Methods For Treatment of Neuropathic Pain
20210171588 · 2021-06-10
Assignee
Inventors
- Paul Louis Chazot (Durham, GB)
- Miles Andrew Nunn (3 Geneve, CH)
- Ilona Genowefa Obara (Newcastle Upon Tyne, GB)
Cpc classification
International classification
Abstract
Methods of treating neuropathic pain are provided. In particular, the invention provides a method for treating neuropathic pain related mechanical hypersensitivity (allodynia and hyperalgesia) and peripheral neuropathic pain.
Claims
1. An histamine binding protein (HBP) for use in a method of treating neuropathic pain.
2. An HBP for use of claim 1, which is derivable from the saliva of a tick.
3. An HBP for use of any one of the preceding claims, wherein the HBP is selected from FS-HBP2, FS-HBP1, MS-HBP1 and D.RET6.
4. An HBP for use of claim 3, wherein the HBP is FS-HBP2.
5. An HBP for use of claim 4, wherein the HBP comprises or consists of the amino acid sequence provided in SEQ ID NO:2.
6. An HBP for use according to any one of the preceding claims, which is a variant of an HBP as described in any one of claims 2 to 5.
7. An HBP for use according to claim 6, wherein the variant has a protein sequence having at least 70% sequence identity to a protein sequence selected from any one of SEQ ID NOs: 2, 4, 6, 8 and 10.
8. An HBP for use according to any one of the preceding claims which is a fragment of a full length HBP or which is a fragment of a variant thereof.
9. A HBP for use according to any one of the preceding claims wherein the HBP is administered at a dosage of more than 3, 5, 6, 7, 8, 9, or 10, 20, 30 or 40 mg mg/kg body weight, optionally wherein the dosage is between 2-10 mg/kg, 4-8 mg/kg or is about 6 mg/kg body weight.
10. A purified nucleic acid molecule encoding an HBP as described in any one of the preceding claims for use in a method of treating neuropathic pain.
11. A purified nucleic acid molecule for use according to claim 10, which comprises or consists of a cDNA sequence encoding the amino acid sequence of SEQ ID NO:2.
12. A purified nucleic acid molecule for use according to claim 11, which comprises or consists of the cDNA sequence of SEQ ID NO:1.
13. A purified nucleic acid molecule for use according to claim 10, which comprises or consists of a cDNA sequence that encodes a variant or active fragment of SEQ ID NO:2 or an active fragment of such a variant.
14. A vector comprising a purified nucleic acid molecule according to any one of claims 10 to 13 for use in a method of treating neuropathic pain.
15. A pharmaceutical composition comprising an HBP, a purified nucleic acid or a vector according to any one of the preceding claims, in conjunction with a pharmaceutically-acceptable carrier, for use in a method of treating neuropathic pain.
16. An HBP, purified nucleic acid, vector or pharmaceutical composition for use according to any one of claims 1 to 15, wherein the neuropathic pain is neuropathic pain in which histamine plays a role.
17. An HBP, purified nucleic acid, vector or pharmaceutical composition for use according to any one of claims 1 to 16, wherein the method involves preventing histamine from binding to its receptors.
18. An HBP, purified nucleic acid, vector or pharmaceutical composition for use according to any one of claims 1 to 17, wherein the method involves treating neuropathic pain that is observed as hypersensitivity to mechanical stimuli.
19. An HBP, purified nucleic acid, vector or pharmaceutical composition for use according to claim 18, wherein the neuropathic pain is allodynia and/or hyperalgesia.
20. An HBP, purified nucleic acid, vector or pharmaceutical composition for use according to any one of claim 18 or 19, wherein the treatment results in improvements in the sensations of one or more of touch (including light touch), “pins and needles”, numbness, itching and excruciating pain.
21. An HBP, purified nucleic acid, vector or pharmaceutical composition for use according to any one of claims 1 to 20, wherein the neuropathic pain is peripheral neuropathic pain.
22. An HBP, purified nucleic acid, vector or pharmaceutical composition for use according to claim 21, wherein the patient being treated for peripheral neuropathic pain has a condition or disease selected from damaged, irritated or diseased peripheral nerves, peripheral neuropathy, peripheral nerve injury-induced neuropathic pain, diabetes mellitus, diabetic neuropathy, diabetic polyneuropathy, pre-diabetes, metabolic dysfunction, infectious disease (e.g. HIV infection or leprosy), an immune disorder (e.g. Guillain-Barré syndrome), an inflammatory disorder, an inherited neuropathy, an inherited channelopathy (e.g. inherited erythromelalgia), injury to the sciatic nerve, spinal stenosis, trigeminal neuralgia, postherpetic neuralgia, peripheral nerve injury pain, post-amputation pain, painful polyneuropathy, painful radiculopathy, spinal cord injury, post-traumatic neuropathy, postsurgical neuropathy, cervical polyradiculopathy, lumbar polyradiculopathy and complex regional pain syndrome type 2.
23. An HBP, purified nucleic acid, vector or pharmaceutical composition for use according to any one of claims 1 to 20, wherein the neuropathic pain is central neuropathic pain.
24. An HBP, purified nucleic acid, vector or pharmaceutical composition for use according to claim 23, wherein the patient being treated for central neuropathic pain has a condition or disease selected from a cerebrovascular disease affecting the central somatosensory pathways, a neurodegenerative disease and a spinal cord lesion or disease.
25. An HBP, purified nucleic acid, vector or pharmaceutical composition for use according to any one of claims 1 to 20, wherein the neuropathic pain is a mixture of both peripheral and central.
26. An HBP, purified nucleic acid, vector or pharmaceutical composition for use according to claim 25, wherein the patient being treated for peripheral and central neuropathic pain has Parkinson's disease.
27. An HBP, purified nucleic acid, vector or pharmaceutical composition for use according to any one of claims 1 to 26, wherein the patient being treated for neuropathic pain is also being treated with chemotherapy or has previously undergone treatment with chemotherapy.
28. An HBP, purified nucleic acid, vector or pharmaceutical composition for use according to any one of claims 1 to 27, wherein the method does not have any negative side effects.
29. A method of treating neuropathic pain which comprises administering an HBP, purified nucleic acid, vector or pharmaceutical composition as described in any one of the preceding claims to a patient in need thereof, for example, wherein the neuropathic pain and/or the patient is as described in any one of the preceding claims.
30. Use of an HBP, purified nucleic acid or vector as described in any one of the preceding claims in the manufacture of a medicament for the treatment of neuropathic pain, for example, wherein the neuropathic pain and/or the patient being treated is as described in any one of the preceding claims.
31. Use of an HBP as a research tool in the study of neuropathic pain.
Description
BRIEF DESCRIPTION OF THE FIGURES
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EXAMPLES
Example 1
Assessment of the Efficacy of Recombinant Histamine Binding Protein EV131 in Neuropathic Pain
[0103] The native protein EV131 occurs in tick saliva where its anti-inflammatory activity counters the host's immunological response at the tick feeding site.
[0104] Methods: Neuropathic pain in adult male C57BL/6J (B6) mice (n=6/group) was induced by chronic constriction injury (CCI) of the sciatic nerve. The effect of EV131 (1, 3 and 10 mg/kg i.p.) on mechanical and thermal hypersensitivity was determined 0.5, 1 and 24 h after EV131 (4 injections every 24 h for 4 days) and compared to saline controls. Mechanical and thermal hypersensitivity was assessed using von Frey filaments and Hargreaves' tests.
[0105] Results: In neuropathic mice, EV131 at the dose of 10 mg/kg, but not 1 and 3 mg/kg, reduced mechanical hypersensitivity (AUC [g.Math.days]−10 mg: 10.47±1.75 [SEM], 3 mg: 4.41±1.23 [SEM], 1 mg: 4.03±1.37 [SEM], saline: 2.22±1.21 [SEM]). EV131 did not affect thermal hypersensitivity. The results are shown in
[0106] Conclusions: Our findings provide the first evidence for the analgesic effects of EV131 upon neuropathic pain related mechanical hypersensitivity and thus emphasize the importance of the histamine system in the modulation of peripheral neuropathies.
Example 2
Assessment of the Efficacy of Recombinant Histamine Binding Protein EV131 in Neuropathic Pain
[0107] Subjects
[0108] C57BL/6J mice. Adult male C57BL/6J mice (8 weeks of age; 20-25 g; Charles River Laboratories, Kent, UK) were allowed to acclimate to the colony room (Durham University, UK or Comparative Biology Centre, Newcastle University, UK) for at least 7 days after arrival and were housed in polyethylene cages (4 per cage), controlled for temperature (21° C.) and humidity (55%) under a regular 12-h day/night cycle (lights on at 8:00 A.M.; lights off at 8:00 P.M.). Standard laboratory rodent chow and water were available ad libitum. Animals were habituated to testing procedures for at least 3-4 days before experiments. The handling and testing of the animals were conducted during the light phase, between 9:00 A.M. and 6:00 P.M. All efforts were made to minimize animal suffering and to reduce the number of animals used in the study. Experimental protocols were performed under UK Home Office licence, with local ethical approval, and in accordance with current UK legislation as defined in the Animals (Scientific Procedures) Act 1986. The ARRIVE guidelines have been followed in reporting this study.
[0109] Drugs: Preparation and Administration
[0110] Systemic injections. For systemic (i.p.) admistration EV131 (SEQ ID NO: 10) was prepared in phosphate-buffered saline as a stock solution of 5.8 mg/mL and then prepared immediately before injections in a vehicle (saline) solution at required concentrations. Mice were weighed and injected intraperitoneally (i.p.) with EV131 (1-40 mg per kg body weight) or equivalent vehicle (saline) solution without EV131 as a control group. EV131/vehicle was injected four times (every 24 h for four days).
[0111] Intraplantar injections. For intraplantar (i.pl.) administration, EV131 (SEQ ID NO: 10; 0.0075-0.25 mg per 50 μl; 50 μl per one injection) was prepared from a stock solution of 5.8 mg/mL immediately before injections in a vehicle (saline) solution. Intraplantar (i.pl.) injections were given over 1 min in a volume of 50 μl without anesthesia into the plantar surface of the animal hind paw ipsilateral to sciatic nerve injury. Control animals received 50 μl of equivalent vehicle solution without EV131. EV131/vehicle was injected four times (every 24 h for four days).
[0112] Pain Model
[0113] Sciatic nerve injury. Mice were subjected to peripheral neuropathy induced by chronic constriction injury (CCI) of the sciatic nerve as described by Bennett and Xie [18], with slight modifications for mice [19,20]. The sciatic nerve injury was performed under isoflurane anesthesia delivered via a nose cone (2% isoflurane with oxygen as the carrier gas). The skin was shaved and an incision was made just below the right hipbone, parallel to the sciatic nerve. The biceps femoris and the gluteus superficialis were separated, and the right sciatic nerve was exposed. Proximal to the sciatic trifurcation, the injury was produced by three loose ligations around the sciatic nerve. The ligatures (4/0 silk) were tied loosely around the nerve with 1 mm spacing, until they elicit a brief twitch in the respective hindlimb, which prevented from applying a too strong ligation. The total length of nerve affected was 3-4 mm. Muscle and skin were closed in two separate layers. For sham surgery, the sciatic nerve was exposed as described above but no contact was made with the nerve. Both mechanical threshold at the lateral plantar surface of the hind paw and thermal sensitivity were assessed before nerve injury (as basal pain threshold) and then testing began on day 7 after surgery and continued for 3 days post surgery (total of 4 days of behavioural testing).
[0114] Behavioural Testing
[0115] In all experiments the observer was not aware of the dose of EV131 given in the intraplantar/intraperitoneal injections. Each animal was subjected to von Frey testing followed by the Hargreaves test.
[0116] Mechanical stimulation. Mechanical sensitivity was assessed by measuring the withdrawal threshold of the paw ipsilateral to the site of injury in response to mechanical stimuli using von Frey filaments (Ugo Basile, Gemonio, Italy). Animals were placed in a plastic cage with a wire net floor and were allowed to habituate before testing began. Animals were also habituated over a period of 3-4 consecutive days by recording a series of baseline measurements. The filaments were applied in ascending order, each five times at an interval of 2-3 seconds to the plantar surface of the hind paw as described previously [21,22] and the smallest filament eliciting a foot withdrawal response was considered the threshold stimulus. The strength of the von Frey stimuli used in the present experiments ranged from 0.04 to 6.0 g.
[0117] Thermal stimulation. Mice were tested for the latency of paw withdrawal response to a noxious thermal stimulus using a radiant heat-emitting device (IITC Life Science Inc., USA) as described by [23]. Mice were placed individually into Plexiglass chambers on an elevated glass platform and allowed to habituate to the apparatus before testing began. Animals were also habituated over a period of 3-4 consecutive days by recording series of baseline measurements. A radiant heat source of constant intensity was applied to the plantar surface of the paw through the glass plate and the latency to paw withdrawal was measured. Hind paw received 3 stimuli and the inter-stimulus interval was at least 3 min to prevent injury. Withdrawal latencies were defined as the mean of readings for hind paw. A cutoff of 20 s was employed to avoid tissue injury.
[0118] Statistical Data Analysis
[0119] Data analysis and statistical comparisons were performed using GraphPad Prism™, version 7.00 for Windows, GraphPad Software, CA, USA, www.graphpad.com.
[0120] Results are presented in the graphs as mean±SEM. Each group included 5-14 animals. Statistical analysis was performed by one- or two-way analyses of variance (ANOVA) with Bonferroni's multiple comparison post-hoc tests or by unpaired Student's t-test when two groups were compared. A value of p<0.05 was considered to be statistically significant.
[0121] Results
[0122] In neuropathic mice subjected to chronic constriction injury (CCI) model of neuropathic mice, EV131 when injected i.p. at the dose of 10, 20 and 40 mg/kg, but not 1 and 3 mg/kg, significantly reduced mechanical hypersensitivity (AUC [g.Math.days]−40 mg: 21.3±0.9 [SEM], 20 mg: 18.3±0.7 [SEM], 10 mg: 15.2±1.4 [SEM], 3 mg: 5.1±0.4 [SEM], 1 mg: 3.9±0.4 [SEM], saline: 2.7±0.3 [SEM]; F(5,32)=102.2, P<0.0001; see
TABLE-US-00001 TABLE 1 SEQUENCE COMPARISON OF FS-HBP1 (top line) (residues 19-190 of SEQ ID NO: 4), FS-HBP2 (middle line) (residues 20 to 190 of SEQ ID NO: 2), and MS-HBP1 (bottom line) (residues 19 to 200 of SEQ ID NO: 6). D K P V W A D E A A N G E H Q D A w K H N Q P D W A D E A A N G A H Q D A W K S N P T W A N E A K L G S Y Q D A W K S = = = = = = = = = = = L Q K L V E E N Y D L I K A T Y K N L K A D V E N V Y Y M V K A T Y K N L Q Q D Q N K R Y Y L A Q A T Q T T = = = = = D P V W G N D F T C V G T A A Q N L N E D P V W G N D F T C V G V M A N D V N E D G V W G E E F T C V S V T A E K I G = = = = = = = = = D E K N V E A W F M F M N N A D T V Y Q D E K S I Q A E F L F M N N A D T N M Q K K K L N A T I L Y K N K H L T D L K = = = = H T F E K A T P D K M Y G Y N K E N A I F A T E K V T A V K M Y G Y N R E N A F E S H E T I T V W K A Y D Y T T E N G I = = = = = = = T Y Q T E D G Q V L T D V L A F S R Y E T E D G Q V F T D V I A Y S K Y E T Q G T R T Q T F E D V F V F S = = = = = = = D D N C Y V I Y A L G P D G S G A G D D N C D V I Y V P G T D G N E E G D Y K N C D V I F V P K E R G S D E G D = = = = = = = Y E L W A T D Y T D V P A S C L E K Y E L W T T D Y D N I P A N C L N K Y E L W V S E D K I D K I P D C C K = = = = = = = = F N E Y A A G L P V R D V Y T F N E Y A V G R E T R D V F T F T M A Y F A Q Q Q E K T V R N V Y T D = = = = = = S D C L P E S A C L E S S C K P A P A Q N = = Identical residues are marked “=” below the three lines of sequence. The sequences were aligned as described above.
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