WO2021191094A1

WO2021191094A1 - Erbb-targeted therapies for neuropathic pain

Info

Publication number: WO2021191094A1
Application number: PCT/EP2021/057166
Authority: WO
Inventors: Susan Fleetwood-Walker; Rory Mitchell
Original assignee: University Court Of The University Of Edinburgh; Merck Patent Gmbh
Priority date: 2020-03-24
Filing date: 2021-03-22
Publication date: 2021-09-30

Abstract

The invention relates to pharmaceutical combinations of ErbB1 and ErbB2 inhibitors, and the use of such combinations for the treatment of neuropathic pain.

Description

ErbB-Targeted Therapies for Neuropathic Pain

Background of the invention

Chronic and/or neuropathic pain after nerve injury is a major health problem worldwide. Neuropathic pain (NP) is caused by a primary lesion or disease of the somatosensory system, often in its peripheral aspects (Jensen, Baron, Haanpaa, et al. (2011) Pain, 152:2204-5). Not uncommonly, its severity, chronicity and the poor side-effect to benefit ratio of current pharmacotherapy for NP (Dworkin. (2002) Clin J Pain, 18:343-9; Finnerup, Sindrup, Jensen. (2010) Pain, 150:573-81 ) lead to severely impaired physical and psychological functioning among sufferers (Jensen, Chodroff, Dworkin. (2007) Neurology, 68:1178-82). In the general population, the incidence of chronic NP is estimated to be almost 7%, with more than 5% experiencing moderate to severe pain and distress (Bouhassira, Lanteri-Minet, Attal, Laurent, Touboul. (2008) Pain, 136:380-7), making it a common and formidable health problem worldwide.

Neuropathic pain is a complex, chronic pain state involving damage, injury or dysfunction in sensory nerve fibers. These damaged nerve fibers send incorrect signals to other pain centers. The impact of nerve fiber injury includes a change in nerve function both at the site of injury and areas around the injury. Some neuropathic pain studies suggest that the use of non-steroidal anti-inflammatory drugs, such as Aleve (naproxen) or Motrin (ibuprofen), may ease pain, although analgesic efficacy is very limited. Some patients may require a stronger painkiller, such as those containing morphine or other opioids, but these show limited efficacy and frequently treatment-limiting side-effects (Finnerup, Sindrup, Jensen. (2010) Pain, 150:573-581 ). Anticonvulsant and antidepressant drugs similarly show modest efficacy and often intolerable side-effects (Finnerup, Sindrup, Jensen. (2010) Pain, 150:573-581 ).

In cases that are difficult to treat, a pain specialist may use invasive or implantable device therapies to manage the pain. Electrical stimulation of the nerves involved in neuropathic pain generation may produce limited control of the pain symptoms. Overall, neuropathic pain often responds poorly to standard pain treatments and as a result of sensitisation in pain pathways may get worse instead of better over time. For some people, it can lead to serious disability and limited quality of life. Current treatments are characterized by a very unsatisfactory side- effect to benefit ratio; identifying a major unmet need.

Recent progress in the field of neuropathic pain has been spurred by an observation of remarkable pain relief in a rectal cancer patient treated with the anti-epidermal growth factor receptor (EGFR, ErbB1) antibody, cetuximab, despite tumour progression (Kersten, Cameron. (2012) BMJ Case Rep.; Kersten, Cameron, Mjaland. (2013) Scand J Pain, 4:3-7).

The EGFR (ErbB1 ) is one of a family of 4 receptor tyrosine kinases (RTKs), which display sequence homology and a common structural organisation (Olayioye, Neve, Lane, Hynes. (2000) EMBO J.,19(13):3159-3167). ErbB2 is thought to lack a physiological ligand yet act as the preferred heterodimerisation partner for all other ErbB receptors (Graus-Porta, Beerli et al. (1997) EMBO J., 16(7): 1647-1655; Tzahar, Pinkas-Kramarski et al. EMBO J.,16(16):4938-4950) and can enable constitutive functional activity (Garrett, McKern et al. (2003) Mol Cell, 11 (2):495-505), while ErbB3 displays minimal levels of tyrosine kinase catalytic activity (around 1000-fold less than ErbB1) (Shi, Telesco et al. (2010) Proc Natl Acad Sci U S A, 107(17):7692-7697).

Kersten et al. (2015) could observe effects similar to what they found with cetuximab also with panitumumab (a further ErbB1 -neutralising antibody) and the small molecule EGFR inhibitors gefitinib and erlotinib (Kersten, Cameron et al. (2015) Br J Anaesth.;115(5):761- 76; WO2013005108; WO2014095088).

However, since an approved therapy based on these approaches is not available at present, there is still a need to develop further therapies for neuropathic pain.

Description of the invention

The inventors have found out that, surprisingly, the combination of an ErbB1 and an ErbB2 inhibitor acts in a synergistic way in the treatment of neuropathic pain. Therefore, the present invention relates to pharmaceutical combinations of ErbB1 and ErbB2 inhibitors, and the use of such combinations for the treatment of neuropathic pain.

Specifically, the ErbB1 inhibitor and/or the ErbB2 inhibitor can be a small molecule, or physiologically acceptable salts thereof, or an antibody, or antigen binding fragments thereof. More precisely, the ErbB1 inhibitor can be a small molecule, or a physiologically acceptable salt thereof, and the ErbB2 inhibitor can be an antibody, or an antigen binding fragment thereof, or vice versa, or both inhibitors can either be a small molecule, or a physiologically acceptable salt thereof, or an antibody, or an antigen binding fragment thereof.

Preferably, the ErbB1 inhibitor is erlotinib, gefitinib, AG 1478, falnidamol, EGFRi 324674, cetuximab, panitumumab or other highly selective ErbB1 -inhibiting agents. More preferably, the ErbB1 inhibitor is erlotinib or falnidamol.

Preferably, the ErbB2 inhibitor is mubritinib, tucatinib, trastuzumab or other highly selective ErbB2-inhibiting agents. More preferably, the ErbB2 inhibitor is mubritinib, or tucatinib.

Alternatively, the ErbB1 inhibitor and the ErbB2 inhibitor can be one molecule, i.e. a dual ErbB1/ErbB2 inhibitor, which again can be a small molecule, or physiologically acceptable salts thereof, wherein the same active principle acts on both targets, or the small molecule may have two moieties each harbouring a distinct active principles, which moieties may be connected by a linker. Or the dual ErbB1/ErbB2 inhibitor is a bispecific antibody, or antigen binding fragments thereof, or other fusion proteins having two different binding specificities.

If separate molecules, the two inhibitors can be administered simultaneously or sequentially. When administered simultaneously, the two inhibitors may be administered in one pharmaceutical composition or as separate pharmaceutical compositions. When administered sequentially, in one embodiment the ErbB1 inhibitor is administered first. In another embodiment, the ErbB2 inhibitor is administered first. The present invention relates in particular to a method for the treatment of neuropathic pain, comprising administering to a subject an ErbB1 inhibitor and an ErbB2 inhibitor, or a physiologically acceptable salt, or an antigen binding fragment each thereof, as described above and below.

In an embodiment of the treatment, the ErbB1 inhibition is effected by the blockage of the ErbB1 ligand binding domain or the inhibition of the ErbB1 kinase domain or down- regulation of ErbB1 expression at the cell surface.

In another embodiment of the treatment, the ErbB2 inhibition is effected by the blockage of the ErbB2 ligand binding domain or the inhibition of the ErbB2 kinase domain or down-regulation of ErbB2 expression at the cell surface.

In yet another embodiment of the treatment, the ErbB1 and ErbB2 inhibition is effected by the blockage of the ErbB1/ErbB2 dimerization.

In yet another embodiment of the treatment, the ErbB1 inhibitor and the ErbB2 inhibitor are administered simultaneously, or sequentially.

In yet another embodiment of the treatment, the ErbB1 and ErbB2 inhibitory functionalities are combined in one and the same molecule (dual ErbB1/ErbB2 inhibitor, for example: lapatinib, afatinib, sapatinib, epertinib, poziotinib, allitinib, pyrotinib, canertinib, varlitinib, tesevatinib or dacomitinib. In a specific embodiment, the dual ErbB1/ErbB2 inhibitor is lapatinib or afatinib.

In yet another embodiment of the treatment, the neuropathic pain is cancer related, non cancer related, non-compressive, compressive, toxic, metabolic, traumatic, autoimmune, infectious, and congenital or hereditary neuropathic pain.

In another aspect, the present invention relates to a pharmaceutical combination comprising an inhibitor of ErbB1 and an inhibitor of ErbB2, or a dual ErbB1/ErbB2 inhibitor, as described above. In an further aspect, the present invention relates to a pharmaceutical composition, comprising a pharmaceutical combination, or a dual ErbB1/ErbB2 inhibitor as described above. In the case of a small molecule ErbB1 and/or ErbB2 inhibitor, or a dual ErbB1/ErbB2 inhibitor, such small molecule may also be a physiologically acceptable salt, solvates, or solvate of a salt of the said inhibitor. Suitable salts are inorganic or organic salts of all physiologically or pharmacologically acceptable acids, for example halides, in particular hydrochlorides or hydrobromides, lactates, sulfates, citrates, tartrates, maleates, fumarates, oxalates, acetates, phosphates, methylsulfonates, benzoates or p-toluenesulfonates.

The pharmaceutical composition will typically further comprise adjuvants, which may be organic or inorganic substances suitable for enteral (for example oral), parenteral (for example intravenous) or topical administration and do not react with the ErbB1 and 2 inhibitors, for example water, vegetable oils, benzyl alcohols, polyethylene glycols, gelatine, carbohydrates, such as lactose, sugar alcohols or starch, magnesium stearate, talc, or Vaseline.

The pharmaceutical composition may further comprise adjuvants, such as lubricants, preservatives, stabilisers and/or wetting agents, emulsifiers, salts for modifying the osmotic pressure, buffer substances, dyes, flavours and/or aroma substances.

In another aspect, the invention also relates to a set (kit) consisting of separate packs of (a) an effective amount of an ErbB1 inhibitor as described above and below, or a physiologically acceptable salt, or an antigen binding fragment thereof, and (b) an effective amount of an ErbB2 inhibitor as described above and below, or a physiologically acceptable salt, or an antigen binding fragment thereof.

The set comprises suitable containers, such as boxes, individual bottles, bags, ampoules or blister packages.

The pharmaceutical combinations of ErbB1 and ErbB2 inhibitors according to the invention can be adapted for administration via any desired suitable method, for example by oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) methods. Such medicaments can be prepared using all processes known in the pharmaceutical art by, for example, combining the active ingredient with the excipient(s) or adjuvant(s).

Compounds and compound mixtures adapted for oral administration can be administered as separate units, such as, for example, capsules or tablets; powders or granules; solutions or suspensions in aqueous or non-aqueous liquids; edible foams or foam foods; or oil-in-water liquid emulsions or water-in-oil liquid emulsions.

Thus, for example, in the case of oral administration in the form of a tablet or capsule, the compound or compound mixtures can be combined with an oral, non-toxic and pharmaceutically acceptable inert excipient, such as, for example, ethanol, glycerol, water and the like. Powders are prepared by comminuting the compound to a suitable fine size and mixing it with a pharmaceutical excipient comminuted in a similar manner, such as, for example, an edible carbohydrate, such as, for example, starch or mannitol. A flavour, preservative, dispersant and dye may likewise be present.

Capsules are produced by preparing a powder mixture as described above and filling shaped gelatine shells therewith. Glidants and lubricants, such as, for example, highly disperse silicic acid, talc, magnesium stearate, calcium stearate or polyethylene glycol in solid form, can be added to the powder mixture before the filling operation. A disintegrant or solubiliser, such as, for example, agar-agar, calcium carbonate or sodium carbonate, may likewise be added in order to improve the availability of the compound or compound mixtures after the capsule has been taken.

In addition, if desired or necessary, suitable binders, lubricants and disintegrants as well as dyes can likewise be incorporated into the mixture. Suitable binders include starch, gelatine, natural sugars, such as, for example, glucose or beta-lactose, sweeteners made from maize, natural and synthetic rubber, such as, for example, acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes, and the like. The lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like. The disintegrants include, without being restricted thereto, starch, methylcellulose, agar, bentonite, xanthan gum and the like. The tablets are formulated by, for example, preparing a powder mixture, granulating or dry-pressing the mixture, adding a lubricant and a disintegrant and pressing the entire mixture to give tablets. A powder mixture is prepared by mixing the compound comminuted in a suitable manner with a diluent or a base, as described above, and optionally with a binder, such as, for example, carboxymethylcellulose, an alginate, gelatine or polyvinylpyrrolidone, a dissolution retardant, such as, for example, paraffin, an absorption accelerator, such as, for example, a quaternary salt, and/or an absorbant, such as, for example, bentonite, kaolin or dicalcium phosphate. The powder mixture can be granulated by wetting it with a binder, such as, for example, syrup, starch paste, acadia mucilage or solutions of cellulose or polymer materials and pressing it through a sieve. As an alternative to granulation, the powder mixture can be run through a tableting machine, giving lumps of non-uniform shape which are broken up to form granules. The granules can be lubricated by addition of stearic acid, a stearate salt, talc or mineral oil in order to prevent sticking to the tablet casting moulds. The lubricated mixture is then pressed to give tablets. The compounds and compound mixtures according to the invention can also be combined with a free- flowing inert excipient and then pressed directly to give tablets without carrying out the granulation or dry-pressing steps. A transparent or opaque protective layer consisting of a shellac sealing layer, a layer of sugar or polymer material and a gloss layer of wax may be present. Dyes can be added to these coatings in order to be able to differentiate between different dosage units.

Oral liquids, such as, for example, solution, syrups and elixirs, can be prepared in the form of dosage units so that a given quantity comprises a prespecified amount of the compound. Syrups can be prepared by dissolving the compounds and compound mixtures in an aqueous solution with a suitable flavour, while elixirs are prepared using a non-toxic alcoholic vehicle. Suspensions can be formulated by dispersion of the compound in a non-toxic vehicle. Solubilisers and emulsifiers, such as, for example, ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavour additives, such as, for example, peppermint oil, or natural sweeteners or saccharin or other artificial sweeteners, and the like, can likewise be added.

The dosage unit formulations for oral administration can, if desired, be encapsulated in microcapsules. The formulation can also be prepared in such a way that the release is extended or retarded, such as, for example, by coating or embedding of particulate material in polymers, wax and the like.

Pharmaceutical combinations adapted for topical administration can be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils.

Pharmaceutical combinations adapted for rectal administration can be administered in the form of suppositories or enemas. Compounds and compound mixtures adapted for nasal administration in which the carrier substance is a solid comprise a coarse powder having a particle size, for example, in the range 20-500 microns, which is administered in the manner in which snuff is taken, i.e. by rapid inhalation via the nasal passages from a container containing the powder held close to the nose. Suitable formulations for administration as nasal spray or nose drops with a liquid as carrier substance encompass active-ingredient solutions in water or oil. Pharmaceutical combinations adapted for administration by inhalation encompass finely particulate dusts or mists, which can be generated by various types of pressurised dispensers with aerosols, nebulisers or insufflators. Pharmaceutical combinations adapted for vaginal administration can be administered as pessaries, tampons, creams, gels, pastes, foams or spray formulations. Pharmaceutical combinations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions comprising antioxidants, buffers, bacteriostatics and solutes, by means of which the formulation is rendered isotonic with the blood of the recipient to be treated; and aqueous and non-aqueous sterile suspensions, which may comprise suspension media and thickeners. The formulations can be administered in single-dose or multidose containers, for example sealed ampoules and vials, and stored in freeze-dried (lyophilised) state, so that only the addition of the sterile carrier liquid, for example water for injection purposes, immediately before use is necessary. Injection solutions and suspensions prepared in accordance with the recipe can be prepared from sterile powders, granules and tablets.

A therapeutically effective amount of a compound or compound mixture of the present invention depends on a number of factors, including, for example, the age and weight of the recipient, the precise condition that requires treatment, and its severity, the nature of the formulation and the method of administration, and is ultimately determined by the treating doctor or vet. However, an effective amount of an ErbB1 or 2 inhibitor for the treatment of the diseases according to the invention is generally in the range from 0.1 to 100 mg/kg of body weight of the recipient (mammal) per day and particularly typically in the range from 1 to 10 mg/kg of body weight per day. Thus, the actual amount per day for an adult mammal weighing 70 kg is usually between 70 and 700 mg, where this amount can be administered as an individual dose per day or more usually in a series of part-doses (such as, for example, two, three, four, five or six) per day, so that the total daily dose is the same. An effective amount of a salt or solvate or of a physiologically functional derivative thereof can be determined as a fraction of the effective amount of the compounds and compound mixtures according to the invention per se.

Examples

Example A: Pharmaceutical compositions.

AT. Injection vials.

A solution of 100 mg of an antibody ErB1 or 2 inhibitor according to the invention and 6 mg of disodium hydrogenphosphate in 10 mL of bidistilled water is adjusted to pH 6.5 using hydrochloric acid, sterile filtered, transferred into an injection vial, lyophilised and sealed under sterile conditions.

A2: Tablets.

1 g of a compound or a compound mixture according to the invention, 4 g of lactose, 1.2 g of potato starch, 0.2 g of talc and 100 mg of magnesium stearate is pressed to give tablets in a conventional manner in such a way that each tablet contains 10 mg of active ingredients.

Example B: ErbB1/ErbB2 combination treatment

B1 : Methods and Materials

B1.1: CCI and CIPN neuropathic pain models.

Animals were housed under a 12h light-dark cycle and given access to food and water ad libitum. Most experiments were carried out on adult Sprague-Dawley rats (200-350g in weight, which were male unless otherwise indicated). The sciatic nerve chronic constriction injury (CCI) preparation was used as a model of chronic neuropathic pain in rats (Bennett and Xie (1988) Pain, 33(1 ):87-107). Animals were anaesthetised with a 4% isoflurane/oxygen mixture (Zeneca, Cheshire, UK) before exposure of the sciatic nerve, proximal to the trifurcation at mid-thigh level. Four loose ligatures of chromic catgut (SMI AG, Hunningen, Belgium) were tied around the nerve, separated by 1 mm. CCI rats consistently developed ipsilateral thermal reflex hypersensitivity, which peaked between days 8 and 12 post-surgery, when experiments were carried out. Some experiments were carried out in adult male C57/BI6J mice (25-35g in weight), using an oxaliplatin model of chemotherapy-induced peripheral neuropathy (CIPN) (Zhao, Isami et al. (2012) Mol Pain, 8:55). Oxaliplatin (Abeam) in 5% glucose vehicle was injected intraperitoneally (under light isoflurane anaesthesia) at a dose of 7mg/kg (IOOmI/mouse) and experiments carried out 3-7days later when mechanical hypersensitivity was fully developed.

B1.2: Quantitative sensory testing in vivo.

Mechanical and thermal nociceptive sensitivity were assessed using standard behavioural tests, during which, the investigator was blinded to drug treatment. Mechanical nociception was assessed as Paw Withdrawal Threshold (PWT; in g) from force-calibrated von Frey nylon filaments (Stoelting, Illinois). Nociceptive heat sensitivity was measured as Paw Withdrawal Latency (PWL; in sec) using Hargreaves’ infrared apparatus (Linton Instrumentation), set to a maximum temperature of 52°C and a cut-off time of 20sec. Testing was always separated by at least 5min to avoid sensitisation of responses. Nociceptive cold sensitivity was measured as Suspended Paw Elevation Time using a shallow water bath at the temperature of iced water (~4°C) (Garry, Moss et al. (2003) Curr. Biol., 13(4):321-328). Animals were initially habituated to the sensory testing environments to establish consistent responses.

B1.3: Drug administration in vivo.

Small molecule agents were obtained from MedChem Express, Selleckchem, Key Organics or Axon MedChem. Murine EGF, purified from submaxilliary gland and recombinant human neuregulinl-bΐ EGF domain (NRG1 ) were obtained from Sigma- Aldrich and R & D Systems, respectively. For intraperitoneal administration (200pl/rat and IOOmI/mouse) agents were dissolved in 10% dimethylsulphoxide, 40% polyethylene glycol-400 and 50% propylene glycol. For intraplantar administration (50pl/rat) agents were dissolved in normal saline with 0.3% dimethylsulphoxide. Vehicle controls showed no discernible change in pain-associated behavioural responses. Doses of pharmacological agents were selected on the basis of those showing clear efficacy/target coverage (producing at least 80-90% maximal effects) in literature reports, wherever possible, or their reported potency in vitro compared to that of the benchmark, erlotinib.

B1.4: Immunofluorescence Histochemistry.

DRGs from spinal segments L4-6 were dissected, rapidly embedded in OCT cryo- sectioning medium (Thermo Scientific) and frozen on dry ice. 18pm sections were cut by cryostat and mounted onto poly-L-lysine coated slides (Thermo Scientific). Sections were washed in Tris-Buffered Saline (TBS) pH7.60 and then incubated for 1 h at room temperature in blocking buffer (10% normal donkey serum, 4% fish skin gelatin, 0.2% Triton X-100 in TBS) prior to overnight incubation at 4°C with a combination of primary antibodies in buffer (4% normal donkey serum, 4% fish skin gelatin and 0.2% Triton X- 100 in TBS). The primary antibodies used were al documented to show specificity for their target and in the case of ErbB receptor antibodies to lack cross-reactivity with other ErbB family members. Sections were washed in TBS and incubated for 1 h at room temperature with a combination of extensively cross-adsorbed, fluorescent secondary antibodies or Alexa Fluor-488 conjugated isolectin IB4 (Invitrogen; 121411 ; 1 :3000) in buffer (4% normal donkey serum, 4% fish skin gelatin in TBS). Donkey anti-mouse, rabbit, chicken or guinea pig secondary antibodies, labelled with 633/647, 568, 488 or 405 nm-emitting fluorophores in appropriate permutations, and all highly cross-adsorbed against other relevant species, were obtained from Biotium via Sigma-Aldrich and were used at a dilution of 1 :600. Sections were washed three further times in TBS and then mounted in ProLong® Gold Antifade (Life Technologies). Standard primary antibody omission or blocking peptide controls, wherever possible, confirmed that non-specific staining was minimal. Both concentration-dependence of staining and association with peripherin- positive or NF-200-positive cellular profiles where appropriate corroborated specificity.

B1.5: Proximity Ligation Assay.

Evidence for in situ protein:protein interactions was obtained using the Duolink Proximity Ligation Assay (Sigma-Aldrich) (Fredriksson, Gullberg et al. (2002) Nat Biotechnol., 20(5):473-477, Soderberg, Gullberg et al. (2006) Nat Methods, 3(12):995-1000, Liu, Chen et al. (2013) Mol Cell Proteomics, 12(5): 1335-1349, Rivera-Oliver, Moreno et al. (2019) Mol. Neurobiol., 56(2):797-811 ). The primary antibodies for potential protein partners used were ab30 (Abeam, mouse monoclonal anti-EGFR1 antibody, binding to the ErbB1 EGF-binding domain, dilution 1 :1000, no cross reaction with ErbB2, 3, 4) and 06-562 (Merck Millipore, rabbit polyclonal anti ErbB2 antibody, dilution 1 :1000, no cross reaction with ErbB1 , 3, 4). Oligonucleotide-derivatised secondary antibody probes were donkey anti-rabbit PLUS and donkey anti-mouse MINUS. Probe ligation, rolling circle amplification (RCA) and detection of the RCA product by Duolink Orange fluorophore (emission 576nm) were all carried out according to the manufacturer’s instructions. An optimised procedure was developed to integrate conventional immunofluorescence counterstaining (with chicken anti-peripherin and donkey anti-chicken 488) into the protocol.

B1.6: Non-linear curve-fitting and statistical analysis.

All data analysis was carried out using GraphPad Prism. Non-linear curve-fitting used a sigmoidal dose-response (variable slope) model. Data in two-group format were analysed statistically by Student’s t-test. Comparisons between more than two groups were made by One-Way ANOVA with Tukey’s or Dunnett’s test, or by Two-Way ANOVA with Bonferroni’s test.

B2: Experimental Evidence

B2.1: ErbB1 involvement in hypersensitive pain behaviours and nociceptor activation following nerve injury.

Using the CCI neuropathic pain model, marked nociceptive hypersensitivity was observed in the ipsilateral (but not contralateral) hindlimb 8-12days following nerve injury. This was manifest as a reduction in Paw Withdrawal Threshold (PWT) from von Frey filament mechanical stimuli (Fig. 1A), a reduction in Paw Withdrawal Latency (PWL) from Hargreaves’ noxious thermal stimuli (Fig. 1 B) and an increase in suspended paw elevation time from 4°C noxious cold stimuli (Fig. 1 C). In each case, intraperitoneal administration of the highly selective inhibitor of ErbB1 tyrosine kinase function, erlotinib (Moyer, Barbacci et al. (1997) Cancer Res., 57(21 ):4838-4848) at a dose of 10mg/kg ip, produced marked reversal of ipsilateral hypersensitivity, which was complete at peak effect and remained statistically significant for 140-160m in. No discernible effects were observed on responses from the contralateral limb or from vehicle injection. This time course is consistent with evidence that plasma levels of erlotinib increase rapidly after administration and began to decline steeply after around 2 hours (Pollack, Savage et al. (1999) J. Pharmacol Exp. Then, 291 (2):739-748). The reversal of CCI-induced mechanical hypersensitivity by erlotinib was dose-dependent (with a statistically significant effect at as little as 1 mg/kg and an IC50 of 1 .4 [1 .3/1 .5] mg/kg ip (mean [95% confidence interval]), (see Table 1 below). This effect was also replicated by further highly selective ErbB1 inhibitors, gefitinib (Moasser, Basso et al. (2001 ) Cancer Res., 61 (19):7184-7188, Pedersen, Pedersen et al. (2005) Br. J. Cancer 93(8):915-923) and AG 1478 (Traxler, Green et al. (1999) J. Med. Chem., 42(6): 1018-1026), with effects similar to erlotinib at corresponding doses (see Table 1 below). Reversal of nerve injury- induced mechanical hypersensitivity by erlotinib was similarly observed in female CCI rats (mean reversal over 20-140min post-injection of 88.9+9.2%, n=4, p<0.01 ).

As most selective ErbB1 inhibitors are based around a common quinazoline core, two further, structurally distinct selective agents, falnidamol (BIBX 3182) (Solca, Baum et al. (2004) J. Pharmacol. Exp. Then, 311(2):502-509) and EGFRi 324674 (Zhang, Liu et al. (2006) J. Am. Chem. Soc., 128(7):2182-2183) were tested. These contain distinct pyrimidopyrimidine and 4,6-substituted-pyrimidine cores, respectively and at appropriate doses they also produced robust reversal of hypersensitivity through 20-140m in post administration, respectively (n=4, in each case), confirming target-specific efficacy (see Table 1 below). Similar results (demonstrating dose-dependence in each case) were observed in a mouse model of oxaliplatin-induced peripheral neuropathy (Zhao, Isami et al. (2012) Mol. Pain, 8:55), with IC50 values [95% Cl] of 3.2 [3.0/3.3] mg/kg ip for erlotinib, 2.4 [2.3/2.6] mg/kg ip for gefitinib and 3.0 [2.7/3.4] mg/kg ip for AG 1478 (n=4 at 4-5 different doses in each case). The doses of erlotinib producing analgesia in the neuropathic pain models here are similar to, or less than, those necessary to reverse EGF-induced autophosphorylation of ErbB1 in liver and tumour xenografts of athymic mice, consistent with appropriate target-associated potency and coverage (Moyer, Barbacci et al. (1997) Cancer Res., 57(21 ):4838-4848; Pollack, Savage et al. (1999) J. Pharmacol. Exp. Then, 291(2):739-748). Repeated testing with erlotinib (10mg/kg ip) showed no significant attenuation of its ability to reverse CIPN-induced hypersensitivity over 7 daily treatments, similar to observations in the CCD (chronic DRG compression) model (Wang, Liu et al. (2019) Mol. Pain, 15:1744806919857297). In order to provide an unbiased biomarker of activity in nociceptive afferents levels of phospho-CaMkinase I la (Thr286) were measured in DRG cells. This marker reflects Ca²⁺- dependent autophosphorylation of the enzyme to an autonomous form of the enzyme, but both activity and Thr286-phosphorylation diminish rapidly, within minutes, following stimulus removal (Lou, Lloyd et al. (1986) Proc. Natl. Acad. Sci. U S A, 83(24):9497-9501 ; Schworer, Colbran et al. (1986) J. Biol. Chem., 261 (19):8581 -8584; Lengyel, Voss et al. (2004) Eur. J. Neurosci., 20(11):3063-3072; Chang, Parra-Bueno et al. (2017) Neuron, 94(4):800-808 e804). Figure 1D shows that phospho-CaMKII (Thr286) staining in small, peripherin-positive DRG cells was significantly increased ipsilateral to CCI and that this was almost completely reversed by erlotinib (1 Omg/kg ip, 1 hr), indicating that CCI-induced activity in nociceptors is highly dependent on ErbB1 functional activity.

Table 1 : Effects of intraperitoneal injection of a variety of highly selective ErbB 1 inhibitors on CCI-induced hypersensitivity in the von Frey Paw Withdrawal Threshold test. Experiments were carried out in animals that displayed marked ipsilateral hypersensitivity to von Frey filaments following CCI carried out 8-12days previously. Drugs were injected (in 0.2ml per animal) under light isoflurane anaesthesia and after a 20min delay for recovery, Paw Withdrawal Threshold (PWT) testing was carried out at 20m in intervals up to 3hr post-injection. The percentage reversal of hypersensitivity (ipsilateral compared to contralateral pre-drug PWT) was meaned over 20-140min post-drug administration. A range of highly selective ErbB1 inhibitors showed robust reversal of hypersensitivity, displaying both concentration-dependence and efficacy from structurally diverse agents. ^** indicates significant reversal of hypersensitivity (One-Way ANOVA with Dunnett’s post- hoc test or Student’s t-test, as appropriate). No significant changes in contralateral PWT were observed for any of the drugs or on either side following vehicle.

B2.2: ErbB1 is selectively localised in small, peripherin-positive DRG neurons.

As there was some disparity in previous reports over ErbB1 expression in subpopulations of DRG cells (Huerta, Diaz-Trelles et al. (1996) Anat. Embryol. (Berl.), 194(3):253-257; Xian and Zhou (1999) Exp. Neurol., 157(2):317-326; Andres, Meyer et al. (2010) Mol. Pain, 6: 98; Martin, Smith et al. (2017) J. Clin. Invest., 127(9):3353-3366; Wang, Liu et al. (2019) Mol. Pain, 15:1744806919857297), extensively characterised ErbB1 -specific antibodies were used to address this. Figure 2A shows results using two antibodies documented to label ErbB1 , but not other ErbB family members, revealing very high correlation between staining for ErbB1 and peripherin in small DRG cells that are likely to represent unmyelinated nociceptors (Goldstein, House et al. (1991) J. Neurosci. Res., 30(1 ):92-104; Fornaro, Lee et al. (2008) Neuroscience 153(4): 1153-1163). In DRG from naive and CCI animals similarly, both ErbB1 -specific antibodies stained 80-90% of peripherin-positive small cells with minimal staining outwith this population. A third ErbB1 antibody (mouse monoclonal 8G6.2); raised against a distinct intracellular epitope in ErbB1 ), confirmed these results, staining 84.8+1.2% of peripherin-positive DRG cells from naive animals (CCI not tested; mean+SEM, n=4). In contrast, co-staining with the myelinated neuronal marker, NF-200 (Goldstein, House et al. (1991 ) J. Neurosci. Res., 30(1 ):92-104; Fornaro, Lee et al. (2008) Neuroscience, 153(4): 1153-1163), the satellite glial cell marker, glutamine synthetase (Miller, Richards et al. (2002) Brain Res., 945(2):202-211 ) and the microglial/recruited macrophage marker, CD68 (Hu and McLachlan (2003) Exp. Neurol., 184(2): 590-605) was minimal (Fig. 2B). The pattern and levels of ErbB1 expression in DRG were not significantly altered following CCI.

Within the population of ErbB1 -expressing small DRG cells, both presumed peptidergic C-fibres, staining for TRPV1 (Cavanaugh, Lee et al. (2009) Proc. Natl. Acad. Sci. U S A, 106(22):9075-9080) and presumed non-peptidergic fibres, labelled with isolectin, IB4 (Dong, Han et al. (2001 ) Cell, 106(5):619-632) were strongly represented, with 87.1 ±2.1 % of TRPV1 (and peripherin)-positive cells, and 88.6+2.1 % of IB4 (and peripherin)-positive cells co-staining for ErbB1 (means+SEM, n=12). Similar values were found in CCI animals. These observations are consistent with the idea of ErbB1 playing an important role in nerve injury-induced hypersensitivity throughout the whole C-fibre nociceptor population.

As ErbB1 may heterodimerise with other ErbB family members (Olayioye, Neve et al. (2000) EMBO J., 19(13):3159-3167), also the expression in DRG of ErbB2-4 was investigated, using highly characterised, isotype-specific antibodies. ErbB2 was found to be widely and selectively expressed in small, peripherin-positive DRG cells, like ErbB1 , with around 90% of peripherin-positive cells expressing ErbB2 in both naive and CCI animals (Fig. 2C). Similar results were obtained for ErbB2 expression in ErbB 1 -positive cells (Fig. 2C) and were confirmed with a second highly characterised ErbB2-specific antibody (mouse monoclonal, UMAB36) which showed staining in 76.2+6.3% and 78.5+4.4% of ErbB1 -positive cells in naive and CCI DRG, respectively (means+SEM, n=4). ErbB3- and ErbB4-specific antibodies showed minimal staining in DRG of naive and CCI animals; with any staining rarely present in small, peripherin-positive cells (Fig. 2C). These observations suggested that ErbB2, in addition to ErbB1 , could potentially represent an analgesic target in nociceptors; acting either independently or in concert with ErbB1 as a heterodimer; an idea supported by evidence that ErbB2 is the preferred heterodimerisation partner for all of the other ErbB family members (Graus-Porta, Beerli et al. (1997) EMBO J., 16(7): 1647-1655; Tzahar, Pinkas-Kramarski et al. (1997) EMBO J., 16(16):4938-4950).

B2.3: Functional evidence for a role of ErbB2 within C-fibre nociceptors in CCI-induced hypersensitivity.

Using paw withdrawal threshold in the von Frey test, it could be shown that several ErbB2 blockers caused marked reversal of CCI-induced mechanical hypersensitivity. The highly selective ErbB2 inhibitors, mubritinib and tucatinib (ARRY-380), both of which show very low affinity for ErbB1 (Nagasawa, Mizokami et al. (2006) Prog. Neurobiol., 168:86-103; Moulder, Borges et al. (2017) Clin. Cancer Res., 23(14):3529-3536), each at a dose of 15mg/kg ip, produced marked reversal of CCI-induced hypersensitivity, with complete reversal at peak in each case and both remaining statistically significant for 180min (Fig. 3A). The mean+SEM percentage reversals of hypersensitivity from 20-140m in following administration were 91.5+4.7% (n=6) and 87.1+6.9% (n=4), respectively. This validated ErbB2, in addition to ErbB1 , as a potential analgesic target for neuropathic pain. Two dual inhibitors of both ErbB1 and ErbB2, lapatinib (a reversible blocker targeting ErbB1 >ErbB2>ErbB4 (Rusnak, Lackey et al. (2001 ) Mol. Cancer Then, 1 (2):85-94) at a dose of 20mg/kg ip, and afatinib (an irreversible covalent blocker targeting ErbB1 >ErbB2>ErbB4 (Li, Ambrogio et al. (2008) Oncogene, 27(34):4702-4711 )) at a dose of 2.5mg/kg ip, were both similarly effective, reversing hypersensitivity completely at peak effect and remaining statistically significant for 180 and 140min, respectively (Fig. 3B). The mean+SEM percentage reversals of hypersensitivity from 20-140m in following administration were 94.4+6.9% (n=6) for lapatinib and 71.7+11.5% (n=4) for afatinib, respectively. Animals treated with inhibitors of ErbB1 , ErbB2 or combinations of these were closely observed and showed no apparent changes in locomotor activity, motor co ordination or any evidence of sedation.

B2.4: Evidence for ErbB1 :ErbB2 interaction and the role of this in CCI-induced hypersensitivity.

In order to evaluate any direct interaction between ErbB1 and ErbB2 in small DRG cells, a Proximity Ligation Assay was used, which relies on hybridisation between DNA-tagged secondary antibodies to produce an amplified fluorescent signal if the epitopes recognised by two, species-distinct, primary antibodies are in close proximity (Fredriksson, Gullberg et al. (2002) Nat. Biotechnol., 20(5):473-477). Fig. 3B shows that ErbB1 :ErbB2 proximity ligation in peripherin-positive DRG cells from naive animals yielded a low fluorescent signal, which was strongly and significantly increased following CCI. Control experiments using rabbit ErbB1 and mouse ErbB3 antibodies showed a minimal proximity ligation signal from DRG cells of naive or CCI rats. These findings provide explicit evidence for close interaction of ErbB1 and ErbB2 in nociceptive C-fibres following nerve injury.

In the classical paradigm of ErbB receptor activation, ligand binding induces receptor dimerisation, which leads to activation of the intrinsic tyrosine kinase function of the monomers and autophosphorylation or transphosphorylation of intracellular tyrosine residues (Weiss and Schlessinger (1998) Cell, 94(3):277-280). ErbB2 lacks any known direct ligand, but readily heterodimerises with each of the other ErbB monomers (Graus- Porta, Beerli et al. (1997) EMBO J., 16(7): 1647-1655; Tzahar, Pinkas-Kramarski et al. (1997) EMBO J., 16(16):4938-4950), so may act as a subservient, signalling co-receptor in ErbB1 :ErbB2 heterodimers. ErbB2 shows prominent auto-/trans-phosphorylation at Tyr1221/1222 upon activation (Ricci, Lanfrancone et al. (1995) Oncogene, 11 (8): 1519- 1529). Using a phospho-specific antibody that specifically recognises ErbB2 phosphorylated at Tyr1221/1222, a marked increase in staining in small, ErbB 1 -positive DRG cells following CCI could be shown, which was reversed by erlotinib treatment (each at a dose of 10mg/kg ip, 1 hr) (Fig. 3C). This indicates that ErbB2 auto-/trans- phosphorylation occurs through an ErbB1 -dependent mechanism following nerve injury; most likely representing the function of ErbB1 :ErbB2 heterodimers.

Given the accumulating evidence for ErbB1 :ErbB2 co-engagement following nerve injury, it was investigated whether ErbB1 and ErbB2 blockers might interact functionally in producing analgesia. Very low doses of erlotinib (0.33mg/kg) and mubritinib (1 mg/kg) were selected that produced just discernible analgesia, (only 9.9+3.1 % and 11.4+3.7% reversal of mechanical hypersensitivity when averaged over 20-140m in, n=4 in each case). Administration of these in combination produced clearly greater than additive analgesia (52.9+11.1 % reversal of hypersensitivity, p<0.01 , n=4, with complete reversal at peak effect and statistically significant effects for up to 100min, Fig. 3D). Formal assessment of synergy using Bliss Additivism effect-based modelling (Berenbaum (1981) Adv. Cancer Res., 35:269-335; Borisy, Elliott et al. (2003) Proc. Natl. Acad. Sci. U S A, 100(13):7977-7982), for combinations of erlotinib through the range 0.1-3.3mg/kg with mubritinib 1.0mg/kg, showed consistently greater observed responses than those predicted from combining individual effects, with more than 3.8-fold reduction in ECso for observed compared to predicted combination dose-response curves (p<0.001 by Extra Sum of Squares F-test; Fig. 3E). Two further, structurally distinct selective ErbB1 and ErbB2 inhibitors, were administered separately at low dose and then in combination, to independently confirm the concept of synergistic analgesic effects from dual ErbB1/ErbB2-targetting. Falnidamol (0.33mg/kg) and tucatinib (1.0mg/kg) individually produced 9.0+1 .6% and 13.8+3.8% reversal of mechanical hypersensitivity, whereas in combination they produced 54.9+6.1 % reversal over 20-140min following administration, clearly much greater than the 21.6+4.1 % predicted from Bliss Additivism modelling (p<0.01 by unpaired t-test, n=4, Fig. 3F). These observations fully support the idea that ErbB1 and ErbB2 in C-fibre nociceptors co-operate in leading to nerve injury-induced hypersensitivity and suggest that conjoint administration of agents to block both ErbB1 and ErbB2 may represent a favourable strategy for analgesia.

Further studies at the level of peripheral terminals of nociceptive afferents in the skin of the paw supported the idea that ErbB1 and ErbB2 were both involved in bringing about nociceptive hypersensitivity.

B2.5 Evidence that peripheral terminals of nociceptors can be sensitised by an ErbB1- dependent process following CCI.

In naive rats, intradermal injection of EGF or heparin-binding EGF (another ErbB1- selective ligand) was found to have no effect, whereas GDNF or NGF caused lasting hypersensitivity in a paw pressure withdrawal test (Andres, Meyer et al. 2010, Ferrari, Bogen et al. 2010). Nonetheless, as we had found ErbB1 abundantly expressed in the cell bodies of small, nociceptive DRG cells, some deployment to peripheral nerve terminals would be anticipated. To investigate whether peripherally localised ErbB1 might play a role in nerve injury-induced sensitisation of nociceptors, we carried out intraplantar injections in both naive and CCI rats and assessed mechanical hypersensitivity by measuring von Frey PWT scores. Table 2 shows that intraplantar injection of EGF amplified the ipsilateral hypersensitivity in CCI animals, but not in naive controls. No changes were seen in contralateral PWT values and contralateral injection of EGF had no effect on either contralateral or ipsilateral PWT values. These findings suggest that the effect observed upon ipsilateral injection was a local, not systemically mediated, action and that some event brought about by nerve injury (perhaps local neuroinflammatory processes) was required to prime the nociceptors before an active role of ErbB1 was revealed. The effect of EGF was not mimicked by neuregulinl-bΐ EGF domain (NRG1 ; a selective ErbB3/ErbB4 ligand, matching our evidence for minimal expression of these proteins in small nociceptive DRG cells. The EGF-evoked amplification of hypersensitivity was however prevented by co-injection of erlotinib, confirming the anticipated targeting of ErbB1 , and also by mubritinib, consistent with our other evidence for ErbB1 acting by way of ErbB1 :ErbB2 heterodimers. Pilot experiments in CIPN mice showed similar marked amplification of hypersensitivity lasting from 20- 80m in following intraplantar injection of EGF (80ng), but no discernible effect in naive controls.

Table 2: Effects of intraplantar injection of selective ErbB-receptor-targeting drugs on CCI-induced hypersensitivity in the von Frey Paw Withdrawal Threshold test.

Experiments were carried out 8-12days following CCI in animals that displayed marked ipsilateral hypersensitivity to von Frey filaments. Drugs were injected intraplantarly into the interdigital skin (in 50mI per animal) under light isoflurane anaesthesia, and Paw Withdrawal Threshold (PWT) testing recommenced after a 20min delay for recovery and then repeated at 10min intervals up to 70min post-injection. The mean percentage reduction in PWT score over 20-40min post-drug administration was calculated and the statistical significance of changes in ipsilateral or contralateral PWT scores was assessed by One-Way ANOVA with Dunnett’s post-hoc test. Injection of EGF (100ng) ipsilateral to CCI caused significant exacerbation of injury-induced hypersensitivity (**p<0.01 ) from 20- 40min following administration. This effect was reversed by co-injection of the ErbB1 inhibitor, erlotinib (2.2ng) or the ErbB2 inhibitor, mubritinib (7.0ng), which had no discernible effects alone. The ErbB3/4-selective agonist, neuregulinl-bΐ EGF domain (NRG1, (100ng) did not mimic the effect of EGF. Contralateral injection of EGF or NRG1 had no effect on ipsilateral PWT values in CCI animals. Either ipsilateral or contralateral injection of EGF had no effect on contralateral PWT values in CCI animals and was also without effect on baseline PWT values in naive controls. Administration of vehicle alone had no discernible effect.

Figure Legends

Figure 1: The highly selective ErbB 1 inhibitor, erlotinib, reverses nerve injury-induced hypersensitivity in reflex pain behaviours and increases a biomarker of cellular activation in nociceptors. A)-C) show time courses of reflex pain behaviours in the CCI neuropathic pain model, following administration of erlotinib (10mg/kg, ip). All values are shown as mean+SEM. A) shows effects on mechanical hypersensitivity (assessed by von Frey filament Paw Withdrawal Thresholds PWT, n=5). B) shows effects on noxious thermal hypersensitivity (assessed by Flargreaves’ infra-red test Paw Withdrawal Latencies PWL, n=5). C) shows effects on noxious cold hypersensitivity (seen as increased Suspended Paw Elevation Times from 4°C shallow water bath; n=4). Ipsilateral hypersensitivity was significantly reversed by erlotinib for 140-160min in each case; ††p<0.01 by One-Way Repeated Measures ANOVA with Dunnett’s post-hoc test, while no significant effects were observed on contralateral responses. D) shows immunofluorescence staining for an unbiased activity reporter (phospho-CaMKII (Thr286), reflecting increased intracellular Ca²⁺ concentrations) in DRG neurons from naive and CCI animals, as well as the effect of treatment with erlotinib (10mg/kg, ip, 1 hr). Both typical immunofluorescence images and quantitative analysis show that phospho-CaMKII (Thr286) staining in peripherin-positive (nociceptive) DRG neurons was increased following CCI and this was reversed by erlotinib (n=4 in each case). One-Way ANOVA with Tukey’s post-hoc test revealed a significant increase due to CCI (*p<0.05) and its significant reversal by erlotinib (†p<0.05). Scale bars represent 50pm. Typical numbers of positive cells counted per section were 197 for peripherin and 76 for phospho-CaMKII (Thr286). Figure 2: ErbB1 expression in DRG is limited to small, unmyelinated nociceptive neurons and colocalises with ErbB2, but not ErbB3 orERbB4. A) shows typical images of immunofluorescence staining using a highly characterised ErbB1 antibody (mouse monoclonal [EGFR1], ab30) that does not cross-react with other ErbB family members, compared to peripherin (a marker of unmyelinated C-fibre nociceptors). Very high concurrence of staining was observed in DRG from either naive or CCI animals. Numbers of positive cells counted per section were typically 134 for peripherin and 112 for ErbB1. Around 80% of peripherin-positive cells expressed ErbB1 in both naive and CCI conditions and this result was corroborated with a second, similarly specific ErbB1 antibody (rabbit monoclonal [E235], ab32077). The n values were 18 and 4, respectively. B) shows typical images of staining for ErbB1 versus the myelinated afferent marker, NF-200; the satellite glial cell marker, glutamine synthetase; and the microglial/invading macrophage marker, CD68. Minimal co-localisation with ErbB1 (<14%) was observed in each case, in DRG from either naive or CCI animals. Numbers of positive cells counted per section were typically 120 for ErbB1 , 95 for NF-200, 182 for glutamine synthetase, and 66 for CD68, n=4 in each case. C) shows typical examples of staining using highly characterised antibodies (that do not cross-react with other ErbB family members) for ErbB2 (rabbit polyclonal, 06-562), ErbB3 (mouse monoclonal,

H3.105.5) and ErbB4 (mouse monoclonal, H4.77.16) versus peripherin or ErbB1 (ab30). Abundant staining for ErbB2 was seen in a very high proportion (>90%) of peripherin- positive cells with little staining of other cells. There was a similarly high concurrence of ErbB2 staining with ErbB1, again limited to small DRG cells. ErbB2 was expressed in 93.9+1.0% and 96.4+1.1% of ErbB1 -positive cells from naive and CCI DRG, respectively. Only occasional staining was observed for ErbB3 or ErbB4 in DRG and this was almost entirely associated with peripherin-negative cellular profiles. Less than 8% of peripherin-positive cells showed staining for ErbB3 or ErbB4. Numbers of positive cells counted per section were typically 191 for peripherin, 187 for ErbB1, 188 for ErbB2, 11 for ErbB3, and 14 for ErbB4, n=4 in each case. Scale bars represent 50pm. Figure 3: ErbB2 inhibitors reverse nerve injury-induced mechanical hypersensitivity and act synergistically with selective ErbB1 inhibitors, matching evidence for direct ErbB1:ErbB2 interaction in small nociceptive DRG neurons. A) and B) show effects of the highly selective ErbB2 inhibitor, mubritinib (15mg/kg, ip) and tucatinib (15mg/kg, ip) in A), and the dual ErbB1/ErbB2 inhibitor, lapatinib (20mg/kg, ip) and afatinib (2.5mg/kg, ip) in B), on mechanical hypersensitivity following CCI, as assessed by von Frey filament PWT values (n=5 and 4, respectively). Ipsilateral hypersensitivity was significantly reversed for 180min in each case; ††p<0.01 by One-Way Repeated Measures ANOVA with Dunnett’s post-hoc test, while no significant effects were observed on contralateral responses. C) shows typical images from a Proximity Ligation Assay, with specific antibodies for ErbB1 (ab30) and ErbB2 (06-562). Close target proximity is reported as orange fluorescence. Sections were counterstained for peripherin (green) using conventional immunofluorescence. Nerve injury induced a clear (>2-fold) increase in the percentage of peripherin-positive cells that showed ErbB1 :ErbB2 proximity fluorescence compared to naive. Typical numbers of positive cells counted per section were 117 for peripherin and 40 for ErbB1 :ErbB2 proximity ligation, n=4 in each case. The statistical significance of changes was assessed by unpaired two-tailed t-test; **p<0.01, showing a significant CCI-induced increase in ErbB1:ErbB2 heterodimerisation compared to naive. Scale bars represent 50pm. D) shows typical images of immunofluorescence staining for a prominent auto-/trans- phosphorylation site in ErbB2, phospho-ErbB2 (Tyr1221/2), identified by a specific rabbit monoclonal antibody [6B12], compared to ErbB1, identified using ab30, in DRG from naive, CCI and CCI animals treated with erlotinib, 10mg/kg ip, 1 hr. The percentage of ErbB1 -positive cells showing phospho-ErbB2 (Tyr1221/2) staining was more than 2.5-fold increased following CCI and this was reversed by erlotinib. One-Way ANOVA with Tukey’s post-hoc test revealed a significant increase due to CCI (***p<0.01) and its significant reversal following erlotinib treatment (††p<0.01), n=4 in each case. Numbers of positive cells counted per section were typically 189 for ErbB1 and 53 for phospho- ErbB2 (Tyr1221/2). Scale bars represent 50pm. E) shows effects of selective ErbB1 and ErbB2 inhibitors, in combination, on mechanical hypersensitivity following CCI, as assessed by von Frey PWT scores. Low doses of erlotinib (0.33mg/kg, ip) and mubritinib (1.0mg/kg, ip), selected to produce just discernible levels of analgesia alone, were tested in combination. One-Way Repeated Measures ANOVA with Dunnett’s post- hoc test indicated statistically significant attenuation due to erlotinib at 20 and 40min following administration, for mubritinib at 20min and for the combination throughout the 20-100min period; ††p<0.01; n=4 in each case. F) shows formal assessment of analgesic synergy between mubritinib (1.Omg/kg, ip) and erlotinib across a range of erlotinib doses (ip), using Bliss Additivism effect-based modelling to predict expected combination outcomes. Comparison of observed versus expected combination dose- response curves by Extra Sum of Squares F test indicated a significant difference, reflected in a more than 3.8-fold reduction in ECso value (mean [95% Cl] from 1.09 [1.06/1.14]) to 0.28 [0.25/0.31 ]mg/kg, †††p<0.001 (n=4-5 for each point). G) shows the effects of individual and combined treatment with further (structurally distinct) selective ErbB1 and ErbB2 blockers, falnidamol (0.33mg/kg, ip) and tucatinib (1. Omg/kg, ip), respectively, against nerve injury-induced hypersensitivity. The observed analgesic effect was significantly greater than the predicted combination effect according to Bliss Additivism modelling (p<0.001, Student’s t-test, n=4).

Claims

Patent Claims

1. A pharmaceutical combination, comprising an inhibitor of ErbB1 and an inhibitor of ErbB2.

2. The pharmaceutical combination according to claim 1 , wherein the ErbB1 inhibitor is a small molecule, or a physiologically acceptable salt thereof, or an anti-ErbB1 antibody, or an antigen binding fragment thereof.

3. The pharmaceutical combination according to claims 1 or 2, wherein the ErbB2 inhibitor is a small molecule, or a physiologically acceptable salt thereof, or an anti-ErbB2 antibody, or an antigen binding fragment thereof.

4. The pharmaceutical combination according to any of claims 1 - 3, wherein the ErbB1 inhibitor is erlotinib, gefitinib, AG 1478, falnidamol, EGFRi 324674, cetuximab, panitumumab or, or other highly selective ErbB1 -inhibiting agents.

5. The pharmaceutical combination according to any of claims 1 - 4, wherein the ErbB2 inhibitor is mubritinib, tucatinib, trastuzumab, or other highly selective ErbB2-inhibiting agents.

6. A pharmaceutical composition, comprising a pharmaceutical combination according to any of Claims 1 - 5 and, optionally, excipients and/or adjuvants.

7. A set (kit) consisting of separate packs of

(a) an effective amount of an ErbB1 inhibitor according to any one of Claims 1- 5, or a physiologically acceptable salt, or an antigen binding fragment thereof, and

(b) an effective amount of an ErbB2 inhibitor, according to any one of Claims 1- 5, or a physiologically acceptable salt, or an antigen binding fragment thereof.

8. A method for the treatment of neuropathic pain, comprising administering to a subject an ErbB1 inhibitor and an ErbB2 inhibitor, each according to any of claims 1 - 5, or a physiologically acceptable salt, or an antigen binding fragment each thereof.

9. The method according to claim 8, wherein the ErbB1 inhibition is effected by the blockage of the ErbB1 ligand binding domain or the inhibition of the ErbB1 kinase domain or down-regulation of ErbB1 expression at the cell surface.

10. The method according to claims 9 or 10, wherein the ErbB2 inhibition is effected by the blockage of the ErbB2 ligand binding domain or the inhibition of the ErbB2 kinase domain or down-regulation of ErbB2 expression at the cell surface.

11. The method according to any one of claims 8 - 10, wherein the ErbB1 and ErbB2 inhibition is effected by the blockage of the ErbB1/ErbB2 dimerization.

12. The method according to any one of claims 8 - 11, wherein the ErbB1 inhibitor and the ErbB2 inhibitor are administered simultaneously.

13. The method according to any of claims 8 - 11, wherein the ErbB1 inhibitor and the ErbB2 inhibitor are administered sequentially.

14. A method for the treatment of neuropathic pain, comprising administering to a subject a dual ErbB1/ErbB2 inhibitor, or a physiologically acceptable salt, or an antigen binding fragment thereof.

15. The method according to claim 14, wherein the dual ErbB1/ErbB2 inhibitor, for example, is: lapatinib, afatinib, sapatinib, epertinib, poziotinib, allitinib, pyrotinib, canertinib, varlitinib, tesevatinib or dacomitinib.

16. The method according to any one of claims 8 - 15, wherein the neuropathic pain is cancer related or non-cancer related.

17. The method according to any one of claims 8 - 16, wherein the neuropathic pain is non-compressive neuropathic pain, compressive neuropathic pain, toxic neuropathic pain, metabolic neuropathic pain, traumatic neuropathic pain, autoimmune neuropathic pain, infectious neuropathic pain, and congenital or hereditary neuropathic pain.