine production in human skin - Google Patents
Assay for screening a compound for the ability to modulate proinflammatory cytokine production in human skin Download PDFInfo
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- G01N2800/205—Scaling palpular diseases, e.g. psoriasis, pytiriasis
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Abstract
The present invention relates to an assay for assessing the production of proinflammatory cytokines in human skin, and for assessing the effect of test compounds on such production. The invention provides a method of screening a compound for the ability to modulate pro-inflammatory cytokine production in human skin, using a liquid-air interface culture of human skin, exposing the skin to a test compound, exposing the skin to a Th17 activating condition, measuring the production of proinflammatory cytokines, and comparing the production of pro-inflammatory cytokines to that produced in the absence of the test compound.
Description
ASSAY FOR SCREENING A COMPOUND FOR THE ABILITY TO MODULATE
PROINFLAMMATORY CYTOKINE PRODUCTION IN HUMAN SKIN
Field of the Invention
The present invention relates to an assay for assessing the production of proinflammatory cytokines in human skin, and for assessing the effect of test compounds such production.
Background of the Invention
Psoriasis is an autoimmune skin disease. It is chronic and recurrent, and characterized by marked inflammatory changes in epidermis and dermis, as well as hyperproliferation of keratinocytes. It is estimated that approximately 2-3% of the human population is affected by psoriasis. Various methods of treating psoriasis are known, including topical, phototherapeutic, and systemic medications.
Several types of psoriasis are recognized, including plaque psoriasis, nail psoriasis, scalp psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, and psoriatic arthritis. Diagnosis of psoriasis may be accomplished by physical examination of the individual, or examination of a skin biopsy. The severity of psoriasis varies among individuals, and the course of the disease may fluctuate. No cure is presently recognized for psoriasis.
Rodent models of autoimmune skin diseases do not completely recapitulate human pathology. There remains a need to develop assays capable of screening compounds and agents for their effects on autoimmune skin diseases in human skin.
Brief Description of the Drawings
Figure 1 is a schematic of one aspect of the tissue-based assay described herein.
Skin resident T cells are activated by exposure to Thl7 polarizing conditions, leading to production of IL-17 and IL-22. RORyt = retinoic acid-receptor-related orphan receptor gamma T; Stat3 = signal transducer and activator of transcription 3; TCR = T Cell Receptor.
Figure 2A depicts the transwell, liquid-air interface design of the present ex vivo skin assay.
Figure 2B is a timeline for an ex vivo experiment, where D-l is the day the skin tissue is received.
Figure 3A graphs the production of IL17a mRNA transcripts by skin-resident T cells following activation (under three different conditions) in the present ex vivo skin assay. Expression values are relative to that on Day 0 (arbitrarily deemed to be a value of "1"). The uppermost graphed line indicates expression using Thl7 activating medium; middle graphed line = TCR only medium; lowest graphed line = medium containing recombinant IL17a and IL22. Each data point represents the average expression value of duplicate cultures from a single skin donor.
Figure 3B graphs the production of IL22 mRNA transcripts by skin-resident T cells following activation (under three different conditions) in the present ex vivo skin assay. Expression values are relative to that on Day 0 (arbitrarily deemed to be a value of "1"). The uppermost graphed line indicates expression using Thl7 activating medium; middle graphed line = TCR only medium; lowest graphed line = IL17a and IL22 medium. Each data point represents the average expression value of duplicate cultures from a single skin donor.
Figure 4 provides photographs of skin samples cultured on different media in the present ex vivo skin assay, at different time points. Ballooning degeneration is shown at Day Six in Thl7 and IL17/IL22 media (black arrows); but not in TCR or LPS media. Med = base medium without any activators added; LPS = medium with Lipopolysaccharide (a control for general immune activation).
Figure 5 is a timeline schematic for an experiment to test compounds for the ability to inhibit expression of pro-inflammatory cytokines. Test compounds may be added to the liquid medium or applied topically in an appropriate vehicle.
Figure 6A graphs IL17a mRNA levels at 48 hours for human skin samples using the present ex vivo assay system and four different media. Medium = base medium without any activators or test compound. The other three culture conditions included pre- treatment for three days with a test compound before TH17 activation on Day 0 using Thl7 medium. DMSO = medium containing DMSO as a vehicle control; Dig(Sal) = medium containing Digoxin-21-Salicylidene as test compound; Compound A = medium containing a small molecule compound as test compound.
Figure 6B graphs IL-17F mRNA levels at 48 hours for human skin using the present ex vivo assay system. Conditions were as described for Figure 6A.
Figure 7A graphs IL17a mRNA levels at 48 hours for human skin samples using the present ex vivo assay system and four different media. Four different conditions were tested. Medium = base medium without any activators or test compound. The other three conditions included TH17 activation and topical pre-treatment of the skin with a control or a test compound: Veh = medium containing vehicle as a control for test compounds; Compound B = small molecule test compound; Compound A = small molecule test compound.
Figure 7B graphs IL17F mRNA levels at 48 hours for human skin samples using the present ex vivo assay system. Four conditions were tested, as described for Figure 7A.
Summary of the Invention
In a first aspect, the invention provides a method of screening a compound for the ability to modulate pro-inflammatory cytokine production in human skin, using a liquid-air interface culture of human skin, exposing the skin to a test compound, exposing the skin to a Thl7 activating condition, measuring the production of proinflammatory cytokines, and comparing the production of pro-inflammatory cytokines to that produced in the absence of the test compound.
A further aspect of the invention is a method of screening a compound for use as a treatment for psoriasis, by providing a liquid-air interface culture of human skin, exposing the skin to a test compound, exposing the skin to a Thl7 activating condition, measuring the production of pro-inflammatory cytokines, and comparing the production of pro-inflammatory cytokines to that produced in the absence of test compound, where a reduction in the production of pro-inflammatory cytokines indicates the compound is useful as a treatment for psoriasis.
Detailed Description of the Invention
Molecular Pathways involved in psoriasis
In recent years, research has progressed into identifying the molecular pathways involved in the pathogenesis of psoriasis. However, it is still unknown whether the main cause of psoriasis is keratinocyte impairment or an immunologic defect.
Additionally, the precise roles of systemic and cutaneous factors in the pathogenesis of the disease remain to be defined.
Psoriasis has been described as mediated by T cells producing interferon gamma (IFNy) and Tumor Necrosis Factor alpha (TNFa). Psoriatic skin lesions have been reported to have increased gene and protein expression of IL-23, IL-21, IL-22 and IL-17 (Boniface et al., Clin. Exp. Immunol. 150:407 (2007)). Elevated IL-17 and IL-22 production has been reported in psoriatic skin.
Recent studies have shown that, in psoriatic lesions, the inflammatory cell infiltrate consists not only of T cells but also of effector cells of innate immunity, including neutrophils, macrophages, and plasmocytoid DCs and CDllc+ DCs, which produce various inflammatory mediators such as cytokines and adhesion molecules.
Investigations into the role of T cell subsets in psoriasis indicate that T helper 17 (Thl7) cells play a role in immunological activation in psoriasis. Thl7 cells are a subset of CD4 T helper cells characterized by production of interleukin-17, particularly IL-17A and IL-17F, and interleukin 22 (IL-22).
Furthermore, it has been demonstrated that innate dermal gamma delta T cells
(γδΤ cells) are a source of IL-17 and produce significant amounts of IL-22 after stimulation with IL-23. Cai et al., Immunity, 2011, 35:596-610.
IL-17A and IL-22 both enhance the innate immune response of tissue fibroblast and epithelial cells; however, their functions do not overlap completely. IL-22 induces proliferation of keratinocytes and augments healing responses, whereas IL-17A induces much stronger proinflammatory effects and more recruitment of neutrophils.
Therapies to ameliorate psoriasis have been developed by targeting key mediators of inflammation. Among them, the development of systemic treatments directed against cytokines; such as, anti-TNFa and anti-cytokine drugs targeting
interleukin-12 and interleukin-23, and downstream products of helper T cells including I L-17A and IL-22. Human monoclonal antibodies against the subunit of p40 of I L-12 and I L-23 have been proposed for the treatment of psoriasis. I L-23/IL-17 pathway
It has been shown that the I L-23/IL-17 pathway plays a critical role in the pathogenesis of human psoriasis, as improvement in clinical scores has been associated with reduction of Thl7 responses.
Recently, the involvement of IL-17A in the pathophysiology of human psoriasis was validated by the systemic neutralization of I L-17, which resulted in a significant improvement in clinical measures, as well as reversal of epidermal hyperplasia, keratinocyte proliferation, and dermal infiltration of leukocytes. The study suggests that inhibition of I L-17 pathway genes, which are highly expressed in keratinocytes, are related to epidermal improvement. Krueger et al., J Allergy Clin Immunol 130:145-54 (2012).
The activation of skin resident T cells and subsequent production of proinflammatory cytokines IL-17 and I L-22 are pivotal steps in the development and progression of autoimmune skin diseases, including psoriasis. Rodent models of autoimmune skin diseases do not completely recapitulate human pathology, nor can animal models replace target validation studies in human tissues.
One aspect of the present invention is the development of a tissue-based assay suitable for use in evaluating therapeutics, including topically-delivered therapeutics, for the ability to modulate expression of pro-inflammatory cytokines. The present tissue- based assay is suitable for use with human skin.
Naive I cells, upon activation, differentiate into cytokine-expressing effector T he!per ceils such as Thl, Th2, and Thl7. Differentiation of T cells into Thl7 ce!is has been reported as driven by IGF- beta and IL6 (Bettelli et aL, Mature 441:235-238 (2006); Veidhoen et aL, immunity 24 :179-89 (2006).
I n the model described herein, skin-resident T cells are activated under Thl7- polarizing conditions to mimic T cell-mediated immune responses within human skin.
Resident T cells are activated in situ in freshly excised healthy human skin, resulting in Thl7-associated cytokine production, including production of IL-17 and IL-22.
IL-17 production is highly dependent on the transcription factor RORg. The present assay was validated using a specific small molecule inhibitor of RORg.
Pretreatment of ex vivo human skin cultures with RORg inhibitor decreased activation- induced H17a and H17f transcript expression.
Present methods
In view of the above, topical and systemic drugs that inhibit the production of IL- 17 in the skin represent possible therapies against psoriasis.
One aspect of the present invention is a tissue-based assay using ex vivo human skin to mimic in vivo T cell-mediated inflammatory processes and cytokine production. This tissue-based assay is used to evaluate test compounds and biological agents as potential therapeutics for the treatment of inflammatory skin diseases, such as psoriasis, based on suppression of induced pro-inflammatory cytokine production. In particular, such diseases are Thl7 mediated diseases.
A further aspect of the present invention is a method of screening compounds and biological agents to identify those able to suppress the production of proinflammatory cytokines. The method comprises providing a liquid-air interface culture of human skin; exposing said skin to both a test compound and Thl7-polarizing conditions, and assessing the effect of said test compound on the transcription or production of proinflammatory cytokines in the skin tissue. In one embodiment the test compound is administered to the cultured skin tissue prior to exposing the skin tissue to the Thl7- polarizing conditions. In one embodiment the cytokine assessed is IL17a and/or IL17f, and the production of mRNA transcripts is measured. In one embodiment, the effect of the test compound on cytokine production or transcription is compared to cytokine production in a control assay lacking test compound. In one embodiment the test compound is delivered topically.
Test compounds identified as able to suppress Th-17 induced pro-inflammatory cytokine production are identified as potential anti-psoriasis medicaments.
Suitable small molecule test compounds include but are not limited to inhibitors of RORy and RORyt (a specific isoform of RORg).
The invention further relates to a method for preventing, treating, or ameliorating a medical condition caused by increased production of pro-inflammatory cytokines, including Thl7- induced IL17 production, comprising administering to a mammalian subject a therapeutically effective amount of a compound identified by the present assay as an inhibitor of pro-inflammatory cytokine production, including a compound identified as an inhibitor of Thl7-induced IL17 production. Administration of such a compound may be by topical administration.
In one embodiment, the compound is an inhibitor of RORyt and the medical condition is psoriasis.
Terms
As used herein the terms "modulate" or "modulates" refer to an increase or decrease in the amount, quality or effect of a particular activity.
As used herein, 'biological agents' means complex biological molecules such as antibodies, monoclonal antibodies, proteins, polypeptides and nucleotides.
As used herein, the term 'psoriasis' includes plaque psoriasis, nail psoriasis, scalp psoriasis, guttate psoriasis, inverse psoriasis, pustular psoriasis, erythrodermic psoriasis, and psoriatic arthritis.
As used herein, a 'treatment' for, or a 'method of treating', a medical condition, such as a psoriatic condition, refers to a method of reducing, ameliorating or delaying the signs, symptoms, or progression of that medical condition. As used herein, 'treatment' does not imply a cure. A treatment need not be effective in every member of a population, e.g., a population of patients with psoriasis, to have clinical utility, as is recognized in the medical and pharmaceutical arts.
As used herein, Thl7 activating conditions refer to tissue culture conditions which result in the differentiation of naive T cells resident in the tissue into effector Th 17 helper cells. As used herein, 'Thl7 activation' is used interchangeably with the term 'Thl7 stimulation'.
As used herein, 'subjects' and/or 'patients' includes human subjects and patients.
Various routes of administering a therapeutic compound to a subject are known in the art, including but not limited to topical, intradermal, intramuscular,
intraperitoneal, intravenous, subcutaneous, intranasal, and oral routes. Administration can be systemic or local.
As used herein, 'topical' administration of a compound or medicament refers to application to and absorption through epithelial or mucocutaneous linings. In one aspect, topical application consists of or comprises application to the cutis or external integument of a subject, such as application to the epidermis of skin, including application to psoriatic lesions. Appropriate vehicles and pharmaceutical carriers for use in topical application are known in the art.
The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a compound or agent, and a pharmaceutically acceptable carrier. In a specific embodiment, the term
"pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
An 'effective amount' of the compound or agent for treatment of a disease or condition can be determined by standard clinical techniques.
Example 1: Materials and Methods
Skin culture: Healthy human skin obtained from elective abdominoplasty surgery underwent minimal processing (removal of sub-dermal fat and sectioning to fit 10 mm transwells), and was then cultured in transwells at the interface of air and a liquid culture medium (liquid-air interface culture). See Figure 2A showing the transwell configuration.
Skin was cultured in the upper chamber of a 0.4 urn PCF membrane transwell (Millicell #PIHP01250). The medium used on day -1 was Cornification medium + hydrocortisone (final concentration of 0.4 ug/ml). From day 0 and onward, medium used was Cornification medium without hydrocortisone. Cornification medium is: equal parts F12 and Dulbecco's Modified Eagle's Medium (DMEM) containing 4 mM L-glutamine, 5
ug/ml Insulin, 5 ug/ml Transferrin, 0.02 nM Triiodothyromine, 100 uM Ethanolamine, 100 uM Phosphorylethanolamine, 180 uM Adenine, 52.6 nM Selenium, 1.88 uM CaCI2, 2% FBS.
Activation of skin cell cultures: After at least one day of resting (undisturbed culturing on the starting mediumj, skin-resident T cells in the human skin cultures were activated under various conditions.
Thl7-polarizing conditions ("Thl7 conditions") were achieved using cornification media (without hydrocortisone) containing: Cluster of Differentiation 3 (CD3) 1 ug/ml (BD Pharmingen); Cluster of Differentiation 28 (CD28) 2 ug/ml (R&D Systems); anti-l nterferon gamma (IFNg) mAb 1 ug/ml (R&D Systems); anti-interleukin 4 (IL4) mAb 1 ug/ml (R&D Systems); interleukin-6 (I L6) 10 ng/ml (R&D Systems); interleukin-lbeta (ILlb) 10 ng/ml (R&D Systems); Transforming Growth Factor beta (TGFb) 1 ng/ml (R&D Systems); and interleukin 21 (I L21) 10 ng/ml (R&D Systems).
See Figure 2B for a time-line schematic. T cell stimulation was maintained by a daily refresh of medium (where refresh refers to complete replacement of the medium by aspiration of the medium and delivery of 1 ml of the replacement medium.
TCR medium or 'TCR only conditions' refers to cornification medium containing CD3 1 ug/ml (BD Pharmingen) and CD28 2 ug/ml (R&D Systems).
Recombinant human IL-17 and I L-22 medium, or 'recombinant IL17/I L22 conditions' refers to cornification medium containing 10 ng/ml of I L-17a and lOng/m l of I L-22 human recombinant proteins (from R&D Systems).
On Day 0 the starting liquid medium was aspirated from the lower chamber and replaced with 1 ml of the relevant experimental medium. Media were 'refreshed' by aspiration and replacement with fresh medium.
Measurement of cytokine expression: Following culture as described above, levels of pro-inflammatory cytokine transcript expression were measured. Skin samples were harvested from duplicate wells (2 wells per condition) each day over the time course of the experiment. Each skin sample was cut in half; one piece was used for histology and the other for qPCR.
This scheme is depicted in Figure 2B, where D-l (D negative one) represents the day the skin tissue is received for culture; Thl7-polarizing conditions are established on Day 0; and samples are harvested for qPCR at DO (for baseline measurements), Dl, D2, D3, D4, D5 and D6.
Cytokine transcript expression was measured using quantitative Reverse-
Transcriptase Polymerase Chain Reaction (qRT-PCR), as is known in the art. Extracted RNA was analyzed for message expression using TAQ.MAN(tm) probes
Histology: Skin samples harvested for histology were stained using
Hematoxylin/Eosin (H&E) staining as is known in the art.
Example 2: Production of IL17a and IL22
Activation of skin-resident T cells under Thl7-polarizing conditions led to rapid induction of H17a and /Viz/transcript expression.
Figures 3 and 4 provide results obtained using skin cultures as described in the Materials and Methods, above. In Figure 3A and 3B, each point represents measurement of duplicate samples (N=2) from the same donor; no error (SEM) is given.
In samples harvested from the transwell cultures, intradermal transcript expression was measured by qPCR. Messenger RNA transcripts for pro-inflammatory cytokines IL17a and IL-22 were found to be produced at significant levels within 24 hours of T cell activation; see Figures 3A and 3B, where values given are relative to expression at day 0 (before activation, arbitrarily set to an value of 1).
Figure 3A shows the relative expression of IL17a (and Figure 3B shows relative expression of IL22) at Day 0, Dl, D2, D3, D4, and D6 (no Day 5 measurement was taken in order to reduce the experiment size) produced by skin-resident T cells in human skin samples activated as described above under: Thl7 conditions (uppermost graphed line), TCR only (middle graphed line), or recombinant IL-17a/IL22 (lower graphed line).
Interleukin expression was normalized to beta-actin as is known in the art.
Figure 4 demonstrates that, after several days of culture, the induced proinflammatory cytokine expression led to ballooning degeneration of the skin. Tissue integrity was assessed by Hematoxylin/Eosin staining at each of three time-points (Day 1,
Day 3 and Day 6), in each of five culture conditions. In Figure 4, "Med" refers to base
medium (cornification medium without additions, refreshed similar to the other media). At Day 6, ballooning degeneration was observed when pro-inflammatory cytokines were present (Day 6; black arrows). LPS medium (lipopolysaccharide 30 ng/ml in medium) was used as a control for general immune activation.
Induced expression of IL-17 and IL-22 in the present assay system led to tissue damage of the cultured human skin, similar to what occurs in the psoriasis disease state (Figure 4). Nonetheless, tissue integrity (structural intactness) was maintained at earlier time-points, indicating that topically-applied test compounds can be screened using the present system.
Example 3: Modulation of 1117 by test compounds
The ability of a test compounds to modulate the expression of pro-inflammatory cytokines was assessed using the ex vivo skin culture method as described above. Test compounds were: Dimethyl Sulfoxide (DMSO; vehicle control), Digoxin-21-Salicylidene in DMSO Dig/sal; positive control) or Compound A in DMSO vehicle; where final
concentration is 10 uM in cornification medium, with a final DMSO concentration of 0.2%.
Four different conditions were tested. Medium = base medium (cornification medium without additions, refreshed similar to the other media). The other three culture conditions included pre-treatment for three days with one of the test
compounds, before a final day (Day 0) of treatment with combined test compound and
TH17 activation (using Thl7 medium). See schematic at Figure 5.
The digoxin derivative, Digoxin-21-Salicylidene, was used as a positive control tool compound because it was previously described as a specific small molecule inhibitor of RORgt (Huh et al., Nature, 472:486-90, 2011).
Because IL-17 and IL22 cytokine expression was shown to be strongly induced within days of Thl7 stimulation (see Examples above), in the present example, medium supplemented with test compound (or vehicle control) was added each of days D-3, D-2 and D-l (prior to Thl7 activation).
On Day 0, Thl7-polarizing medium was added (also including test compound), and then skin cultures were left untouched (no further refreshment of medium) for 48 hours
(until Day 2). At Day 2, samples were harvested for qPCR and histology. See
experimental design in Figure 5.
The tissue integrity of the samples taken at Day 2 was confirmed by H&E staining (data not shown) and the level of H17a and /Viz/transcript expression was analyzed by qPCR. Results are shown in Figures 6 and 7.
Figure 6A graphs IL17a mRNA levels at Day 2, relative to expression in the medium-only sample. Figure 6B graphs IL-17F mRNA levels at Day 2, relative to expression in the medium-only sample. Use of Dig(sal) was shown to suppress levels of both IL17a mRNA and IL17f mRNA relative to the vehicle (DMSO) control. Use of
Compound A was also shown to suppress levels of both IL17a mRNA and IL17f mRNA relative to the vehicle (DMSO) control. Compound A was also shown to be approximately as potent as the Dig/Sal inhibitor in suppressing IL-17a and IL17f production relative to vehicle control.
In Figures 6A and 6B, each data point is the average of triplicate wells +/- SEM from the same skin donor. The data are representative of four independent experiments run on four different skin donors and yielding similar results.
Example 4: Topical application of test compounds
The present ex vivo assay system was validated for use with topical application of test compounds. See schematic at Figure 5. Test Compounds A (1%) and B (1%) were each dissolved in a simple ethanolic formulation of 60% EtOH: 40% H20 and applied 0.13 mg per cm2 to the epidermis (air-interface) of the ex vivo human skin cultures for four days (D-3, D-2, D-l and DO). A vehicle control (ethanolic formulation without test compound) was also used. Additionally, a medium-only (no test compound, no Thl7 activation) was also conducted. On Day 0, skin was also stimulated with Thl7-polarizing conditions and forty-eight hours later (D2), skin was harvested and the level of Thl7- associated cytokine transcripts assessed by qPCR (Figure 7) (relative to expression in medium-only sample).
Figure 7A graphs the relative expression of IL17a mRNA, and Figure 7B graphs the relative expression of IL17f mRNA, measured at D2. It is shown that topical application of test Compound A inhibits H17a and Ill7f transcript expression from skin-resident T cells.
Data shown is the average +/- SEM of triplicate wells from a single skin donor. These data are representative of three independent experiments run on three different skin donors yielding similar results.
Thus the above experiments show that T cells can be activated in situ in healthy skin cultures, and that pre-treatment with small molecule test compounds result in significant cytokine inhibition when applied either in the medium or topically.
Claims
1. A method of screening a compound for the ability to modulate proinflammatory cytokine production in human skin, comprising:
(a) providing a liquid-air interface culture of human skin;
(b) exposing said skin to a test compound;
(c) exposing said skin to Thl7 activating condition;
(d) measuring the production of pro-inflammatory cytokines; and
(e) comparing the production of pro-inflammatory cytokines to that produced in the absence of said test compound.
2. A method according to claim 1, wherein said pro-inflammatory cytokine is measured by assessing levels of mRNA transcripts.
3. A method according to claim 1, wherein said pro-inflammatory cytokine is selected from IL-17a, IL-17f and IL-22.
4. A method according to claim 1, wherein said test compound is added to the liquid medium of the human skin culture.
5. A method according to claim 1, wherein said test compound is applied topically to the human skin.
6. A method according to claim 1 wherein said Thl7 activating condition comprises addition of Cluster of Differentiation 3 (CD3), Cluster of Differentiation 28 (CD28), interleukin-lbeta (ILlb), interleukin-6 (IL6), Transforming Growth Factor beta (TGFb), interleukin 21 (IL21), anti-interleukin 4 (IL4) monoclonal antibody (mAb), and anti-lnterferon gamma (IFNg) mAb to the liquid medium.
7. A method of screening a compound for use as a treatment for psoriasis, comprising:
(a) providing a liquid-air interface culture of human skin;
(b) exposing said skin to a test compound;
(c) exposing said skin to Thl7 activating condition;
(d) measuring the production of pro-inflammatory cytokines; and
(e) comparing the production of pro-inflammatory cytokines to that produced in the absence of said test compound,
wherein a reduction in the production of pro-inflammatory cytokines indicates the compound is useful as a treatment for psoriasis.
8. A method according to claim 2, wherein said pro-inflammatory cytokine is measured by assessing levels of mRNA transcripts.
9. A method according to claim 2, wherein said pro-inflammatory cytokine is selected from IL-17, IL-17a, IL-17f and IL-22.
10. A method according to claim 2, wherein said test compound is added to the liquid medium of the human skin culture.
11. A method according to claim 2, wherein said test compound is applied topically to the human skin.
12. A method according to claim 2 wherein said Thl7 activating condition comprises addition of Cluster of Differentiation 3 (CD3), Cluster of Differentiation 28 (CD28), interleukin-lbeta (ILlb), interleukin-6 (IL6), Transforming Growth Factor beta (TGFb), interleukin 21 (IL21), anti-interleukin 4 (IL4) monoclonal antibody (mAb), and anti-lnterferon gamma (IFNg) mAb to the liquid medium.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR3071508A1 (en) * | 2017-09-26 | 2019-03-29 | Genoskin | EX VIVO MODEL OF INFLAMMED HUMAN SKIN AND USES THEREOF FOR SCREENING ANTI-INFLAMMATORY COMPOUNDS |
| WO2020186010A1 (en) | 2019-03-12 | 2020-09-17 | Epm Group, Inc. | Cannabinoid acid ester compositions and uses thereof |
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| US20130035333A1 (en) * | 2010-02-23 | 2013-02-07 | The Regents Of The University Of California | Use of cinnabarinic acid as a modulator of immune responses in autoimmune disorders |
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2014
- 2014-05-06 WO PCT/US2014/036900 patent/WO2014182655A1/en active Application Filing
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR3071508A1 (en) * | 2017-09-26 | 2019-03-29 | Genoskin | EX VIVO MODEL OF INFLAMMED HUMAN SKIN AND USES THEREOF FOR SCREENING ANTI-INFLAMMATORY COMPOUNDS |
| WO2019063122A1 (en) | 2017-09-26 | 2019-04-04 | Genoskin | Ex-vivo model of inflamed human skin and uses thereof for screening anti-inflammatory compounds |
| JP2020535402A (en) * | 2017-09-26 | 2020-12-03 | ゲノスキン | Ex vivo model of inflamed human skin and its use for screening of anti-inflammatory compounds |
| JP7199427B2 (en) | 2017-09-26 | 2023-01-05 | ゲノスキン | An ex vivo model of inflamed human skin and its use for screening anti-inflammatory compounds |
| WO2020186010A1 (en) | 2019-03-12 | 2020-09-17 | Epm Group, Inc. | Cannabinoid acid ester compositions and uses thereof |
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