Abstract
The present invention relates to an approach for the treatment of adverse neurological sensations in a certain body surface area such as the skin, in particular treatment of pain or itching. The invention is based on the finding that administration of a targeting molecule which specifically binds a cell or receptor responsible for the adverse sensation in the respective body surface area of a patient, and which is coupled/conjugated to a photosensitive inhibitor or cytotoxic agent can enable the irradiation dependent ablation of cells responsible for the sensation. This approach allows a targeted and specific treatment of body surface areas by irradiation. Provided are conjugate compounds for use in the photoablation treatment of the invention and pharmaceutical compositions which comprise these compounds.
Claims
1. A conjugate compound for use in therapy for targeting and inhibiting/killing a target cell in a target body surface area of a subject, wherein the conjugate compound, preferably a protein conjugate compound, comprises (i) a binding domain which specifically binds to the target cell, and (ii) a photosensitive inhibition/cytotoxin group, and wherein the therapy comprises the steps of: (a) Administering said conjugate compound to the subject, and (b) Irradiating said target body surface area of the subject with an appropriate excitation light in an amount to effectively activate said photosensitive inhibition/cytotoxin group and to induce cellular inhibition or cell death of the target cell.
2. The conjugate compound for use according to claim 1, wherein the target cell is a neuron, preferably a sensory neuron.
3. The conjugate compound for use according to claim 1, wherein the binding domain specifically binds to a receptor expressed on the target cell.
4. The conjugate compound for use according to claim 3, wherein the binding domain is a receptor ligand, or a receptor binding fragment thereof, or a receptor binding antibody, or a receptor binding fragment thereof.
5. The conjugate compound for use according to claim 1, wherein the photosensitive cytotoxin group is a phthalocyanine dye IRDye 700DX, or a derivative thereof, such as a benzylguanine modified derivative.
6. The conjugate compound for use according to claim 1, wherein the therapy is for alleviating a neurological sensation in the target body surface area of the subject, for example a neurological sensation selected from noxious or innocuous stimuli, such as all forms of mechanical (touch) sensation, pain and/or itching.
7. The conjugate compound for use according to claim 1, wherein conjugate compound comprises a pruritogen as a binding domain which specifically binds to the cell, for example interleukin-31 (IL31) or mutant IL31, or derivatives or fragments of these compounds.
8. The conjugate compound for use according to claim 7, wherein the mutant IL-31, is an IL31 binding to IL31 receptor (I131RA and OSMR), but eliciting a reduced IL31 signaling, such as a mutation in human IL31 at position K134, for example IL31.sup.K134A.
9. The conjugate compound for use according to claim 1, wherein the binding domain which specifically binds to the target cell is capable to bind to an expression product of a TrkB gene, preferably the NTRK2 gene, in the target cell.
10. The conjugate compound for use according claim 9, wherein the TrkB ligand is a protein binding to the TrkB/p75 receptor complex, and preferably is selected from Brain-derived neurotrophic factor (BDNF) or Neurotrophin 4 (NT-4).
11. The conjugate compound for use according to claim 1, wherein the binding domain which specifically binds to the target cell is capable to bind to an expression product of a TrkA gene in the target cell.
12. The conjugate compound for use according to claim 11, wherein the compound that is capable to bind to an expression product of the TrkA gene comprises a TrkA-ligand or an anti-TrkA-antibody or anti-TrkA-T cell receptor (TCR), or chimeric antigen receptor (CAR); or wherein the compound comprises a nucleic acid having a nucleic acid sequence that is complementary to, or can under stringent conditions hybridize to, an mRNA produced by the TrkA locus.
13. The conjugate compound for use according claim 12, wherein the TrkA ligand is a protein binding to the TrkA receptor, and preferably is Nerve Growth Factor (NGF), more preferably a mutant NGF, such as a mutant human NGF, for example an NGF mutated at position R121, such as NGF.sup.R121W.
14. The conjugate compound for use according to claim 1, wherein the conjugate compound in step (a) is administered locally or systemically to the subject, such as a subcutaneous injection, and intraneural injection, or topical administration, such as by applying a cream, ointment, salve, or other topical formulations.
15. A conjugate compound comprising (i) a binding domain which specifically binds to the target cell, and (ii) a photosensitive inhibition/cytotoxin group , wherein the binding domain is selected from a TrkA ligand, a TrkB ligand, or a pruritogen.
16. A pharmaceutical composition comprising a conjugate compound according to claim 15, together with a pharmaceutically acceptable carrier and/or excipient.
Description
[0088] The following figures, sequences, and examples merely serve to illustrate the invention and should not be construed to restrict the scope of the invention to the particular embodiments of the invention described in the examples. All references as cited herein are hereby incorporated in their entirety by reference.
[0089] FIG. 1: IL31.sup.SNAP labelling and photoablation. (a) Representative Coomassie (upper panel) and fluorescence (lower panel) gel showing the binding of IL31.sup.SNAP with the fluorescent substrate BG549 at a 1:3 molar ratio. First and fourth lanes represent the binding of 10 and 20 pmol IL31.sup.SNAP respectively, with BG549-Second and last lanes represent the protein IL31.sup.SNAP alone, 10 and 20 pmol respectively. (b) Primary keratinocyte culture from wild type and (c) IL31RA.sup./ mice labelled with 1 M IL31.sup.SNAP coupled with 3 M BG549 (in red). Nuclei were stained with Dapi (in blue). Scale bar 20 m. (d) Representative back skin cryosection (25 m) of wild type and (e) IL31RA.sup./ mice injected with 5 M IL31.sup.SNAP coupled with 15 M BG549 (in red) and Dapi for nuclear staining (in blue). Scale bar 50 m. (f) Scratching evoked by intradermal injection of 5 M SNAP, IL31 or IL31.sup.SNAP in wild type (n=4) and IL31RA.sup./ mice (n=4). Error bars indicate SEM. *p<0.05 (t-test). (g, h, i) Propidium Iodide staining to assess cell death (in red) 24 hours after photoablation performed on primary wild type keratinocytes labelled with 1 M IL31.sup.SNAP+3 M BGIR700 (g), with 3 M IR700 only (h), or IL31RA.sup./ keratinocytes labelled with 1 M IL31.sup.SNAP+3 M BGIR700 (i). Insets represent the brightfield images of the stained cells. Scale bar 50 m. (j, k) TUNEL assay staining to assess apoptosis (in red) after 3 consecutive days of photoablation performed on the back skin of wild type (j) and IL31RA.sup./ (k) mice injected with 5 M IL31SNAP+15 M BGIR700. Insets represent the brightfield image of the same skin area. Scale bar 50 m. (l) Scratching behavior evoked by 3 days of injection with 5 M IL31.sup.SNAP (black line) and 5 M IL31.sup.SNAP+15 M IR700 (red line). Baseline refers to spontaneous scratching before the first injection. Number of scratch bouts was counted over a 30 minutes recording time. *p<0.05 (One-Way Anova).
[0090] FIG. 2: Functional analysis of IL31.sup.K138A-SNAP. (a) Primary keratinocyte cultures from wild type and (b) IL31iRA.sup./ mice labelled with 1 M IL31.sup.K138A-SNAP+3 M BG549 (in red). Nuclei were stained with DAPI (in blue). Scale bar 20 m. (c) Representative western blots showing the expression level of AKT, phospho AKT, MAPK, phospho-MAPK, STAT3, phospho STAT3 and Actin (loading control) in skin injected with vehicle (PBS, lane i), 5 M IL31.sup.SNAP (lane 2) and 5 M IL31.sup.K138A-SNAP (lane 3). (d) Levels of each protein were expressed as the ratio between the phosphorylated form and the total counterpart and then normalized to the vehicle-treated sample. (e) Scratching response evoked by 3 consecutive days of injection of 5 M IL31.sup.SNAP (black line) and 5 M IL31.sup.K138A-SNAP (red line). Baseline refers to spontaneous scratching before the first injection. Number of scratching bouts was counted over a 30 minutes recording time. Error bars indicate SEM. *p<0.05 (One-Way Anova). (f) Scratching response evoked by the injection of vehicle (PBS, N=4) and different pruritogens (5 M IL31, 10 mM Histamine, 1 mM LY344864, and 12.5 mM Chloroquine CQ) after mice were injected for 3 consecutive days with 5 M IL31.sup.K138A-SNAP+15 M BGIR700, with (red bars, n=5) and without (black bars, n=4) near IR illumination. The number of scratching bouts was counted over a 30 minute recording time. Error bars indicate SEM. *p<0.05 (t-Test). (g) Thermal sensation was evaluated using the Hot Plate test after 3 days of injection of 5 M IL31.sup.K138A-SNAP+15 M BGIR700 into the hind paw of the mice, with (red bars, n=4) and without (black bars, n=4) near IR illumination. Baseline refers to the thermal latency before the first injection was performed. Bar graphs represent the latency expressed in seconds of the paw withdrawal in response to heat. Error bars indicate SEM. (h) Mechanical sensation was evaluated using the Von Frey test after 3 days of injection of 5M IL31.sup.K138A-SNAP+15 M BGIR700 into the hind paw of the mice, with (red bars, n=4) and without (black bars, n=4) near IR illumination. Baseline refers to the mechanical threshold before the first injection was performed. Bar graphs represent the force expressed in grams required to trigger a 50% response. Error bars indicate SEM.
[0091] FIG. 3: IL31 .sup.K138A-SNAP guided photoablation prevents and reverses symptoms of atopic dermatitis. (a-d) Prevention of atopic dermatitis-like symptoms. (a) Number of scratch bouts in response to 14 days of Calcipotriol treatment in mice pre-injected for 3 consecutively days with 5 M IL31.sup.K138A-SNAP+15 M BGIR700, with (red line, n=4) or without (black line, N=4) near IR illumination. The number of scratch bouts was counted over a 30-minute recording time. Baseline refers as spontaneous scratching before injections were performed. Error bars indicate SEM. * p<0.05 (One-Way Anova). (b) Skin thickness expressed in millimeter (mm) and measured at day 14 of Calcipotriol treatment in mice injected with IL31.sup.K138A-SNAP+IR700, with (red bar, n=4) and without (black bar, n=4) near IR illumination. Error bars indicate SEM. *p<0.05 (t-Test). (c) Hematoxylin & Eosin staining of 6 m-paraffin sections of back skin collected after 14 days of Calcipotriol treatment showing difference in skin histology (epidermal thickness and dermal infiltration of eosinophilic material) between mice treated with IL31.sup.K138A-SNAP+IR700, with (Top panel) IR light and without (Lower panel, No light) near IR illumination. Scale bars 200 m. (d) Representative skin pictures of mice after 14 days of Calcipotriol treatment treated with IL31.sup.K138A-SNAP+IR700 with near IR illumination (Top panel) and without (Lower panel) (e-h) Rescue of atopic dermatitis symptoms after treatment with IL31.sup.K138A-SNAP at days 6-8 of Calcipotriol application. (e) Scratching bouts with (red line, n=6) and without (black line, N=6) near IR illumination. Error bars indicate SEM. *p<0.05 (One-Way Anova;). (f) Skin thickness at day 21 of Calcipotriol treatment (n=6 both groups). Error bars indicate SEM. *p<0.05. (g) Hematoxylin & Eosin staining of 6 m-paraffin sections of back skin after 21 days of Calcipotriol treatment. Scale bars 200 m. (h) Representative skin images at 21 days of Calcipotriol treatment. (i-j) Prevention of atopic dermatitis-like symptoms using topical delivery of IL31.sup.K138A-SNAP (i) Scratching behavior evoked by Calcipotriol. Error bars indicate SEM. *p<0.05 (One-Way Anova). (j) Representative skin pictures of mice after 10 days of Calcipotriol application. (k-l) Rescue of atopic dermatitis symptoms using topical application of IL31.sup.K138A-SNAP at day 5-7 of Calcipotriol applications. (k) Scratching behavior (red circles, n=7, black circles, n=8) for 3 consecutive days Error bars indicate SEM. *p<0.05 (One-Way Anova). (l) Representative skin pictures after 14 days of Calcipotriol application.
[0092] FIG. 4: TrkB positive sensory neurons are myelinated low threshold mechanoreceptors. (a-e) Double immunofluorescence of DRG sections from TrkBCreERT2::Rosa26RFP mice with (a) NF200, (b) Ret, visualized using TrkBCreERT2::Rosa26RFP::RetEGFP triple transgenic mice, (c) IB4, (d) CGRP, and (e) TH. (f) Section from the glabrous skin of TrkBCreERT2::Rosa26ChR2YFP (red) stained with anti-S100 a marker for Meissner's corpuscles (green) and DAPI (blue) showing TrkB+ innervation. (g) TrkB+ lanceolate endings in a section of the back hairy skin of TrkBCreERT2::Rosa26SnapCaaX labeled with Snap Cell TMRstar (red), NF200 (green) and DAPI (blue). (h) Section through the lumbar spinal cord of TrkBCreERT2::AvilmCherry mice stained with IB4. (i-k) Double immunofluorescence of human DRG sections stained with antibodies against TrkB and (i) NF200, (j) Ret and (k) TrkA. (l) Section from human glabrous skin stained with antibodies against TrkB (red) and NF200 (green), and DAPI (blue). (m) Quantification of staining on mouse DRG sections; TrkB+ cells account for 10% of all DRG neurons and all co-express NF200 (NF) or NF200+ReteGFP, while they are negative for IB4, CGRP (CG) and TH. (n) Size distribution for human DRG neurons expressing TrkB, NF200 and TrkA. (o-q) in-vitro skin nerve preparation from TrkBCreERT2::Rosa26ChR2 mice showing (o) the minimal force required to elicit an action potential in the indicated fibre type, (p) the conduction velocities of the fibre types and (q) representative responses to 10 Hz stimulation with blue light. Red bar represents TrkB+ afferents, n number indicated in brackets. Scale bars, A-E and H 50 m, F, G and I-L 40 m. Error bars represent SEM. TrkB positive sensory neurons are myelinated low threshold mechanoreceptors.
[0093] FIG. 5: Diphtheria toxin mediated ablation of TrkB+ sensory neurons. Immunostaining of DRG sections of TrkB.sup.CreERT2::Avil.sup.iDTR mice with an antibody against the diphtheria toxin receptor (red) from (A) untreated mice and (B) after i.p injections of diphtheria toxin. (C) Quantification of DRG sections indicating a 90% decrease in TrkB.sup.DTR and Trke.sup.mRNA cells after ablation and 40% reduction in NF200.sup.+ neurons without affecting other subpopulations. (D-J) Behavioral responses in littermate control mice (Avil.sup.iDTR, black bars) and TrkB.sup.CreERT2::Avil.sup.iDTR mice (white bars) showing no differences in responses before and after ablation in the (D) acetone drop test (t-test; p>0.05), (E) hot plate test (t-test; p>0.05), (F) grip test (t-test; p>0.05), (G) pin-prick test (t-test; p>0.05), (H) tape test (t-test; p>0.05). (I) Ablated mice show a reduction in sensitivities to cotton swab (t-test, p<0.001). Scale bars in A, B 50 m, error bars indicate SEM.
[0094] FIG. 6: TrkB+ neurons are necessary and sufficient to convey mechanical allodynia after nerve injury. Mechanical hypersensitivity in both control Avil.sup.iDTR (black bar) and TrkB.sup.CreERT2::Avil.sup.iDTR (white bar) mice 48 hours after CFA injections as measured by (A) von Frey filaments (t-test, p>0.05) and (B) dynamic brush stimuli (t-test; p>0.05). All mice received two diphtheria toxin injections 7 days and to days before CFA treatment. (C) Paw withdrawal frequencies in contralateral (black bar) and CFA injected ipsilateral (white bar) paw of TrkB.sup.CreERT2::Rosa26.sup.ChR2 mice upon stimulation with 473 nm blue light shows no significant difference under baseline conditions and 48 hours after CFA injection (Mann-Whitney test; p>0.05). (D) von-Frey mechanical thresholds indicating that ablation of TrkB+ neurons abolished the development of mechanical allodynia after SNI in TrkB.sup.CreERT2::Avil.sup.iDTR mice (white circles) as compared to Avil.sup.iDTR controls (black circles) (n=7 for both sets, Two-way RM ANOVA; p<0.001 followed by a Bonferroni post-hoc test). (E) Reduced dynamic brush allodynia in ablated TrkB.sup.CreERT2::Avil.sup.iDTR mice (white bar) as compared to littermate controls (black bar; t-test p<0.05). (F) Nociceptive behavior evoked by optogenetic stimulation of ipsilateral (black bars) and contralateral (white bar) paw of TrkB.sup.CreERT2::Rosa26.sup.ChR2 mice after SNI (Two-way RM ANOVA; p<0.001). (G-H) Cross section of the lumbar spinal cord from TrkB.sup.CreERT2::Rosa26.sup.ChR2 mice labelled for c-fos after 1 minute exposure to a 15 Hz blue light 7 days post SNI (G) Represents the contralateral uninjured and (H) the injured ipsilateral dorsal horn. (I) shows quantification of the number of c-fos positive cells in each lamina of the lumbar spinal cord within a 40 m section (black bar contralateral, white bar ipsilateral). Error bars indicate SEM. Scale bars in G and H, 40 m.
[0095] FIG. 7: BDNF.sup.SNAP labelling and IR700 mediated photoablation in vitro. (a-c) BDNF.sup.SNAP labeling of HEK293T cells transfected with (a) TrkB/p75NTR, (b) TrkA/p75NTR, or (c) TrkC/p75NTR. (d) Labeling (inset) and quantification of dissociated DRG from TrkB.sup.CreERT2::Rosa26.sup.RFP mice with BDNF.sup.SNAP shows substantial overlap of BDNF.sup.SNAP binding to TrkB+ cells. (e)
[0096] Staining of HEK293T cells transfected with TrkB/p75NTR with propidium iodide 24 hours after treatment with BDNFSNAP-IR700 and near infrared illumination. (f) Staining of mock transfected HEK293T cells with propidium iodide 24 hours after photoablation following treatment with BDNFSNAP-IR700. Scale bars 50 m.
[0097] FIG. 8: Optopharmacological targeting of TrkB+ neurons with BDNF.sup.SNAP. (a-b) BDNF.sup.SNAP-IR700 mediated photoablation of the paw of SNI mice results in a dose dependent reversal of mechanical hypersensitivity as assayed with von Frey filaments (a) (n=10, two-way RM ANOVA; p<0.05 followed by a Bonferroni post-hoc test) and (b) dynamic brush stimuli (t-test; p<0.05). (c) Hypersensitivity to cotton swab is also reversed by photoablation (t-test: p<0.05). (d) BDNF.sup.SNAP-IR700 mediated photoablation reverses mechanical allodynia in the streptozotocin (STZ) model of diabetic neuropathy (n=5, two-way RM ANOVA; p<0.05 followed by a Bonferroni post-hoc test. Open circles; 5 M BDNF.sup.SNAP-IR700 at 200 J/cm.sup.2, closed circles, 5 M IR700 at 200 J/cm.sup.2). (e) BDNF.sup.SNAP-IR700 mediated photoablation reverses mechanical allodynia in the paclitaxel (PTX) model of chemotherapy induced neuropathy (n=5, two-way RM ANOVA; p<0.05 followed by a Bonferroni post-hoc test. Open circles; 5 M BDNF.sup.SNAP-IR700 at 200 J/cm.sup.2, closed circles, 5 M IR700 at 200 J/cm.sup.2). (f-i) BDNF.sup.SNAP-IR700 mediated photoablation in the paw does not affect baseline sensory behavior responses to (f) acetone drop test (t-test; p>0.05), (g) hot plate test (t-test; p>0.05), (h) pin-prick test (t-test; p>0.05) and (i) cotton swab test (t-test; p>0.05). White bars 5 M BDNF.sup.SNAP-IR700 at 200 J/cm.sup.2, black bars 5 M IR700 at 200 J/cm.sup.2. Baseline indicates pre-ablation and pre-treatment. Error bars indicate SEM.
[0098] FIG. 9: BDNF.sup.SNAP-IR700 photoablation promotes local retraction of TrkB+ afferents. (a-d) Substantial loss of TrkB.sup.CreERT2 positive afferents (red), but persistence of other fibers (green) upon BDNF.sup.SNAP-IR700 mediated photoablation. (a) Innervation of paw hairy skin prior to ablation, arrows show lanceolate endings. (b) Loss of TrkB.sup.CreERT2 afferents after ablation, arrows show PGP9.5 fibers. (c) High magnification image of a hair follicle after ablation. Note the absence of TrkBCreERT2 fibers (red) but PGP9.5 positive circumferential and longitudinal lanceolate endings (green). (d) Reinnervation of skin by TrkB.sup.CreERT2 afferents at 24 days post ablation. (e) DRG section from photoablated TrkB.sup.CreERT2 mouse labelled for RFP (red) and NF200 (green). (f) Quantification of the proportion of hair follicle innervation and DRG neurons positive for TrkB following photoablation in the paw. (g) Quantification of loss of other cells types in the skin upon photoablation. (h-l) Behavioral sensitivity following BDNF.sup.SNAP-IR700 mediated ablation in the sciatic nerve. (h) Acetone drop test (t-test; p>0.05), (i) radiant heat test (t-test; p>0.05), and (j) pin-prick test (t-test; p>0.05) are not altered by nerve photoablation. However sensitivity to (k) cotton swab (t-test; p<0.05) in control animals, and (1) light evoked behavior in TrkB.sup.CreERT2::Rosa26.sup.ChR2 mice with SNI, are reduced by nerve photoablation (Two-way RM ANOVA; p<0.001). White bars 5 M BDNF.sup.SNAP-IR700 at 200 J/cm.sup.2, black bars 5 M IR700 at 200 J/cm.sup.2. Baseline indicates pre-ablation and pre-treatment. Error bars indicate SEM. Scale bars a-d 40 m, e 10 m.
[0099] FIG. 10. NGF.sup.SNAP-IR700 mediated photoablation and identification of a painless NGF mutant. (a) NGF.sup.SNAP-IR700 mediated photoablation prevents thermal hyperalgesia in the CFA model of inflammatory pain. Blue arrow indicates CFA application. (b) NGF.sup.SNAP-IR700 mediated photoablation reverses mechanical hypersensitivity following CFA injections. Red arrow indicates photoablation. (c) The NGFR.sup.121W-SNAP mutant binds to cells expressing TrkA and p75. (d) Photoablation of HEK293 cells expressing TrkA and p75 upon application of NGF.sup.R121W-SNAP and illumination. (e) Wildtype NGF.sup.SNAP induces mechanical hypersensitivity when injected in the paw (black bars), while NGF.sup.R121W-SNAP does not (white bars).
[0100] FIG. 11. NGFR.sup.121W-SNAP-IR700 mediated photoablation to control acute and inflammatory pain. (a). Injection of NGFR.sup.121W-SNAP-IR700 into the paw and subsequent near-IR illumination substantially increases paw withdrawal thresholds to von Frey filaments. (b) Nociceptive pin prick evoked responses are significantly reduced by NGFR.sup.121W-SNAP-IR700 photoablation. (c) Non-nociceptive brush sensitivity is not altered by NGFR.sup.121W-SNAP-IR700 mediated photoablation. (d) CFA induced thermal hyperalgesia is reversed by NGFR.sup.121W-SNAP-IR700 photoablastion. (e) CFA induced mechanical hypersensitivity is reversed by NGFR.sup.121W-SNAP-IR700 mediated photoablation. Red arrow indicates time of photoablation, control indicates injection without illumination.
TABLE-US-00001 SEQIDNO:1showswildtypehumanIL31amino acidsequence: MASHSGPSTSVLFLFCCLGGWLASHTLPVRLLRPSDDVQKIVEELQSL SKMLLKDVEEEKGVLVSQNYTLPCLSPDAQPPNNIHSPAIRAYLKTIR QLDNKSVIDEIIEHLDKLIFQDAPETNISVPTDTHECKRFILTISQQF SECMDLALKSLTSGAQQATT SEQIDNO:2showswildtypehumanNGFamino acidsequence: MSMLFYTLITAFLIGIQAEPHSESNVPAGHTIPQAHWTKLQHSLDTAL RRARSAPAAAIAARVAGQTRNITVDPRLFKKRRLRSPRVLFSTQPPRE AADTQDLDFEVGGAAPFNRTHRSKRSSSHPIFHRGEFSVCDSVSVWVG DKTTATDIKGKEVMVLGEVNINNSVFKQYFFETKCRDPNPVDSGCRGI DSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRR A
EXAMPLES
I: ITCHING
Example 1: Generation and Characterization of IL31.SUP.SNAP
[0101] The inventors produced recombinant IL31 with a C-terminal fusion of SNAP (IL31.sup.SNAP) in E. Coli. Following purification and refolding from inclusion bodies, IL31.sup.SNAP was efficiently labelled with BG derivatized fluorophores (FIG. 1a) indicating that the SNAP tag was successfully incorporated and correctly folded in the fusion protein. To determine whether IL31.sup.SNAP was functional the inventors first performed binding studies in primary keratinocyte cultures. IL31.sup.SNAP was labelled in vitro with BG-Surface549 and applied to keratinocytes from wildtype or IL31 Receptor A (IL31RA) knockout mice (IL31RA.sup./). At a range of concentrations the inventors observed strong fluorescent signal internalized in wildtype keratinocytes that was not present in cells for IL31RA.sup./ mice (FIGS. 1b and c). The inventors further examined IL31.sup.SNAP labelling in vivo by injecting IL31.sup.SNAP-Surface549 intradermally into the back skin of wild type and IL31RA.sup./ mice. Again, fluorescent signal was observed in cells in skin sections from wildtype mice but not from IL31RA.sup./ mice (FIGS. 1d and e). Finally, the inventors determined whether IL31.sup.SNAP was active by quantifying scratching behavior in mice upon intradermal injection. IL31.sup.SNAP evoked robust scratching that was comparable in duration and intensity to native recombinant IL31 in wildtype mice. In IL31RA.sup./ mice, IL31.sup.SNAP and IL31 did not evoke scratching (FIG. 1f). Thus the IL31.sup.SNAP retains the functional properties of native IL31.
Example 2: IL31 Mediated Photoablation
[0102] To manipulate IL31 receptor expressing cells in vivo, the inventors reasoned that IL31.sup.SNAP may allow for targeted photoablation of these cells through delivery of a photosensitizing agent. The inventors synthesized a benzylguanine modified derivative of the highly potent near-infrared photosensitizer IRDye700DX phthalocyanine (IR700) and conjugated it in vitro to IL31.sup.SNAP (20). Application of IL31.sup.SNAP-IR700 to keratinocytes followed by 1 minute illumination provoked substantial cell death (FIG. 1g) that was not evident in keratinocytes only treated with IR700 (FIG. 1h) or in keratinocytes from IL31RA.sup./ mice (FIG. 1i). The inventors further examined photosensitizer induced cell death in skin by injecting IL31.sup.SNAP-IR700 and applying near infrared light to the skin. TUNEL positive apoptotic cells were observed throughout the epidermis and dermis of the illuminated area in wildtype mice (FIG. 1j), but largely absent in skin from IL31RA.sup./ mice (FIG. 1k). The inventors next examined whether IL31.sup.SNAP-IR700 mediated photoablation would impact upon IL31 evoked scratching behavior. Strikingly, a progressive decrease in scratching bouts was observed when IL31.sup.SNAP-IR700 was injected for three consecutively days and the skin illuminated (FIG. 1l).
Example 3: Generation and Characterization of a Non-Signaling IL31 Mutant
[0103] A conceptual problem of using IL31.sup.SNAP therapeutically is that it in itself evokes itch. The inventors therefore sought to engineer IL31.sup.SNAP to obtain a ligand that still binds to IL31 receptor complex but no longer promotes signaling. From a previous structure/function study (21) the inventors selected an IL31 point mutant IL31.sup.K138A that was reported to exhibit reduced signaling in cells expressing IL31 receptors. The K138A mutation denotes the murine IL31 mutations. The corresponding mutation in the human IL31 protein is at position K134 in the human IL31 (SEQ ID NO: 1). The inventors generated a recombinant IL31.sup.K138A-SNAP fusion protein, labelled it with BG-Surface549 and applied it to keratinocytes. Pronounced fluorescence was evident in cells from wildtype mice treated with fluorescent IL31.sup.K138A-SNAP (FIG. 2a), at a similar concentration range to that observed with IL31.sup.SNAP (Supplementary FIG. 2). Importantly, such signal was not present in IL31RA.sup./ keratinocytes (FIG. 2b). The inventors further assessed cellular signaling pathways activated by IL31.sup.SNAP and IL30.sup.K138A-SNAP in the skin by examining levels of phosphorylated Akt, pMAPK and pSTAT3 which have all been previously implicated in IL31 downstream signaling (14, 21, 22). Mice were injected subcutaneously with IL31.sup.SNAP and IL31.sup.K138A-SNAP and skin harvested 1 hour later for immunoblot analysis. The inventors observed increased phosphorylation in each pathway in skin injected with IL31.sup.SNAP, and this increase was absent in skin injected with IL311.sup.K138A-SNAP (FIGS. 2c and d). As a final test for the functional activity of IL31.sup.K138A-SNAP, the inventors assayed its capacity to provoke scratching behavior in mice. In contrast to IL31.sup.SNAP which induced robust scratching when injected intradermal, IL31.sup.K138A-SNAP did not evoke any scratching above baseline levels in mice (FIG. 3e). Thus the engineered ligand IL31.sup.K138A-SNAP may offer a powerful means of targeting cells involved in itch without triggering itch in itself.
Example 4: IL31.SUP.K138A-SNAP.-IR700 Mediated Photoablation and Acute Itch
[0104] To characterize IL31.sup.K138A-SNAP mediated photoablation in vivo, the inventors first tested its efficacy at alleviating IL31 provoked itch. Mice were treated for three consecutively days with IL31.sup.K138A-SNAP-IR700 and the skin was illuminated with near-IR light. Strikingly, IL31-induced scratching behavior was abolished in these animals (FIG. 2f), and this persisted throughout an 8 week observation period (Supplementary FIG. 2l). In control mice that received an IL31.sup.K138A-SNAP-IR700 injection but were not illuminated, the inventors observed no reduction in IL31-evoked scratching (FIG. 2f). The inventors next tested the effects of photoablation on scratching provoked by other acutely applied pruritogens. Intriguingly, IL31.sup.K138A-SNAP guided photoablation had no significant effect on histamine, chloroquine or LY344864 (a serotonin 5-HT1F receptor agonist) evoked itch (FIG. 2f). Finally, to assess the specificity of photoablation to itch sensation, the inventors examined other sensory modalities after treatment. Using the hot plate test to assay thermal sensitivity (FIG. 2g), and calibrated von Frey filaments to measure mechanical sensitivity (FIG. 2h), the inventors observed no difference in response properties of treated mice, indicating that IL31 guided laser ablation is indeed a selective and effective means of disrupting the itch pathway.
Example 5: IL31.SUP.K138A-SNAP.-IR700 Mediated Photoablation and Chronic Inflammatory Itch
[0105] The inventors examined the effects of IL31.sup.K138A-SNAP-IR700 ablation on inflammatory skin conditions using the well characterized Calcipotriol model of atopic dermatitis (23). To assess the effectiveness of treatment the inventors monitored three indicators of clinical progression; scratching behavior, skin integrity and skin histology. The inventors first determined whether pretreatment with IL31.sup.K138A-SNAP-IR700 would abolish the development of the disease, and then investigated whether post-treatment, upon establishment of robust inflammation, could reverse symptoms.
[0106] As previously reported (M. Li et al., Topical vitamin D3 and low-calcemic analogs induce thymic stromal lymphopoietin in mouse keratinocytes and trigger an atopic dermatitis. Proc Natl Acad Sci USA 103, 11736-11741 (2006)), application of Calcipotriol to skin provoked a severe atopic dermatitis-like phenotype that was evident as a progressive increase in the number of spontaneous scratching bouts over time, distinct skin damage, and a thickening of the epidermis and cell infiltration (not shown). Injection of IL31.sup.K138A-SNAP-IR700 and subsequent near IR illumination of the skin for 3 days prior to Calcipotriol application completely abolished the development of all indicators in this model. Thus scratching behavior remained at baseline levels (FIG. 3a), skin thickness and histological characteristics were not altered (FIGS. 3b and c), skin appeared healthy and typical features of dermatitis such as redness and scaling were entirely absent (FIG. 3d). To control for a pharmacological antagonistic effect of IL31.sup.K138A-SNAP the inventors performed identical experiments in the absence of near IR illumination and observed the normal development of dermatitis-like symptoms (FIGS. 3a-d). Similarly, near IR light and IR700 alone were also ineffective at blocking the progression of the condition (not shown).
[0107] For IL31.sup.K138A-guided photoablation to be developed as a clinical tool, it must also be effective in reversing already established skin inflammation. The inventors therefore treated mice with Calcipotriol for 7 days until severe symptoms were evident. IL31.sup.K138A-SNAP-IR700 was then injected subcutaneously and near IR light applied to the skin for three consecutively days. Strikingly, the inventors observed a rescue of all disease indicators over the course of 1 week. Thus scratching behavior returned to baseline levels (FIG. 3e), and skin thickness (FIG. f), morphology (FIG. 3g)) and structure (FIG. 3h) became indistinguishable from healthy mice. Such profound reversal of dermatitis-like symptoms was not evident in control experiments where IL31.sup.K138A-SNAP was applied without subsequent near IR illumination (FIG. 3e-h).
[0108] Finally, to further improve the clinical applicability of IL31.sup.K138A guided photoablation, the inventors sought to develop a formulation that would allow for topical, pain-free application of .sup.IL31K138A-SNAP-IR700. The inventors selected a water-in-oil micro-emulsion preparation based upon previous evidence that this type of formulation can deliver high molecular weight proteins across the dermal barrier (R. Himes, S. Lee, K. McMenigall, G. J. Russell-Jones, Reduction in inflammation in the footpad of carrageenan treated mice following the topical administration of anti-TNF molecules formulated in a micro-emulsion. J Control Release 145, 210-213 (2010)). IL31.sup.K138A-SNAP-IR700 was loaded into the aqueous phase of the micro-emulsion, applied topically and 20 minutes later, skin was illuminated with near IR light. Similar to subcutaneous delivery, topical application of IL31.sup.K138A-SNAP-IR700 both prevented and reversed Calcipotriol provoked dermatitis-like symptoms. This was evident as a return to baseline levels of scratching behavior (FIGS. 3i and k) and a normalization of skin structure and histology (FIGS. 3j and l). Thus molecule guided delivery of a photosensitizer complex allows for on-demand, pain-free control of chronic itch.
[0109] Finally, photo-ablation guided by the mutated IL31 according to the invention is specific for IL31RA expressing cells and does not affect other cell types such as keratinocytes or epidermal Langerhans cells (FIGS. 3q and r).
Materials and Methods:
Animals
[0110] Wild type or IL31RA knock out (IL31RA.sup./) Black 6/J, 8-10 week-old male mice were used for all behavioral studies. 1-3 day-old mice were used for primary keratinocyte culture. All mice were bred and maintained at the EMBL Mouse Biology Unit, Monterotondo, in accordance with Italian legislation (Art. 9, 27. Jan. 1992, no 116) under license from the Italian Ministry of Health, and in compliance with the ARRIVE guidelines.3
Production of Recombinant IL31.sup.SNAP and IL31.sup.K138A-SNAP cDNAs for murine IL31 and SNAP tag were cloned into pETM11 vector and expressed in E. Coli as fusion protein. To generate the mutant IL31.sup.K138A-SNAP mutagenesis was performed, according to the manufacturer's instruction (Agilent, #200555). The proteins were isolated from inclusion bodies, solubilized, refolded, and eluted using a Ni-NTA resin (Qiagen, #30210). Eluted fractions were then pooled, concentrated and stored for further analysis.
Synthesis of BG-IR700
[0111] 3 mg of IRDye700DX N-hydroxysuccinimide ester fluorophore (LI-COR Biosciences GmbH, Bad Homburg, Germany) were dissolved in 150 ul DMSO and treated with 1.5 mg BG-PEG11-NH.sub.2 and 5 ul diisopropylethylamine. After 1 h, the product BG-PEG11-IRDye700DX was purified by HPLC using a Waters Sunfire Prep C18 OBD 5 M; 19150 mm column using 0.1M triethylammonium acetate (TEAA)] (pH 7.0) and 0.1M TEAA in water/acetonitrile 3:7 (pH 7.0) as mobile phases A and B, respectively. A linear gradient from 100% A to 100% B within 30 minutes was used. The fractions containing the product were lyophilized.
Primary Keratinocyte Culture
[0112] Primary keratinocytes were isolated from 1-3 day-old wild type and IL31RA.sup./ mice as previously described (25). Briefly, newborn mouse skin was removed and incubated flat in cold and freshly thawed trypsin overnight, at 4 C., with the dermis side down. The next day, epidermis was peeled off, triturated and keratinocytes were cultured in serum free media (Invitrogen #10744-019) on Collagen I (Sigma #3867)-coated dishes (Ibidi #81151). All experiments were performed on 48-72 hours cultured cells.
In-Vitro and In Vivo Labeling
[0113] For keratinocyte labelling, 1 M IL31.sup.SNAP or IL31.sup.K138A-SNAP was coupled with 3 M BG549surf (NEB #S9112) for 1 hour at 37 C. in CIB buffer (NaCl 140 mM; KCl 4 mM; CaCl.sub.2 2 mM; MgCl.sub.2 1 mM; NaOH 4.55 mM; Glucose 5 mM; HEPES to mM; pH 7.4). Cells were incubated with the coupling reaction for to minutes at 37 C., then washed 3 times in CIB; and imaged using confocal microscope.
[0114] For skin labelling, intradermal injection of 5 M IL31.sup.SNAP coupled with 5 M BG549surf was performed in the nape of the neck. After to min (30 min) skin was collected, fix in PFA 4% overnight, OCT-embedded and cryo-sectioned (25 m).
In-Vitro Photo-Ablation
[0115] 1 M IL31.sup.SNAP and 3 M BG-IR700 were coupled for 1 hour at 37 C. The coupling reaction was applied for 10 minutes at 37 C. on primary wild type and IL31RA.sup./ keratinocytes. Cells were then exposed to near infra-red light (680 nm) at 40 J/cm.sup.2 for 2 minutes. 24 hours after light exposure cell death was assessed by Propidium iodide (PI) staining (Invitrogen #P3566) and cells were imaged with an epifluorescent microscope.
In-Vivo Photo-Ablation The skin at the nape of the neck of wildtype and IL31RA.sup./ mice was shaved and injected with 5 M IL31.sup.SNAP or IL31.sup.K138A-SNAP coupled to 15 M BG-IR700 in a 50 l volume. 20 minutes after the injection, near infra-red light (680 nm) at 120-150 J/cm.sup.2 or at 550-600 J/cm.sup.2 was applied at the injection site for 4 minutes. This procedure was repeated for 3 consecutively days. For other sensory modalities, the photoablation procedure was performed in the hind paw using a 20 l injection volume. For histological analysis, mice were sacrificed after 3 days after the last illumination; skin was collected, fixed in PFA 4% and paraffin-embedded. 6 m sections were stained for the TUNEL assay, following the manufacturer's instructions (Roche, #156792910).
Microemulsion
[0116] The microemulsion was prepared as already described (24). Briefly, all the components were assembled as follow: Caprylic Triglyceride 81gr; Glyceryl Monocaprylate 27gr; Polysorbate80 12gr; Sorbitan Monooleate 8gr. The microemulsion was mixed with the coupling reaction (IL31.sup.K138A-SNAP+IR700) at 1:1 ratio in 10 l volume with 5 M as IL31.sup.K138A-SNAP final concentration.
SDS-Page and Western Blot
[0117] To assess the coupling reaction, 10 and 20 pmol IL31.sup.SNAP was coupled with 30 and 60 pmol BG549 respectively for 1 hour at 37 C. The coupling reactions were then loaded into a precast acrylamide gel (BioRad #456-9034), along with the same concentrations of IL31.sup.SNAP alone. The bands corresponding to the binding of IL31.sup.SNAP with BG549 were visualized by gel fluorescence. All the samples were visualized by Coomassie staining. Back skin from wild-type mice were injected with vehicle (PBS), 5 M IL31.sup.SNAP or IL31.sup.K138A-SNAP. After 1 hour mice were sacrificed and skin was collected and lysated in Ripa Buffer (Sigma, #R0278) with proteases inhibitor cocktail. Protein lysate was quantified by Bradford assay. 30 g total lysate were separated on 10% SDS-Page gel and transferred to a nitrocellulose membranes (Protran #10600007). Membrane were incubated with the following antibodies, anti STAT3 (Cell Signaling #9139), anti phospho STAT3 (Tyr705) (Cell Signaling #9131), anti MAPK (Cell Signaling #4695), anti phospho MAPK (Thr202/Tyr204) (Cell Signaling #9106), anti AKT (Cell Signaling #4691), anti phospho AKT (Ser473) (Cell Signaling #9271). Bands were visualized using the ECL detection system (Amersham #RPN2106); band density was calculated using ImageJ and the levels of phosphorylated proteins were normalized to the total counterpart.
Atopic Dermatitis Model
[0118] 10 M analogue of the vitamin D3 analogue Calcipotriol (Tocris #2700) was topically applied on the shaved back skin of the mice for 10, 14 or 21 days, depending on the experiment. 0.002% DMSO was applied as vehicle control for the same duration of time. Atopic dermatitis development was assessed by histological analysis of the treated skin by Hematoxylin & Eosin staining, skin thickness measurement using a Caliper and scratching behavior over a 30 minute-recording period.
Scratching Behavior
[0119] To evaluate the scratching response, 8-10 week-old male wild-type or IL31RA.sup./ mice were shaved at the nape of the neck, placed in Plexiglas chambers to acclimatize (30 minutes), videotaped for 30 minutes and scratching bouts were counted. One bout was defined as an event of scratching lasting from when the animal lifted the hind paw to scratch until it returned it to the floor or started licking it. For every experiment, spontaneous scratching referred to as baseline, was measured. To characterize the mutant ligand in terms of itching properties, 5 M IL31.sup.SNAP or IL31.sup.K138A-SNAP were injected for 3 consecutively days and scratching bouts were counted every day. To assess the photoablation effect on the scratching response, 5 M IL31 (Peprotech, #210-31), 10 mM histamine (Sigma, #H7250), 1 mM LY344864 (Abcam, #ab120592) or 12.5 mM Chloroquine (Sigma, #C6628) were injected into the back skin of the mice previously injected with 5 M .sup.IL31.sup.K138A-SNAP+15 M IR700 with or without near IR illumination. The injection of pruritogens was done 3 days after the last illumination treatment. For long-term reversal IL31 evoked scratching, IL31 was injected 1 day after the last day of illumination and then every week for 8 weeks. 10 M Calcipotriol-mediated itch was also considered at different time point to monitor dermatitis development, with or without photoablation.
Von Frey Test
[0120] Mice were habituated on an elevated platform with a mesh floor for 30 minutes. The plantar side of the hind paw was stimulated with calibrated von-Frey filaments (North coast medical #NC12775-99) to assess baseline levels of mechanical sensitivity. The stimulation was then repeated at the 3.sup.rd day after the last photoablation performed on the same paw considered for the baseline. As a control group, the animals were injected but not illuminated. The 50% paw withdrawal thresholds were calculated using the Up-Down method (26).
Hot Plate Test
[0121] Mice were injected for three consecutive days with IL31.sup.K134-SNAP coupled with BG-IR700 with or without near IR light illumination. 3 days after the last injection, mice were placed on top of a hot plate (Ugo Basile #35150) that was preset to 52 C. and the latency to response as distinguished by flicking or licking of the hind paw was observed. In order to avoid injury to the mice, a cutoff of 30 seconds was set.
Statistical Analysis
[0122] All statistical data are presented as Standard error of the mean (SEM) along with the number of samples analysed (n). Student's t-test and/or analysis of variance ANOVA were used; Statistical significance was assumed at p<0.05.
II: PAIN
Example 5: TrkB Positive Neurons are a Subset of Mechanoreceptive Neurons
[0123] Using TrkBCreERT2::Rosa26RFP reporter mice the inventors examined colocalization of TrkB with established cellular markers in adult sensory ganglia. Approximately 10% of dorsal root ganglia (DRG) were positive for TrkBCreERT2, corresponding to the 8% of cells which expressed TrkB mRNA (FIG. 1I). Expression was evident in 2 populations of large neurons marked by NF200 and NF200 plus Ret (FIG. 4A, B, I), and not present in nociceptors positive for CGRP or IB4, or C low threshold mechanoreceptors marked by TH (FIG. 4C-E, I). The inventors further investigated the projections of TrkB neurons to the skin and spinal cord. TrkBCreERT2 fibres extended to Meissner corpuscles in the glabrous skin (FIG. 4F) and formed longitudinal lanceolate endings around hair follicles (FIG. 4G). To assay TrkBCreERT2 positive sensory input into the spinal cord a reporter line was generated in which Cre dependent expression of mCherry was driven from the sensory neuron specific Avil locus. TrkBCreERT2::AvilmCherry positive sensory neurons were present in laminae III/IV of the dorsal horn of the spinal cord where they formed column-like structures extending dorsally (FIG. 4H). The authors also examined expression of TrkB in human tissue using a TrkB antibody. In agreement with mouse data, TrkB immunoreactivity was present in human DRG in large neurons co-expressing NF200 and Ret but largely absent from nociceptors expressing TrkA (FIG. 4i-k, n). Similarly, in glabrous skin, TrkB immunoreactivity was detected in NF200 positive fibers innervating Meissner corpuscles (FIG. 4l). Collectively, these data indicate that TrkBCreERT2 marks a population of putative mechanoreceptive neurons in mouse and human.
[0124] To unequivocally establish the identity of TrkBCreERT2 positive sensory neurons the inventors characterized their response properties utilizing a combination of electrophysiology and optogenetic activation. Mice expressing the light-gated ion channel channel-rhodopsin in TrkB positive cells were generated (TrkBCreERT2::Rosa26ChR2) and an ex vivo skin nerve preparation used to identify neuronal subtypes which could be concomitantly activated by light. Strikingly, the inventors determined that all D-hair and RAMs could be stimulated by light (FIG. 40-q) whereas all other subtypes of sensory neurons were not responsive (FIG. 40-q). Thus TrkB marks myelinated neurons that innervate hair follicles and are tuned to detect gentle moving mechanical stimuli.
Example 6: Ablation of TrkB Positive Neurons Affects Exclusively Light Mechanical Sensation
[0125] To determine the role played by TrkBCreERT2 positive D-hairs and RAMs in sensory evoked behavior, the inventors genetically ablated TrkB neurons in the peripheral nervous system. A Cre-dependent diphtheria toxin receptor transgene knocked-in to the sensory neuron specific Avil locus was generated that allowed for selective deletion of TrkB positive neurons only in adult sensory ganglia. Upon systemic injection of diphtheria toxin a 90% ablation of TrkBCreERT2::AviliDTR and TrkB mRNA positive neurons was achieved with a parallel reduction in the number of NF200 positive neurons by 40% and no change in the expression of other markers (FIG. 5A-C).
[0126] The inventors performed a series of behavioral tests in these animals examining sensory responses to a range of thermal and mechanical stimuli. There was no difference in responses to evaporative cooling evoked by acetone application (FIG. 5D), or in thresholds to noxious heat (FIG. 5E) after diphtheria toxin ablation. Similarly, grip strength (FIG. 5F) was unaltered by ablation of TrkBCreERT2 neurons, as were responses to noxious pinprick (FIG. 5G), and static mechanical stimulation of the hairy skin evoked by application of tape to the back (FIG. 5H). Further examined were the responses to dynamic mechanical stimuli by monitoring responses to brushing of the plantar surface of a paw. Using a puffed out cotton swab which exerts forces in the range 0.7-1.6 mN, the inventors observed a significant reduction in responsiveness upon ablation of TrkB positive neurons (FIG. 5I). Intriguingly, these differences were not apparent upon application of stronger dynamic forces using a paint brush (>4 mN, FIG. 6B, D). Thus under basal conditions, TrkB positive sensory neurons are required for behavioral responses to the lightest of dynamic mechanical stimuli.
Example 7: TrkB Sensory Neurons are Necessary and Sufficient to Convey Mechanical Pain in a Neuropathic Pain Model
[0127] On account of the exquisite sensitivity of TrkB positive neurons, the inventors next asked whether they contribute to mechanical hypersensitivity in models of injury induced pain. The inventors took both a loss of function approach using genetic ablation, and a gain of function approach using optogenetic activation of TrkB neurons. First considered was a model of inflammatory pain by injecting Complete Freund's Adjuvant (CFA) into the plantar surface of the paw, and monitoring responses to von Frey filaments and dynamic brush stimuli. Ablation of TrkB neurons in TrkBCreERT2::AviliDTR mice had no effect on either basal mechanical sensitivity or mechanical hypersensitivity after inflammation (FIG. 6A-B).
[0128] It was further examined whether optogenetic activation of TrkB neurons could evoke pain behavior under inflammatory conditions. Using stimulation parameters which evoked robust firing in the ex vivo skin nerve preparation, the inventors observed no discernible behavioral response to light application to the paw either in basal conditions or after inflammation in TrkBCreERT2::Rosa26ChR2 mice (FIG. 6C). Importantly, identical stimulation conditions applied to the auricle of the ear evoked a brief ear twitch in TrkBCreERT2::Rosa26ChR2 mice (not shown), likely reflecting activation of the dense network of mechanoreceptors in this structure.
[0129] Next neuropathic pain was induced in mice using the Spared Nerve Injury (SNI) model. Control mice developed a profound mechanical hypersensitivity in the sural nerve territory of the paw to both von Frey filaments and dynamic brush stimuli (FIGS. 6D and E). Strikingly, upon ablation of TrkBCreERT2::AviliDTR sensory neurons, mice did not develop mechanical allodynia to either punctate or brushing stimuli, and mechanical sensitivity remained at preinjury levels throughout the observation period. The inventors performed further experiments in TrkBCreERT2::Rosa26ChR2 mice to optogenetically activate these neurons. Three days after injury it was observed that selective stimulation of TrkB neurons with light evoked behavior indicative of pain. This was evident as a prolonged paw withdrawal from the stimulation, lifting of the paw and licking of the illuminated area (FIG. 6F) that continued for several minutes after light application. Such behavior persisted throughout the 2 weeks observation period and was never observed in control mice (FIG. 6F).
[0130] As a neuronal correlate of this apparent pain behavior, the inventors examined induction of the immediate early gene C-fos in the dorsal horn of the spinal cord. In TrkBCreERT2::Rosa26ChR2 mice without injury, optical stimulation evoked C-fos immunoreactivity primarily in laminae III and IV of the spinal cord, the region where TrkB neurons terminate (FIGS. 6G and I). Upon nerve injury however, identical stimulation parameters induced C-fos staining in lamina I of the dorsal horn, an area associated with nociceptive processing. Thus under neuropathic pain conditions, TrkB sensory neurons are necessary and sufficient to convey the light touch signal that evokes pain.
[0131] Example 8: Treatment of Mechanical Allodynia In Vivo
[0132] In light of the clinical importance of mechanical allodynia in neuropathic pain patients, it was sought to develop a pharmacological strategy to exploit the striking selectivity of TrkB to the peripheral neurons which provoke this pain state. It was reasoned that BDNF, the ligand for TrkB, may give access to these neurons and allow for their manipulation in wildtype, non-transgenic animals. To this end the inventors produced a recombinant BDNF protein with a SNAP-tag fused to its C-terminus that would enable its chemical derivatization. BDNF.sup.SNAP was labelled in vitro with fluorescent SNAP-Surface647 substrate and applied to HEK293T cells expressing neurotrophin receptors. Fluorescently labelled BDNF.sup.SNAP displayed remarkable selectivity for its cognate receptor complex TrkB/p75 (FIG. 7A), and did not bind to cells expressing related neurotrophin receptors TrkA/p75 (FIG. 7B) or TrkC/p75 (FIG. 7C).
[0133] The inventors further tested whether BDNF.sup.SNAP would recognize native TrkB receptors in DRG neurons. BDNF.sup.SNAP was conjugated to Qdot 655 quantum dots and applied to dissociated DRG from TrkBCreERT2::Rosa26RFP mice. a >95% overlap between BDNF.sup.SNAP and TrkBCreERT2 positive cells (FIG. 7D) was observed indicating that recombinant BDNF.sup.SNAP is a highly selective means of targeting TrkB neurons.
[0134] To manipulate TrkB neurons in vivo, it was reasoned that BDNF.sup.SNAP may allow for targeted photoablation of these neurons through delivery of a photosensitizing agent. The inventors synthesized a benzylguanine modified derivative of the highly potent near-infrared photosensitizer IRDye700 DX phthalocyanine (IR700) and conjugated it in vitro to BDNF.sup.SNAP. In initial experiments BDNF.sup.SNAP-IR700 was applied to HEK293T cells expressing TrkB/p75 and cell death assayed following near infrared illumination. In cells expressing TrkB/p75 the inventors observed substantial cell death 24 hours after brief illumination (FIG. 7E) that was not evident upon mock transfection or treatment with IR700 alone (FIG. 7F).
[0135] The inventors next sought to assess the therapeutic potential of this approach by investigating the effects of BDNF.sup.SNAP-IR700 mediated photoablation in wildtype mice with neuropathic pain. Upon establishment of robust mechanical allodynia three days after SNI, a range of concentrations of BDNF.sup.SNAP-IR700 was injected into the ipsilateral paw of injured mice and the skin illuminated with different light intensities. Strikingly, the inventors observed a concentration and illumination dependent rescue of both von Frey withdrawal thresholds (FIG. 8a) and dynamic brush or cotton swab evoked allodynia (FIG. 8b and c) that persisted for more than 3 weeks after a single treatment regime. It was examined whether such pronounced effects were also evident in other types of neuropathic pain. Indeed, in both the streptozotocin model of painful diabetic neuropathy, and the paclitaxel model of chemotherapy induced neuropathic pain, a marked reversal of mechanical hypersensitivity that peaked around 10 days post treatment and returned to injury levels by day 20 (FIG. 8d and e) was observed. To determine the selectivity of this approach, the inventors further assessed the effects of BDNF.sup.SNAP-IR700 mediated photoablation on behavioral responses under basal conditions. No deficits in sensitivity to cold, heat, or pinprick upon treatment were observed (FIG. 8f to h). Responses to cotton swab were also unaffected by photoablation (FIG. 3i), perhaps because the skin area that is stimulated in this test extends beyond the zone of illumination.
[0136] To investigate the mechanism by which BDNF.sup.SNAP-IR700 reverses mechanical allodynia, a TrkB.sup.CreERT2::Ros26.sup.SNAPCaaX reporter mouse line was used to identify TrkB positive afferents, and a PGP9.5 antibody to label all fibers, in order to examine the innervation density of hypersensitive skin over the course of phototherapy. Prior to photoablation, TrkB positive lanceolate endings were detected around hair follicles (FIG. 9a) and innervating Meissner corpuscles in the plantar surface of the paw. At 7 days after photoablation (13 days post-SNI) when behavioral reversal of mechanical hypersensitivity was most pronounced, a selective loss of TrkB fibers but persistent innervation by PGP9.5 fibers in hairy and glabrous skin was observed (FIG. 9b, f, g). Indeed, many hair follicles displayed a complete loss of TrkB innervation but still contained PGP9.5 positive circumferential and longitudinal lanceolate endings demonstrating the remarkable specificity of ablation (FIG. 9c). At 24 days post-photoablation when mechanical hypersensitivity had reverted, TrkB positive fibers were again seen innervating their appropriate end organs in both glabrous and hairy skin (FIG. 9d). Importantly, there was no apparent reduction in innervation of control tissue injected with unconjugated IR700 and illuminated (FIG. 9f). It was further investigated whether loss of TrkB.sup.CreERT2 neurons was also evident at the level of the cell soma by analyzing the number of TrkB.sup.CreERT2 positive neurons in the DRG. No differences in the proportion of TrkB neurons 10 days after photoablation were observed (FIG. 9e and f), indicating that the loss of fibers likely reflects local retraction from their peripheral targets.
[0137] TrkB is also expressed by other cells in the skin in addition to sensory fibers. The inventors sought to identify these cell types and determine whether they are lost upon photoablation and contribute to the behavioral phenotype. TrkB was not detected in Merkel cells, keratinocytes, or dendritic and dermal antigen presenting cells, and BDNF.sup.SNAP-IR700 mediated photoablation did not alter their numbers in the skin (FIG. 9g). Expression of TrkB was however evident in cells labelled with CD34, a marker of mast cells and epithelial and endothelial progenitor cells. Moreover, photoablation significantly reduced the number of CD34 positive cells in the skin (FIG. 9g). To determine whether it is loss of these cells or TrkB+ afferents which influences sensory behavior, BDNF.sup.SNAP-IR700 was injected into the sciatic nerve at mid-thigh level and the nerve illuminated to ablate TrkB sensory fibers but spare CD34 cells in the skin. In these animals, behavioral responses to cooling, heating and pinprick were normal (FIG. 9h-j), however, sensitivity to cotton swab was reduced (FIG. 9k), paralleling the results using genetic ablation. It was further investigated whether optogenetically evoked pain behavior in SNI mice is dependent upon CD34 + cells or TrkB+ fibers in the skin. Upon photoablation of TrkB+ fibers in the sciatic nerve a significant reduction in light driven nocifensive behavior in TrkB.sup.CreERT2::Rosa26.sup.ChR2 mice (FIG. 9l) was observed. Thus, TrkB+ sensory afferents, rather than other cells in the skin likely underlie behavioral sensitivity to light touch under basal conditions and after nerve lesion.
[0138] In summary, the results identify the first relay station in the neuronal pathway that confers pain from gentle touch under neuropathic pain states. It was demonstrated that TrkBCreERT2 positive sensory neurons detect the lightest touch under basal conditions but after nerve injury are both necessary and sufficient to drive mechanical allodynia.
[0139] The invention further describes a new technology based upon ligand mediated delivery of a phototoxic agent to target these neurons and reverse mechanical hypersensitivity in neuropathic pain states. This approach is analogous to clinically approved capsaicin patches, in which a high concentration of capsaicin is applied to the skin and leads to retraction of nociceptive fibers. Instead, here the invention targets directly the neurons responsible for mechanical allodynia, allowing for local, on demand treatment of pain through application of light.
Example 9: Treatment of Inflammatory Pain using NGF to Target Nociceptive Sensory Neurons
[0140] A similar experiment as with BDNF was performed with nerve growth factor (NGF). The reasoning here was that TrkA, the receptor for NGF is expressed exclusively by nociceptive sensory neurons, thus their ablation should allow for treatment of acute and inflammatory pain. In initial experiments, a recombinant NGF.sup.SNAP protein was produced and shown to bind to TrkA positive cells and not TrkB or TrkC. NGF.sup.SNAP was conjugated to IR700 and injected into the paw of mice which was then illuminated with near IR light in the treatment group, while a control group received no illumination. Complete Freund's adjuvant (CFA) was injected into the paw, and responses to thermal stimuli monitored for a period of 50 days. In the control group NGF itself produced a robust thermal hyperalgesia which was further increased by CFA injections. In animals illuminated with near IR light, thermal hyperalgesia did not develop (FIG. 10a). In a further experiment, CFA was injected first and then the paw was subjected to NGF.sup.SNAP-IR700 mediated photoablation and mechanical hypersensitivity was monitored. In control animals which received no illumination, robust mechanical hypersensitivity developed which was maintained throughout the 25 day observation period. In animals which received near IR light, and substantial reduction in mechanical withdrawal thresholds was observed and mechanical sensitivity returned to baseline levels (FIG. 10b).
[0141] A conceptual problem with using NGF.sup.SNAP as a means of targeting TrkA positive nociceptors is that it in itself evokes pain and sensitization. To circumvent this problem, the authors generated an engineered NGF.sup.SNAP with Arginine mutated to Tryptophan at position 121 (NGF.sup.R121W-SNAP). This molecule was found to bind specifically to Hek293 cells expressing TrkA and p75 receptors (FIG. 10c) and to evoke cell death when conjugated to IR700 and applied to these cells and illuminated (FIG. 10d). Importantly, when NGF.sup.R121W-SNAP was injected into the paw of mice it did not provoke mechanical hypersensitivity, while wildtype NGF.sup.SNAP had a strong sensitizing effect (FIG. 10e). Thus NGF.sup.R121W-SNAP is a painless NGF derivative that binds to TrkA receptors but does not activate pain signaling pathways.
[0142] To determine whether NGF.sup.R121W-SNAP can be used as a photosensitizing agent to control pain, the authors conjugated it to IR700 and injected it into skin for subsequent near IR light illumination. Acute behavioral responses were first examined. NGF.sup.R121W-SNAP mediated photoablation was found to significantly elevate painful mechanical withdrawal latencies to von Frey filaments, while non-illuminated controls showed no change (FIG. 11a). Similarly, responses to painful pinprick were reduced by photoablation (FIG. 11b) while non-nociceptive responses to brush were not affected (FIG. 11c). Finally, the authors examined the efficacy of photoablation under inflammatory pain conditions. Using the CFA model of inflammatory pain, it was found the NGF.sup.R121W-SNAP-IR700 mediated photoablation led to prolonged recovery of both thermal hyperalgesia (FIG. 11d) and mechanical hypersensitivity (FIG. 11e) in this model. Thus NGF.sup.R121W-SNAP can be used to control acute nociceptive pain and hypersensitivity that results from an inflammatory stimulus.
