USE OF NMN FOR THE PREVENTION AND/OR TREATMENT OF PAIN, AND CORRESPONDING COMPOSITIONS

Abstract

The invention relates to nicotinamide mononucleotide, a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, for the use thereof in the prevention and/or treatment of pain, in particular nociceptive pain; the invention relates as well to compositions that comprise the same.

Claims

1. Nicotinamide mononucleotide (NMN), a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, for the use thereof in the prevention and/or treatment of pain.

2. Nicotinamide mononucleotide (NMN), a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, for the use thereof according to claim 1 in which the NMN derivative may be selected from among alpha nicotinamide mononucleotide (α-NMN), dihydronicotinamide mononucleotide (denoted as NMN-H), the compound having the formula (I): ##STR00072## or a stereoisomer thereof, a salt thereof, a hydrate thereof, a solvate thereof, or a pharmaceutically acceptable crystal thereof, in which: X is selected from among O, CH.sub.2, S, Se, CHF, CF.sub.2 and C═CH.sub.2; R.sub.1 is selected from among H, azido, cyano, (C.sub.1-C.sub.8) alkyl, (C.sub.1-C.sub.8) thio-alkyl, (C.sub.1-C.sub.8) heteroalkyl, and OR; wherein R is selected from H and (C.sub.1-C.sub.8) alkyl; R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are selected independently of one another, from among H, halogen, azido, cyano, hydroxyl, (C.sub.1-C.sub.12) alkyl, (C.sub.1-C.sub.12) thio-alkyl, (C.sub.1-C.sub.12) heteroalkyl, (C.sub.1-C.sub.12) haloalkyl, and OR; wherein R is selected from among H, (C.sub.1-C.sub.12) alkyl, C(O)(C.sub.1-C.sub.12)alkyl, C(O)NH(C.sub.1-C.sub.12)alkyl, C(O)O(C.sub.1-C.sub.12)alkyl, C(O)aryl, C(O)(C.sub.1-C.sub.12)alkyl aryl, C(O)NH(C.sub.1-C.sub.12)alkyl aryl, C(O)O(C.sub.1-C.sub.12)alkyl aryl, and C(O)CHR.sub.AANH.sub.2; wherein R.sub.AA is a side chain selected from a proteinogenic amino acid; R.sub.6 is selected from among H, azido, cyano, (C.sub.1-C.sub.8) alkyl, (C.sub.1-C.sub.8) thio-alkyl, (C.sub.1-C.sub.8) heteroalkyl, and OR; wherein R is selected from H and (C.sub.1-C.sub.8) alkyl; R.sub.7 is selected from among H, P(O)R.sub.9R.sub.10, and P(S)R.sub.9R.sub.10; in which R.sub.9 and R.sub.10 are selected independently of one another, from among OH, OR.sub.11, NHR.sub.13, NR.sub.13R.sub.14, a (C.sub.1-C.sub.8) alkyl, a (C.sub.2-C.sub.8) alkenyl, a (C.sub.2-C.sub.8)alkynyl, (C.sub.3-C.sub.10) cycloalkyl, a (C.sub.5-C.sub.12) aryl, (C.sub.1-C.sub.8)alkyl aryl, (C.sub.1-C.sub.8) aryl alkyl, (C.sub.1-C.sub.8) heteroalkyl, (C.sub.1-C.sub.8) heterocycloalkyl, a heteroaryl, and NHCHR.sub.AR.sub.A′C(O)R.sub.12; in which: R.sub.11 is selected from among a group: (C.sub.1-C.sub.10) alkyl, (C.sub.3-C.sub.10) cycloalkyl, (C.sub.5-C.sub.18) aryl, (C.sub.1-C.sub.10) alkylaryl, substituted (C.sub.5-C.sub.12) aryl, (C.sub.1-C.sub.10) heteroalkyl, (C.sub.3-C.sub.10) heterocycloalkyl, (C.sub.1-C.sub.10) haloalkyl, a heteroaryl, —(CH.sub.2).sub.nC(O)(C.sub.1-C.sub.15)alkyl, —(CH.sub.2).sub.nOC(O)(C.sub.1-C.sub.15)alkyl, —(CH.sub.2).sub.nOC(O)O(C.sub.1-C.sub.15)alkyl, —(CH.sub.2).sub.nSC(O)(C.sub.1-C.sub.15)alkyl, —(CH.sub.2).sub.nC(O)O(C.sub.1-C.sub.15)alkyl, and —(CH.sub.2).sub.nC(O)O(C.sub.1-C.sub.15)alkyl aryl; wherein n is an integer selected from 1 to 8; P(O)(OH)OP(O)(OH).sub.2; halogen, nitro, cyano, C.sub.1-C.sub.6 alkoxy, C.sub.1-C.sub.6 haloalkoxy, —N(R.sub.11a).sub.2, C.sub.1-C.sub.6 acylamino, —OCOR.sub.11b; NHSO.sub.2(C.sub.1-C.sub.6 alkyl), —SO.sub.2N(R.sub.11a).sub.2SO.sub.2; wherein each of R.sub.11a is independently selected from H and a (C.sub.1-C.sub.6) alkyl, and R.sub.11b is independently selected from OH, C.sub.1-C.sub.6 alkoxy, NH.sub.2, NH(C.sub.1-C.sub.6 alkyl) or N(C.sub.1-C.sub.6 alkyl).sub.2; R.sub.12 is selected from among H, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.8 alkynyl, C.sub.1-C.sub.10 haloalkyl, C.sub.3-C.sub.10 cycloalkyl, C.sub.3-C.sub.10 heterocycloalkyl, C.sub.5-C.sub.18 aryl, C.sub.1-C.sub.4 alkylaryl, and C.sub.5-C.sub.12 heteroaryl; wherein the said aryl or heteroaryl groups are optionally substituted with one or two groups selected from among halogen, trifluoromethyl, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkoxy, and cyano; and R.sub.A and R.sub.A′ are independently selected from among H, a (C.sub.1-C.sub.10) alkyl, (C.sub.2-C.sub.10) alkenyl, (C.sub.2-C.sub.10) alkynyl, (C.sub.3-C.sub.10) cycloalkyl, (C.sub.1-C.sub.10) thio-alkyl, (C.sub.1-C.sub.10) hydroxylalkyl, (C.sub.1-C.sub.10) alkylaryl, and (C.sub.5-C.sub.12) aryl, (C.sub.3-C.sub.10) heterocycloalkyl, a heteroaryl, —(CH.sub.2).sub.3NHC(═NH)NH.sub.2, (1H-indol-3-yl)methyl, (1H-imidazol-4-yl)methyl, and a side chain selected from among a proteinogenic amino acid or a non-proteinogenic amino acid; wherein the said aryl groups are optionally substituted with a group selected from among hydroxyl, (C.sub.1-C.sub.10) alkyl, (C.sub.6-C.sub.1) alkoxy, a halogen, a nitro, and a cyano; or R.sub.9 and R.sub.10 form, together with the phosphorus atoms to which they are attached, a 6-membered ring in which —R.sub.9—R.sub.10— represents —CH.sub.2—CH.sub.2—CHR—; wherein R is selected from among H, a (C.sub.5-C.sub.6) aryl group, and (C.sub.5-C.sub.6) heteroaryl group, wherein the said aryl or heteroaryl groups are optionally substituted by a halogen, trifluoromethyl, a (C.sub.1-C.sub.6) alkyl, a (C.sub.1-C.sub.6) alkoxy, and cyano; or R.sub.9 and R.sub.10 form, together with the phosphorus atoms to which they are attached, a 6-membered ring in which —R.sub.9—R.sub.10— represents —O—CH.sub.2—CH.sub.2—CHR—O—; wherein R is selected from among H, a (C.sub.5-C.sub.6) aryl group, and (C.sub.5-C.sub.6) heteroaryl group, wherein the said aryl or heteroaryl groups are optionally substituted by a halogen, trifluoromethyl, a (C.sub.1-C.sub.6) alkyl, a (C.sub.1-C.sub.6) alkoxy, and cyano; R.sub.8 is selected from among H, OR, NHR.sub.13, NR.sub.13R.sub.14, NH—NHR.sub.13, SH, CN, N.sub.3, and halogen; wherein R.sub.13 and R.sub.14 are selected independently of one another, from among H, (C.sub.1-C.sub.5) alkyl, (C.sub.1-C.sub.5) alkyl aryl, and —CR.sub.BR.sub.C—C(O)—OR.sub.D in which R.sub.B and R.sub.C are independently a hydrogen atom, a (C.sub.1-C.sub.6) alkyl, a (C.sub.1-C.sub.6) alkoxy, benzyl, indolyl, or imidazolyl; where the (C.sub.1-C.sub.6) alkyl and the (C.sub.1-C.sub.6) alkoxy may be optionally and independently of one another substituted by one or more of the halogen, amino, amido, guanidyl, hydroxyl, thiol, or carboxyl groups, and the benzyl group is optionally substituted by one or more halogen or hydroxyl groups; or R.sub.B and R.sub.C form, together with the carbon atom to which they are attached, a C.sub.3-C.sub.6 cycloalkyl group optionally substituted by one or more halogens, amino, amido, guanidyl, hydroxyl, thiol, and carboxyl; and R.sub.D is a hydrogen, a (C.sub.1-C.sub.6) alkyl, a (C.sub.2-C.sub.6) alkenyl, a (C.sub.2-C.sub.6) alkynyl, or a (C.sub.3-C.sub.6) cycloalkyl; Y is selected from among CH, CH.sub.2, C(CH.sub.3).sub.2 and CCH.sub.3; represents a single or a double bond along Y; and represents the alpha or beta anomer depending on the position of R.sub.1; or a stereoisomer thereof, a salt thereof, a hydrate thereof, a solvate thereof, or a crystal thereof; or the compound having the formula (II): ##STR00073## or a stereoisomer thereof, a salt thereof, a hydrate thereof, a solvate thereof, or a crystal thereof; in which X′.sub.1 and X′.sub.2 are independently selected from among O, CH.sub.2, S, Se, CHF, CF.sub.2, and C═CH.sub.2; R′.sub.1 and R′13 are independently selected from among H, azido, cyano, a C1-C8 alkyl, a C1-C8 thio-alkyl, a C1-C8 heteroalkyl, and OR, wherein R is selected from H and a C1-C8 alkyl; R′.sub.2, R′.sub.3, R′.sub.4, R′.sub.5, R′.sub.9, R′.sub.10, R′.sub.11, R′.sub.12 are independently selected from among H, a halogen, an azido, a cyano, a hydroxyl, a C.sub.1-C.sub.12 alkyl, a C.sub.1-C.sub.12 thioalkyl, a C.sub.1-C.sub.12 hetero-alkyl, a C.sub.1-C.sub.12 haloalkyl, and OR; wherein R may be selected from among H, a C.sub.1-C.sub.12 alkyl, a C(O)(C.sub.1-C.sub.12) alkyl, a C(O)NH(C.sub.1-C.sub.12) alkyl, a C(O)O(C.sub.1-C.sub.12) alkyl, a C(O) aryl, a C(O)(C.sub.1-C.sub.12) aryl, a C(O)NH(C.sub.1-C.sub.12) alkyl aryl, a C(O)O(C.sub.1-C.sub.12) alkyl aryl, or a C(O)CHR.sub.AANH2 group; wherein R.sub.AA is a side chain selected from a proteinogenic amino acid; R′.sub.6 and R′.sub.8 are independently selected from among H, an azido, a cyano, a C.sub.1-C.sub.8 alkyl and OR, wherein R is selected from H and a C.sub.1-C.sub.8 alkyl; R′.sub.7 and R′.sub.14 are independently selected from among H, OR, NHR, NRR′, NH—NHR, SH, CN, N.sub.3 and halogen; wherein R and R′ are independently selected from H and un (C.sub.1-C.sub.8) alkyl aryl; Y′.sub.1 and Y′.sub.2 are independently selected from among CH, CH.sub.2, C(CH.sub.3).sub.2, or CCH.sub.3; M′ is selected from H or a suitable counter ion; represents a single or double bond depending on Y′.sub.1 and Y′.sub.2; and represents an alpha or beta anomer depending on the position of R′.sub.1 and R′.sub.13; and combinations thereof.

3. Nicotinamide mononucleotide, a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, for the use thereof according to claim 1 in which the precursor is selected from nicotinamide riboside or dihydronicotinamide riboside.

4. Nicotinamide mononucleotide, a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, for the use thereof according to claim 1, in which the pain is not a neuropathic pain.

5. Nicotinamide mononucleotide, a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, for the use thereof according to claim 1, in which the pain is a nociceptive pain.

6. Nicotinamide mononucleotide, a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, for the use thereof according to claim 1, in order to reduce allodynia.

7. Nicotinamide mononucleotide, a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, for the use thereof according to claim 1, in order to reduce hyperalgesia.

8. Nicotinamide mononucleotide, a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, for the use thereof according to claim 4 in which the pain is a visceral pain.

9. Nicotinamide mononucleotide, a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, for the use thereof according to claim 8 in which the pain is a pain caused by a urinary tract infection.

10. Nicotinamide mononucleotide, a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, for the use thereof according to claim 1, in combination with at least one other therapeutic agent.

11. Nicotinamide mononucleotide, a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, for the use thereof according to claim 10, in which the at least one additional therapeutic agent is selected from among antibiotics, antifungals, antivirals, and combinations thereof.

12. Nicotinamide mononucleotide, a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, for the use thereof according to claim 10, in which the at least one therapeutic agent is an analgesic.

13. A composition comprising nicotinamide mononucleotide, a pharmaceutically acceptable precursor thereof, a pharmaceutically acceptable derivative thereof, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient, for the use thereof according to claim 1.

14. A composition according to claim 13 further comprising at least one additional therapeutic agent.

Description

FIGURES

[0292] FIG. 1 is a graph showing the nociceptive score and nociceptive threshold induced by the administration of cyclophosphamide in rats versus animals treated with the carrier.

[0293] FIG. 2 is a graph showing the evolution of the nociceptive score and of the nociceptive threshold in the rats, 2 hours and 4 hours after the administration of cyclophosphamide as compared with the carrier.

[0294] FIG. 3 shows the nociceptive threshold in animals treated with cyclophosphamide to whom the carrier, the NMN, or the ibuprofen was administered at T0, T=2 h, or T=4 h after administration of cyclophosphamide.

[0295] FIG. 4 shows the nociceptive score in the animals treated with cyclophosphamide to whom the carrier, the NMN, or the ibuprofen was administered at T0, T=2 h, or T=4 h after the administration of cyclophosphamide.

[0296] FIG. 5A is a graph showing the baseline nociceptive threshold for each experimental group.

[0297] FIG. 5B is a graph showing baseline nociceptive scores for all of the experimental groups.

[0298] FIG. 6A is a graph showing the nociception threshold for the effects of NMN, Compound A, and Compound B on allodynia induced by CYP at 2 hours.

[0299] FIG. 6B is a graph showing the nociceptive threshold for the effects of NMN, Compound A, and Compound B on allodynia induced by CYP at 4 hours.

[0300] FIG. 7A is a graph showing the effects of NMN on visceral pain induced by CYP at 2 hours (nociceptive scores).

[0301] FIG. 7B is a graph showing the effects of NMN on Visceral pain induced by CYP at 4 hours (nociceptive scores).

[0302] FIG. 8A is a graph showing the effects of Compound A on visceral pain induced by CYP at 2 h (nociceptive scores).

[0303] FIG. 8B is a graph showing the effects of Compound A on Visceral pain induced by CYP at 4 h (nociceptive scores).

[0304] FIG. 9A is a graph showing the effects of Compound B on Visceral pain induced by CYP at 2 h (nociceptive scores).

[0305] FIG. 9B is a graph showing the effects of Compound B on visceral pain induced by CYP at 2 h (nociceptive scores).

EXAMPLES

[0306] In the remainder of this description, the examples provided are intended by way of illustration of the present invention and are in no way intended to limit the scope thereof.

Example 1

[0307] The effectiveness of the use of NMN according to the invention was evaluated in rats in a model of nociceptive pain. More precisely, the model used is a visceral pain model. The administration of cyclophosphamide (CYP) serves as the means to simulate cystitis in rats.

[0308] The positive control is ibuprofen, a non-steroidal anti-inflammatory drug frequently prescribed to relieve nociceptive pain. The negative control is the carrier for NMN and ibuprofen, that is to say, distilled water.

[0309] For this study, 7-week-old female Sprague-Dawley rats were divided into three groups, each group comprising of 6 rats: [0310] a control group who received 5 ml of distilled water (carrier); [0311] a group treated with NMN at 500 mg/kg; and [0312] a group treated with ibuprofen at 300 mg/kg.

[0313] The NMN is in the form of zwitterion.

[0314] After 24 hours of adaptation, a test to measure the threshold of pain tolerance of each animal was carried out with von Frey filaments prior to the start of the study. This measurement prior to any exposure to a painful stimulus makes it possible to obtain the baseline pain tolerance threshold level in the animals and serves as a negative control.

[0315] Von Frey filaments are used as a device for measuring the sensitivity of the skin to touch. The use of von Frey filaments makes it possible to test for allodynia and hyperalgesia in rodents. Briefly put, each filament corresponds to a given force. The filaments are applied in ascending order of increasing force to the animal's skin. The rodents in effect have a reflex to withdraw when an unexpected contact occurs. The withdrawal in response to a force exerted on the rodent is indicative of the animal's threshold of tolerance to pain. The use of von Frey filaments is commonly implemented for measurement of pain in rodents (Deuis J R, Dvorakova L S and Vetter I (2017) Methods Used to Evaluate Pain Behaviors in Rodents. Front. Mol. Neurosci. 10:284.).

[0316] In the present study, eight filaments in ascending order of increasing force, of 1 g, 2 g, 4 g, 6 g, 8 g, 10 g, 15 g, and 26 g, were used and applied three times each to the animal's abdomen, near the bladder. The animal's response was assessed as follows: [0317] score 0: no response [0318] score 1: retraction of the abdomen [0319] score 2: stamping or changing of position of the animal [0320] score 3: twitching, or curving or rounding of the abdomen, or licking of the area stimulated by the von Frey filament.

[0321] For each rat, the results are expressed as follows: [0322] nociceptive threshold: first force level at which a response from the animal is observed (response score greater than or equal to 1). This indicates the lowest threshold of tolerance to pain for measuring allodynia; [0323] nociceptive score: percentage of the maximum response for each filament. This indicates the overall pain response.

[0324] The animals of each of the groups receive either the carrier, or NMN at 500 mg/kg, or ibuprofen at 300 mg/kg administered via the oral route. After administration of the compounds to be tested (carrier, NMN, or ibuprofen), cyclophosphamide is injected via the intraperitoneal route into each rat. The cyclophosphamide induces strong inflammation in the bladder and simulates a pain induced by a urinary tract infection such as cystitis.

[0325] The test with the von Frey filaments is repeated at 2 hr and then subsequently at 4 hr after injection of the cyclophosphamide in order to measure the pain response of the animals.

[0326] The results were analysed by a one-way analysis of variance (ie one-way ANOVA) test supplemented by a Dunnett's test or by a two-way ANOVA analysis. With regard to statistical significance, * signifies that p<0.05, ** signifies that p<0.01, and *** signifies that p<0.001, as compared to the group treated with the carrier.

[0327] As may be seen in FIG. 1A, the administration of cyclophosphamide elicits a response from the rats immediately upon application of the first filament of 1 g force, while the response from the animals prior to exposure shows that the animals exhibit no response to pain until 10 g prior to administration of cyclophosphamide: the injection of cyclophosphamide therefore lowers the allodynia threshold. This result is corroborated by FIG. 1B which shows that the administration of cyclophosphamide reduces the pain tolerance threshold from 10 g to 3 g. In other words, the allodynia threshold is significantly reduced by the injection of cyclophosphamide in rats. The animals who are treated with cyclophosphamide are therefore more sensitive to pain.

[0328] FIGS. 2A and 2B show that the lowering of the allodynia threshold continues, with the animals showing even lower tolerance to pain 4 h after the injection of cyclophosphamide as compared to 2 h after injection.

[0329] FIG. 1A also shows that at equal force, the animals feel greater pain: the injection of cyclophosphamide therefore triggers hyperalgesia. FIG. 2B shows that this effect persists over time, with the pain scores measured in the treated rats after 4 hours being higher than those from the measurement carried out 2 hours after the injection.

[0330] The injection of cyclophosphamide therefore induces on the one hand a reduction in allodynia and an increase in hyperalgesia in the treated animals, with the effects being more accentuated over time.

[0331] FIGS. 3 and 4 show the effects of administration of NMN and ibuprofen on the pain thresholds and scores in the rats treated with cyclophosphamide.

[0332] As shown in FIGS. 3A, 3B, and 3C, the administration of NMN provides the ability to increase the response threshold to nociceptive pain significantly, at T0, T=2 h after injection, and T=4 h after injection. In an expected manner, ibuprofen also provides the ability to significantly increase the threshold of tolerance to pain in rats treated with cyclophosphamide at T0, T=2 hours after injection, and T=4 hours after injection.

[0333] These results show that the administration of NMN provides the ability to reduce allodynia.

[0334] As shown in FIG. 4A, the NMN, the ibuprofen, and the carrier show similar curves of nociceptive scores at T0, at the time instant of injection of cyclophosphamide. This demonstrates that the animals do not develop pain in response to the administration of NMN or ibuprofen. On the other hand, FIGS. 4B and 4C show that the administration of NMN serves as the means for reducing the nociceptive score 2 hours and 4 hours after the administration of cyclophosphamide, in the same way as ibuprofen.

[0335] The administration of NMN and of a composition comprising the same therefore makes it possible to reduce allodynia and hyperalgesia to a significant extent, and in a manner similar to ibuprofen.

Example 2

[0336] Synthesis of the Compound of the Invention

[0337] Materials and Methods

[0338] All the reagents were obtained from commercial suppliers and used without any further purification. Thin layer chromatography was carried out on TLC silica gel 60 F254 plastic sheets (0.2 mm layer thickness) from Merck. Purification by column chromatography was carried out on silica gel 60 (70-230 mesh ASTM, Merck). The melting points were determined either on a digital device (Electrothermal IA 8103) and are not corrected, or on a Kofler heating bench of type WME (Wagner & Munz). The .sup.1H, .sup.19F, and .sup.13C nuclear magnetic resonance (NMR) and infrared (IR) spectra confirmed the structures of all of the compounds. The IR spectra were recorded on a Perkin Elmer Spectrum 100 FT-IR spectrometer; and the NMR spectra were recorded, using CDCl.sub.3, CD.sub.3CN, D.sub.2O or DMSO-d.sub.6 as solvent, on a BRUKER AC 300 or 400 spectrometer at 300 or 400 MHz for the .sup.1H spectra, 75 or 100 MHz spectra for the .sup.13C spectra, and 282 or 377 MHz for the .sup.19F spectra. The chemical shifts (δ) were expressed in parts per million relative to the signal, indirectly (i) with CHCl.sub.3 (δ 7.27) for .sup.1H; and (ii) with CDCl.sub.3 (δ 77.2) for .sup.13C; and directly (iii) with CFCl.sub.3 (internal standard) (δ 0) for .sup.19F. The chemical shifts are provided in ppm and the peak multiplicities are denoted as follows: s, singlet; br s, broad singlet; d, doublet; dd, doublet of doublets; t, triplet; q, quartet; quint, quintet; m, multiplet. High-resolution mass spectra (HRMS) were obtained from the “Service central d'analyse de Solaize” (French National Center for Scientific Research—Solaize) and were recorded on a Waters spectrometer using electrospray ionisation time-of-flight (ESI-TOF) mass spectrometry.

[0339] Protocol

[0340] Step 1: Synthesis of the Compound Having the Formula X-1

[0341] The compound having the formula XIV (1.0 equiv.) is dissolved in dichloromethane. The nicotinamide having the formula XV (1.50 equiv.) and the TMSOTf (1.55 equiv.) are added at ambient temperature. The reaction mixture is heated under reflux and stirred until completion of the reaction. The mixture is cooled to ambient temperature and filtered. The filtrate is concentrated to dryness so as to give crude nicotinamide riboside tetraacetate having the formula X-1.

[0342] Step 2: Synthesis of the Compound Having the Formula X

[0343] The crude NR tetraacetate having the formula X-1 is dissolved in methanol and cooled to −10° C. This is followed by addition of 4.6 M ammonia in methanol (3.0 equivalents) at −10° C. and the mixture is stirred at this temperature until completion of the reaction. Dowex HCR (H.sup.+) is added until a pH of 6-7 is attained. The reaction mixture is heated to 0° C. and filtered. The resin is washed with a mixture of methanol and acetonitrile. The filtrate is concentrated until it becomes dry. The residue is dissolved in acetonitrile and concentrated to solid content dryness. The residue is dissolved in acetonitrile so as to give a solution of crude nicotinamide riboside triflate having the formula X.

[0344] Step 3: Synthesis of the Compound Having the Formula XI

[0345] The solution of crude nicotinamide riboside triflate in acetonitrile is diluted with trimethyl phosphate (10.0 equivalents). The acetonitrile is distilled under vacuum and the mixture is cooled to −10° C. Phosphorus oxychloride (4.0 equiv.) is added at −10° C. and the mixture is stirred at −10° C. until completion of the reaction.

[0346] Step 4 and Step 5: Synthesis of the Compound Having the Formula I-A

[0347] The mixture is hydrolysed by adding a 50/50 mixture of acetonitrile and water, followed by the addition of methyl tert-butyl ether (or tert-butyl methyl ether). The mixture is filtered and the solid is dissolved in water. The aqueous solution is neutralised by adding sodium bicarbonate and extracted with dichloromethane. The aqueous layer is concentrated to dryness so as to give a crude mixture of NMN and di-NMN having the formula I-A.

[0348] Isolation of Di-NMN Having the Formula I-A:

[0349] The NMN and the di-NMN having the formula I-A are separated by purification on Dowex 50wx8 with elution of water. The fractions containing di-NMN are concentrated to solid content dryness. The residue is purified by column chromatography on silica gel (isopropanol/water gradient). The pure fractions are combined and concentrated. The residue is lyophilised so as to give di-NMN as a beige solid.

[0350] Biological Data

[0351] The objective of the present study is to evaluate the effects of oral administration of nicotinamide mononucleotide (NMN), alpha-NMN (compound A) and compound I-A (compound B) at 500 mg/kg on the visceral pain response in the model of acute cystitis induced by cyclophosphamide (CYP) in female Sprague-Dawley rats.

[0352] Materials and Methods

[0353] Animals: Sprague-Dawley female rats, 7 weeks old as of their birth

[0354] Pharmacological treatment: [0355] NMN: 500 mg/kg [0356] alpha-NMN: 500 mg/kg [0357] Compound I-A: 500 mg/kg [0358] Carrier: distilled water [0359] Route of administration: per os [p.o.] (by oral administration), 5 ml/kg [0360] Frequency of administration: once on D0, 15 min prior to the intraperitoneal injection (i.p.) of CYP.

[0361] Acute Cystitis Induced by CYP: The CYP was injected via intraperitoneal injection at 150 mg/kg in a final volume of 5 ml/kg of saline solution.

[0362] Mechanical Stimulation Using Von Frey Filaments

[0363] The rats were placed in individual Plexiglas boxes with a metal wire mesh floor and allowed to adapt to the chamber for a period of at least 30 minutes prior to the commencement of any testing. Eight von Frey filaments with increasing levels of force viz 1, 2, 4, 6, 8, 10, 15, and 26 g were used. Each calibrated filament was applied 3 times to the lower abdominal area, near the bladder.

[0364] Evaluation of Nociceptive Behaviours for Each Application [0365] Score 0=no response [0366] Score 1=retraction of the abdomen [0367] Score 2=stamping or changing of position [0368] Score 3=wheezing or squealing, or abdominal curvature, or licking of the site stimulated with von Frey filaments

[0369] For each rat, the results were expressed as follows: [0370] Nociceptive threshold: first von Frey force level for which the stimulus is perceived as being painful (the score 1 is obtained)=>lowered threshold=allodynia [0371] Nociceptive score: % of the maximum response (total=9 for 3 combined applications) for each filament=>Overall response to pain

[0372] Experimental Groups (6 Rats Per Group): [0373] Group 1: Carrier (5 ml/kg)+CYP [0374] Group 2: NMN (500 mg/kg)+CYP [0375] Group 3: Compound A (500 mg/kg)+CYP [0376] Group 4: Compound B (500 mg/kg)+CYP

[0377] Results

[0378] Baseline nociceptive parameters (prior to CYP injection) for all the experimental groups: The results show (FIGS. 5A and 5B) that the baseline nociceptive responses were similar among all of the experimental groups (prior to the injection of CYP).

[0379] The visceral pain induced by the CYP 2 hours and 4 hours post injection (as compared to the baseline value in the carrier treated group): The results show that as compared to the baseline response, the CYP (150 mg/kg, i.p.) induced a significant decrease in the nociceptive threshold (FIG. 2A) and a significant increase in the nociceptive scores (FIG. 2B) at the two time points.

[0380] Effects of the NMN, Compound A, and Compound B on the CYP-Induced Allodynia (Nociceptive Threshold): The results show that, as compared to the carrier: [0381] The NMN (500 mg/kg, p.o.) resulted in a slight increase in the nociceptive threshold at +2 hr (FIG. 6A) and +4 hr (FIG. 6B) with an effect just above the margin of statistical significance at +4 hr (p=0.063); [0382] Compound A (500 mg/kg, p.o.) resulted in an increase in the nociceptive threshold at +2 hr (FIG. 6A) and +4 hr (significant at +4 hr) (FIG. 6B); [0383] Compound B (500 mg/kg, p.o.) resulted in a significant increase in the nociceptive threshold only at +4 hr (FIG. 6B) and induced no effect at +2 hr (FIG. 6A).

[0384] Effects of the NMN, Compound A, and Compound B on the CYP-Induced Visceral Pain (Nociceptive Scores): The results show (FIGS. 7, 8 and 9) that as compared to the carrier: [0385] The NMN (500 mg/kg, p.o.) led to a significant decrease in the nociceptive scores at +2 hr (FIG. 7A) and +4 hr (FIG. 7B); [0386] Compound A (500 mg/kg, p.o.) led to a decrease in the nociceptive scores at +2 hr (FIG. 8A) and +4 hr (FIG. 8B) which only achieved the level of statistical significance at +4 h; [0387] Compound B (500 mg/kg, p.o.) led to a significant decrease in the nociceptive scores at +4 hr (FIG. 9B) (no effect was observed at +2 hr (FIG. 9A)).

[0388] Summary of Results

[0389] The baseline nociceptive responses were similar in all of the experimental groups (prior to the injection of CYP).

[0390] In comparison with the baseline response, the effects of CYP (150 mg/kg, i.p.) at 2 and 4 hours were characterised by: [0391] A significant decrease in the nociceptive threshold at +2 hours and +4 hours; [0392] A significant increase in the nociceptive scores at +2 hours and +4 hours.

[0393] As compared to the carrier, in the rats injected with CYP, the effects of the NMN (500 mg/kg, p.o.) led to: [0394] a slight increase in the nociceptive threshold at +2 hr and +4 hr with an effect just above the margin of statistical significance at +4 hr (p=0.063) [0395] a significant decrease in the nociceptive scores at +2 hr and +4 hr.

[0396] As compared to the carrier, in the rats who received an injection of CYP, the effects of Compound A (500 mg/kg, p.o.) were characterised by: [0397] An increase in the nociceptive threshold at +2 hr and +4 hr with an effect which only achieved the level of significance at +4 hr; [0398] A decrease in the nociceptive scores at +2 hr and +4 hr which only achieved the level of statistical significance at +4 hr.

[0399] As compared to the carrier, in the rats injected with CYP, the effects of Compound B (500 mg/kg, p.o.) led to: [0400] A significant increase in the nociceptive threshold at +4 hr (no effect was observed at +2 hr); [0401] A significant decrease in the nociceptive scores at +4 hr (no effect was observed at +2 hr).

CONCLUSION

[0402] A single intraperitoneal injection of CYP (150 mg/kg) induced a visceral pain 2 hours and 4 hours after the injection with a more pronounced effect at +4 hours.

[0403] A single oral treatment of NMN (500 mg/kg) relieved the visceral pain induced by the CYP at the two time points, with a higher level of significance obtained at +4 hr.

[0404] The administration of alpha-NMN (Compound A) reduced the visceral pain induced by the CYP at +2 hr and +4 hr, with its effects however, only achieving the level of significance at +4 hr.

[0405] In the rats injected with CYP, the oral treatment with Compound I-A (Compound B) had no beneficial effect at +2 hr but showed significant anti-nociceptive activity at a later time point (that is to say, +4 hr).

[0406] The inventors have therefore demonstrated that the use of NMN and the pharmaceutically acceptable derivatives thereof, such as alpha NMN and Compound I-A, as well as the compositions that comprise the same in accordance with the invention, provide the ability to reduce nociceptive pain, and more particularly visceral pain induced by cystitis.

[0407] The use of NMN and the pharmaceutically acceptable derivatives thereof, such as alpha NMN and Compound I-A, as well as the compositions that comprise the same in accordance with the invention, therefore provide the ability to treat and prevent pain, in particular nociceptive pain.