Reconstituted nipple skin model

Abstract

A skin model comprises keratinocytes capable of overexpressing filaggrin. A method for evaluating the in vitro activity of a formulation or of at least one active agent on the healing of nipple skin and/or on the reduction of nipple skin inflammation, by bringing said active agent or said formulation into contact with a skin model comprising keratinocytes overexpressing filaggrin and also comprising a lesion, and measuring the level of production of at least one biological marker chosen from lactate dehydrogenase (LDH), TNF, filaggrin, TGF1 and 1 integrin.

Claims

1. An in vitro reconstructed nipple skin model comprising keratinocytes of human origin overexpressing filaggrin and a lesion, wherein said keratinocytes constitutively express filaggrin at a level greater than the filaggrin expression level of healthy human primary keratinocytes.

2. The model of claim 1, wherein said model comprises a support matrix.

3. The model of claim 2, wherein said support matrix is inoculated with fibroblasts.

4. A method, comprising: a) contacting an active ingredient or a formulation with the nipple skin model of claim 1; b) measuring the production level of a biological marker in the nipple skin model of step a), wherein said biological marker is selected from the group consisting of: epidermal integrity markers; epidermal barrier markers; epidermal inflammation markers; and healing markers c) measuring a reference production level of said biological marker in a skin model according to claim 1 that has not been contacted with said active ingredient or said formulation, or that has been contacted with a reference active ingredient or formulation, d) preparing a dermatological formulation comprising the candidate active ingredient or candidate formulation if: (i) the production level of said biological marker measured in step b) is less than or equal to the reference production level of said biological marker measured in step c) when said biological marker is an epidermal integrity marker or an epidermal inflammation marker, or (ii) the production level of said biological marker measured in step b) is greater than or equal to the reference production level of said biological marker measured in step c) when said biological marker is an epidermal barrier marker or a healing marker.

5. The method of claim 4, further comprising administering an effective amount of the dermatological formulation to a human patient presenting a nipple skin lesion or nipple skin inflammation.

6. The method of claim 4, wherein step b) comprises measuring the production level of two biological markers.

7. The model of claim 1, wherein the lesion is a linear ulceration.

8. The method of claim 4, wherein said epidermal integrity marker is lactate dehydrogenase (LDH).

9. The method of claim 4, wherein said epidermal barrier marker is filaggrin.

10. The method of claim 4, wherein said epidermal inflammation marker is selected from cytokines.

11. The method of claim 4, wherein said epidermal inflammation marker is TNF.

12. The method of claim 4, wherein said healing marker is selected from integrins.

13. The method of claim 4, wherein said healing marker is integrin 1 or TGF 1.

14. The method of claim 5, wherein said nipple skin lesion is a linear ulceration.

15. The method of claim 4, wherein the production level of said biological marker is measured using an enzymatic test when said biological marker is the product of a gene with an enzymatic activity.

16. The method of claim 15, wherein said marker is lactate dehydrogenase (LDH).

17. The method of claim 4, wherein the production level of said biological marker is measured using analysis of the transcriptome, RT-PCR, quantitative RT-PCR, or nucleic acid chips when said biological marker is a gene.

18. The method of claim 17, wherein said biological marker is lactate dehydrogenase (LDH), TNF, TGF 1, or filaggrin.

19. The method of claim 4, wherein the production level of said biological marker is measured using immunoassays when said biological marker is a polypeptide or the product of a gene.

20. The method of claim 19, wherein said biological marker is integrin 1.

21. The model of claim 1, wherein said lesion is a lesion mimicking a crack.

Description

LEGEND OF THE FIGURES

(1) FIG. 1: Histology of a reconstructed epidermis (on day 17) obtained from the batch of keratinocytes chosen for implementing the nipple model.

(2) FIG. 2: Gene expression of filaggrin in the reconstructed skin after 24 hours of incubation

(3) FIG. 3: Release of LDH into the culture medium 4 hours and 24 hours after injuring the reconstructed nipple skin

(4) FIG. 4: Gene expression of TNF in the reconstructed nipple skin 4 hours after injury

(5) FIG. 5: Gene expression of filaggrin in the reconstructed nipple skin 24 hours after injury

(6) FIG. 6: Analysis of the production of integrin 1 by western blot in the reconstructed nipple skin 24 hours after injury

(7) FIG. 7: Release of LDH into the culture medium 4 hours after injuring the reconstructed nipple epidermis

(8) FIG. 8: Gene expression of TNF in the reconstructed nipple epidermis 24 hours after injury

(9) FIG. 9: Gene expression of TGF 1 into the reconstructed nipple epidermis 24 hours after injury

(10) FIG. 10: Labeling of integrin 1 in reconstructed nipple epidermis 4 hours after injury (blue=nucleus, dapi; red=integrin)

(11) FIG. 11: Scanning electron microscopy (SEM) imaging of the reconstructed nipple epidermal surface 4 hours after injury (magnification1000)

EXAMPLES

Example 1: Development of a Nipple Tissue Model for Evaluating a Breastfeeding Balm

(12) Objectives: Development of a model mimicking the characteristics and morphology of nipple epithelium to implement an experimental model mimicking fissures and cracks to evaluate the efficacy of cosmetic products as soothing (antiinflammatory) and repair treatment.

(13) The nipple is a specialized epidermis that has certain characteristics: it is hyperkeratinized and thickened with extended suprabasal, granular and horny layers.

(14) At the molecular level, there is increased production and a specific distribution of certain markers (high level of production in multiple layers of the epidermis): filaggrin (barrier marker.fwdarw.different tissue response to external stimuli) and keratin 14 (mitosis marker, indicative of a hyperproliferative state of the tissue).

(15) The nipple model was obtained by reconstruction of an epidermis or complete skin (reconstructed epidermis on a collagen matrix) from a batch of keratinocytes overexpressing filaggrin.

(16) A. Selection of the Keratinocyte Batch to be Used to Produce the Nipple Model

(17) Reconstructed epidermises were produced from different batches of keratinocytes and analyzed comparatively by histochemistry and immunohistochemistry.

(18) The batch chosen to produce the nipple model is a batch of keratinocytes overexpressing filaggrin, and after reconstruction of an epidermis, having a large number of granular layers with many keratohyalin granules and extensive labeling of filaggrin.

(19) This keratinocyte batch was used in the following steps to produce tissue models mimicking the nipple.

(20) A slice of a reconstructed epidermis (on day 17) obtained from the batch of keratinocytes chosen for implementing the nipple model is illustrated in FIG. 1.

(21) B. Characterization of a Nipple Model

(22) 1. Materials and Methods

(23) The nipple model was obtained by reconstruction of skin (from the batch of keratinocytes defined previously), on a collagen matrix containing fibroblasts.

(24) At the same time, a standard skin was reconstructed under the same conditions, on a collagen matrix containing fibroblasts, from a batch of standard keratinocytes (not overexpressing filaggrin). This standard tissue serves as a reference for the nipple tissue model.

(25) The reconstructed tissues were incubated for 4 hours and 24 hours and then the gene expression of filaggrin was studied by real-time quantitative PCR (qPCR).

(26) The reconstructed skins were lysed and the total RNAs extracted and then reverse transcribed into cDNA. The expression of the gene of interest (filaggrin) was analyzed by real-time qPCR and compared to the expression of housekeeping gene GAPDH.

(27) 2. Results

(28) The nipple skin model expresses much more filaggrin than a standard skin model (+260%). These results are illustrated by FIG. 2. They confirm that the model implemented approximates nipple physiology in terms of filaggrin production.

(29) C. Study 1: Use of a Reconstructed Nipple Skin Model to Evaluate the Reparative and Antiinflammatory Efficacy of a Breastfeeding Balm

(30) 1. Materials and Methods

(31) The nipple model was obtained by reconstruction of skin from a batch of keratinocytes overexpressing filaggrin (as described previously), on a collagen matrix containing fibroblasts.

(32) A lesion (mechanical wound) was created on the surface of the skin in order to mimic a nipple crack or fissure. The test product (breastfeeding balm) was applied topically immediately after creating the injury. Any balm formulation could be tested by the method of the invention, such as, for example, formulations 1-3 below.

(33) The results presented below are those obtained for the balm whose composition corresponds to formulation 3 (breastfeeding balm 3).

(34) Breastfeeding Balm 1

(35) TABLE-US-00001 Raw material/Brand name % PURIFIED WATER From 5 to 20% STARCH From 5 to 20% GLYCEROL QS 100% BUTTER or OIL From 5 to 20% LANOLIN QS 100%
Breastfeeding Balm 2
Breastfeeding Balm 3

(36) TABLE-US-00002 Raw material/Brand name % PURIFIED WATER QS 100% SHEA BUTTER From 5 to 20% GLYCEROL From 5 to 20% DECYL PENTANOATE From 5 to 20% FATTY PHASE GELLING AGENT From 1 to 5% METHYLGLUCOSEDIOLATE From 1 to 5% PRESERVATIVE SYSTEM From 1 to 5% VEGETABLE WAX From 1 to 5% ACTIVE INGREDIENTS From 1 to 5% EMULSIFIER From 1 to 5% XANTHAN GUM From 0 to 1% pH ADJUSTER From 0 to 1% ANTIOXIDANT From 0 to 1%

(37) The reconstructed skin was then incubated for 4 hours and 24 hours and then the following parameters were analyzed: Release of LDH, Gene expression (real-time qPCR) of TNF and filaggrin, Integrin 1 production (western blot).
a. Quantification of Released LDH:

(38) The LDH released into the reconstructed skin culture medium was quantified by means of a colorimetric test based on the detection of formazan salts whose formation is related to the enzymatic activity of the enzyme. The quantity of LDH was determined using a standard curve and expressed in mU/ml.

(39) b. Real-Time qPCR:

(40) The reconstructed skins were lysed and the total RNAs extracted and then reverse transcribed into cDNA. The expression of the genes of interest (TNF, filaggrin) was analyzed by real-time qPCR and compared to the expression of housekeeping gene GAPDH.

(41) c. Analysis of Integrin 1 by Western Blot:

(42) The reconstructed skins were lysed in the presence of protease inhibitors. The proteins resulting from samples were quantified, separated by polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane. The membranes were then incubated in the presence of a primary anti-integrin 1 antibody (Santa Cruz), and then in the presence of a labeled secondary antibody (Invitrogen). The chemiluminescence signal was then displayed and quantified; its intensity is proportional to the quantity of integrin 1 produced.

(43) 2. Results

(44) a. Evaluation of the Cell Membrane Integrity: LDH Assay

(45) The LDH assay results are indicated in Table 1 and illustrated by FIG. 3.

(46) TABLE-US-00003 TABLE 1 LDH (mU/ml) 4 hours 24 hours Control tissue 64 2.99 86.5 24.74 Injured tissue 407 9 +536% 470.5 191.62 +444% Injured tissue + balm 35 0.86 91% 14.5 7.78 97%

(47) Cell membranes form a functional membrane around cells. When cells are damaged, the membranes are altered and become porous and permeable to certain components.

(48) Cell membrane integrity was evaluated by measurement of LDH (lactate dehydrogenase) in the extracellular medium. This enzyme is normally present in the cytosol and is undetectable in the extracellular space in the absence of cell damage.

(49) The reconstructed skin lesion induced extensive release of LDH into the extracellular space, bearing witness to the impaired integrity of the cell membranes. Topical application of the breastfeeding balm greatly reduced LDH release. The breastfeeding balm protects tissue integrity and accelerates repair thereof after the injury.

(50) b. Evaluation of the Inflammatory Response: Expression of TNF

(51) The TNF expression measurement results are indicated in Table 2 and illustrated by FIG. 4.

(52) TABLE-US-00004 TABLE 1 TNF (Gene expression by relative quantity) Control tissue 1.00 Injured tissue 2.45 +145% Injured tissue + balm 1.76 28%

(53) Tumor Necrosis Factor-alpha is an important inflammation mediator; the inflammatory signal contributes to the amplification of the pain experienced. The reconstructed skin lesion induced a strong increase (+145%) of TNF gene expression, representative of induction of an inflammatory signal.

(54) The topical application of the breastfeeding balm modulated the overexpression of TNF by the lesion (28%).

(55) The breastfeeding balm modulates inflammation and therefore contributes to minimizing pain.

(56) c. Evaluation of Barrier Repair: Expression of Filaggrin

(57) The filaggrin production measurement results are indicated in Table 3 and illustrated by FIG. 5.

(58) TABLE-US-00005 TABLE 1 Filaggrin (Gene expression by relative quantity) Control tissue 1.00 Injured tissue 0.24 76% Injured tissue + balm 0.56 +133%

(59) Filaggrin plays an essential double role in the barrier function: one the one hand, it aggregates to keratin fibers of the cytoskeleton, thus reducing corneocytes into flattened disks; this intracellular network provides strength and protection to the stratum corneum. On the other hand, filaggrin is the precursor for NMF (Natural Moisturizing Factor). NMF is highly hygroscopic and is essential to retaining water in corneocytes, it plays a major role in maintaining skin pH, thus regulating key biochemical events such as protease activity, barrier permeability and antimicrobial defences, functions that are fundamentally linked and co-regulated. The reconstructed skin lesion led to a substantial reduction (76%) of filaggrin gene expression, showing significant impairment of the barrier function.

(60) The application of the breastfeeding balm modulated filaggrin repression (+133%). The breastfeeding balm tends to counteract the harmful effect of the lesion on the barrier and thus contributes to accelerating barrier repair.

(61) d. Evaluation on Healing Initiation: Expression of Integrin-1

(62) The integrin-1 production measurement results are illustrated by FIG. 6.

(63) The integrins, a group of membrane proteins, are important factors in healing regulation. On the one hand, integrins help the migration of keratinocytes, an early step in the healing process; and on the other hand, they are bound to macromolecules of the dermal matrix, thus ensuring contact of the epidermis with the newly synthesized dermal extracellular matrix to permit cohesion and communication between the two compartments (dermis and epidermis).

(64) The integrin 1 subunit is a 130 kDa transmembrane protein that forms noncovalent complexes with different subunits to form functional and active receptors.

(65) These receptors coordinate the activation of intracellular signalling pathways particular involved in healing.

(66) Western analysis of integrin 1 production (FIG. 6) shows that: in control and injured tissues, a single band of 130 kDa is observed; this band corresponds to the integrin 1 subunit alone; in the presence of the breastfeeding balm, the 1 subunit alone (130 kDa band) decreases and aggregates of higher molecular weight (220 kDa) are formed; these aggregates correspond to the formation of complexes (subunit 1 lined to an subunit) or functional heterodimer receptors representative of the activation of integrin 1.

(67) The breastfeeding balm makes integrin 1 functional and therefore permits initiating the healing process.

(68) 3. Conclusion

(69) A nipple model obtained by skin reconstruction from a batch of keratinocytes overexpressing filaggrin was used in order to evaluate the reparative and antiinflammatory efficacy of a breastfeeding balm after injury of the nipple mimicking a crack.

(70) In the injured nipple model, the breastfeeding balm has a protective effect (protection of the integrity of cell membranes/LDH), reparative effect (induction of healing/integrin 1; restoration of the barrier/filaggrin and soothing effect (antiinflammatory/TNF)

(71) These results confirm the usefulness of the model implemented as a model mimicking nipple skin and allowing the evaluation of the reparative and soothing efficacy of a formulation dedicated to nipple care, in particular in the case of appearance of cracks or fissures.

(72) D. Study 2: Use of a Reconstructed Nipple Epidermis Model to Comparatively Evaluate the Reparative and Antiinflammatory Efficacy of Cosmetic Breastfeeding Products in Comparison to Breast Milk

(73) 1. Materials and Methods

(74) To mimic the nipple, a reconstructed epidermis, obtained from a batch of keratinocytes overexpressing filaggrin (as described previously), was used.

(75) Two cosmetic products designed to care for cracks related to breastfeeding were studied: a breastfeeding balm (corresponding to the breastfeeding balm 3 formulation) and an ointment based on lanolin (product 2). At the same time, breast milk, described for its reparative properties, was also evaluated.

(76) A lesion (mechanical wound) was created on the surface of the nipple epidermis in order to mimic a nipple crack or fissure.

(77) The test products were applied topically immediately after creating the lesion.

(78) The reconstructed skin was then incubated for 4 hours and 24 hours and then the following parameters were analyzed: Release of LDH; Gene expression (real-time qPCR) of TGF 1 and TNF; Immunofluorescence labeling of integrin 1 (only product 1).
a. Quantification of Released LDH:

(79) The LDH released into the reconstructed epidermis culture medium was quantified by means of a colorimetric test based on the detection of formazan salts whose formation is related to the enzymatic activity of the enzyme. The quantity of LDH was determined using a standard curve and expressed in mU/ml.

(80) b. Real-Time qPCR:

(81) The reconstructed epidermises were lysed and the total RNAs extracted and then reverse transcribed into cDNA. The expression of the genes of interest (TNF, TGF 1) was analyzed by real-time qPCR and compared to the expression of housekeeping gene GAPDH.

(82) c. Immunofluorescence Labeling of Integrin 1 (Evaluated Only for Product 1).

(83) The reconstructed epidermises were fixed, coated in paraffin and sliced. The sections were incubated in the presence of a primary antibody directed against the extracellular domain of integrin 1 (Santa Cruz), and the in the presence of a secondary antibody conjugated to fluorochrome Alexa Fluor 555 (Invitrogen). The nuclei were counterstained with DAPI.

(84) The labeling was imaged by confocal microscope (Leica).

(85) 2. Results

(86) a. Evaluation of the Cell Membrane Integrity: LDH Assay

(87) The results are presented in Table 4 and illustrated in FIG. 7.

(88) TABLE-US-00006 TABLE 1 LDH (mU/ml) Control tissue 7.8 0 Injured tissue 123.11 0.86 Injured tissue + product 1 12.45 6.98 90% Injured tissue + product 2 106.85 6.06 13% Injured tissue + breast milk .sup.63.15 21.94 49%

(89) The reconstructed nipple epidermis lesion induced extensive release of LDH into the extracellular space, representative of the impaired integrity of the cell membranes.

(90) The topical application of product 1 greatly reduced the release of LDH (90% inhibition). Product 2 tested slightly reduced LDH release (13%) and breast milk led to an intermediate reduction of LDH release (49%).

(91) Product 1 and breast milk, to a lesser extent, protect tissue integrity and accelerate their recovery after injury.

(92) b. Evaluation of the Inflammatory Response: Expression of TNF

(93) The results are presented in Table 5 and illustrated in FIG. 8.

(94) TABLE-US-00007 TABLE 5 TNF (Gene expression by relative quantity) Control tissue 1.00 Injured tissue 35.21 Injured tissue + product 1 8.38 76% Injured tissue + product 2 29.03 17% Injured tissue + breast milk 16.28 54%

(95) The reconstructed epidermis lesion induced a strong increase in TNF gene expression, representative of induction of an inflammatory signal.

(96) Product 1 very clearly inhibited TNF expression induced by the lesion (76%); product 2 induced a slight reduction (17%) and breast milk induced an intermediate reduction (54%).

(97) Product 1 and, to a lesser extent, breast milk, modulate inflammation and contribute to pain relief.

(98) c. Evaluation on Healing Initiation: Expression of TGF 1

(99) The results are presented in Table 6 and illustrated in FIG. 9.

(100) TABLE-US-00008 TABLE 6 TGF 1 (Gene expression by relative quantity) Injured tissue 1.00 Injured tissue + product 1 2.05 +105% Injured tissue + product 2 1.18 +18% Injured tissue + breast milk 1.59 +59%

(101) TGF 1 is a growth factor that controls numerous cell functions; it is notably involved in the regulation of healing and in stimulating the re-epithelization phase early in the epidermal healing phase.

(102) Product 1 induced a substantial increase (+105%) in TGF 1 expression in injured tissues, product 2 led to a slight increase (+18%) and breast milk led to an intermediate increase (+59%).

(103) Product 1 and, to a lesser extent, breast milk, contribute to initiating the healing process for a reparative effect.

(104) d. Evaluation on Healing Initiation: Expression of Integrin 1

(105) The results are illustrated in FIG. 10.

(106) In the control tissues, the results show an intense labeling of integrin 1 associated with the plasma membrane of keratinocytes (yellow arrows). This membrane location for integrin 1 shows the availability/functionality of the receiver.

(107) The tissue lesion leads to a strong decrease of integrin 1 labeling; this latter has practically disappeared, which shows an alteration of the receptor.

(108) In the presence of a breastfeeding balm (product 1), membrane labeling of integrin 1 is recovered (yellow arrows) which bears witness to the restoration of receptor functionality.

(109) The breastfeeding balm (product 1) restores the production of integrin 1, therefore restoring intercellular contacts to initiate the repair process.

(110) 3. Conclusion

(111) A nipple model obtained by reconstruction of an epidermis from a batch of keratinocytes overexpressing filaggrin was used in order to comparatively evaluate the reparative and antiinflammatory efficacy of cosmetic products intended for nipple care in comparison with breast milk. The reconstructed nipple epidermis was injured on the surface in order to mimic a lesion.

(112) On this injured nipple epidermis model, efficacy differences were found between the two products tested and breast milk (known for its reparative properties) in terms of protective action (protection of cell membrane integrity/LDH), repair (initiation of wound healing/TGF 1, integrin 1) and healing (anti-inflammatory/TNF).

(113) These results confirm the usefulness of the model implemented as a model mimicking nipple epidermis, allowing the evaluation of different formulas and the identification of effective formulas for care of nipple cracks/fissures.

(114) E. Study 3: Use of a Reconstructed Nipple Epidermis Model to Show the Protective and Reparative Effect of a Breastfeeding Balm.

(115) The preceding steps showed the usefulness of the nipple model implemented for evaluation of the activity or identification of a formulation to treat nipple skin lesions relying on measurement of markers of a reparative, protective or antiinflammatory effect.

(116) In order to validate the conclusions obtained by the analysis of the marker production level, we sought to show the effect of a lesion in the nipple epidermal model and the reparative effect of a product by using the electron microscope technique.

(117) 1. Materials and Methods

(118) A reconstructed epidermis, obtained from a batch of keratinocytes overexpressing filaggrin (as described previously), was used as a nipple model.

(119) A lesion (mechanical wound) was created on the surface of the nipple epidermis in order to mimic a nipple crack or fissure.

(120) A breastfeeding balm corresponding to the breastfeeding balm 3 formulation was then applied topically.

(121) After incubation for 4 hours, the epidermises were fixed, dehydrated and covered with a layer of gold before being observed in the scanning electron microscope (Zeiss).

(122) 2. Results

(123) The results are illustrated in FIG. 11.

(124) Epidermal keratinocytes form a corneal envelope during the final steps of their differentiation. This corneal envelope ensures the cohesion and solidarity of the stratum corneum and plays a vital role in the epidermis barrier function.

(125) SEM observation of the reconstructed tissue surface shows, in the control tissues, an intact cutaneous surface, homogenous and smooth (corresponding to the corneal envelope) where cell outlines are distinguished (corneocytes; blue arrow).

(126) The lesion caused a deep wound with destruction of the corneal envelope.

(127) The application of the breastfeeding balm after the lesion visibly accelerates the repair of this wound: the surface is more regular and smoother, showing restoration of the corneal envelope.

(128) 3. Conclusion

(129) These SEM observations directly confirm the reparative and protective effect of the breastfeeding balm shown by the production of different markers during the preceding steps.

(130) Thus these results confirm: the usefulness of the model implemented as a model mimicking cracked nipple skin or epidermis permitting evaluating or identifying the reparative or protective effect of a formulation, the choice of markers (in the preceding steps) and their validity as markers representative of a reparative and protective effect.