STROMAL CELLS DERIVED FROM NEONATAL FELINES AND USES THEREOF

Abstract

The present invention relates to a pharmaceutical composition comprising a population of stromal cells derived from neonatal felines and a pharmaceutically acceptable vehicle, in particular a solution comprising a cryoprotectant. The invention also relates to a population of neonatal stromal cells or a pharmaceutical composition comprising such a population for use in cell therapy, in particular allogeneic cell therapy, and more particularly for treating feline stomatitis. The invention also relates to a ready-to-use injectable solution comprising stromal cells derived from neonatal felines.

Claims

1. Pharmaceutical composition comprising a population of feline neonatal stromal cells (NSCs) and a pharmaceutically acceptable carrier.

2. Pharmaceutical composition according to claim 1, wherein said feline NSCs come from a sample of neonatal tissue.

3. Pharmaceutical composition according to claim 1, wherein said feline NSCs come from felines in the gestation period or from felines at the end of gestation.

4. Pharmaceutical composition according to claim 1, wherein said feline NSCs are placental feline NSCs.

5. Pharmaceutical composition according to claim 1, comprising between 5.Math.10.sup.4 and 4.Math.10.sup.7 cells/ml.

6. Pharmaceutical composition according to claim 1, characterised in that it is in frozen form.

7. A method of performing cell therapy in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a population of NSCs or a pharmaceutical composition of claim 1.

8. The method of claim 7, wherein the cell therapy is an allogenic cell therapy.

9. The method of claim 7, wherein the cell therapy is a xenogeneic cell therapy.

10. A method of treating a feline stomatitis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a population of NSCs or a pharmaceutical composition of claim 1.

11. The method of claim 10, wherein the feline stomatitis is a feline chronic gingivostomatitis (CGS).

12. The method of claim 7, wherein a single dose of 3.Math.10.sup.6 to 2.Math.10.sup.7 feline NSCs, is administered.

13. The method of claim 10, wherein the treatment of the feline stomatitis is carried out in a feline not having undergone dental avulsion or having undergone a partial dental avulsion.

14. Ready-to-use injectable solution comprising a single dose of 5.Math.10.sup.5 to 2.Math.10.sup.7 feline NSCs in solution with a cryoprotectant.

15. The pharmaceutical composition of claim 1, wherein pharmaceutical composition comprises a cryoprotectant.

16. The pharmaceutical composition according to claim 2, wherein the neonatal tissue is from one or more of: one or more placentas; one or more umbilical cords; a sample of neonatal biological fluid.

17. The pharmaceutical composition according to claim 16, wherein the neonatal biological fluid is blood of one or more umbilical cords and/or amniotic fluid.

18. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition comprises i) between 5.Math.10.sup.4 and 4.Math.10.sup.7 cells/ml; or ii) 1.Math.10.sup.7 cells/ml.

19. The method of claim 12, wherein the single dose is i) from 5.Math.10.sup.6 to 1.5.Math.10; or ii) 1.Math.10.sup.7 feline NSCs.

20. The ready-to-use injectable solution of claim 14, wherein the single dose comprises 1.Math.10.sup.7 feline NSCs.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0130] FIG. 1A shows the analysis of the feline NSCs by flow cytometry according to the parameters FSC-A/FSC-H allowing to select the unique cells. An analysis according to the parameters FSC-A/SSC-A allows to then select the NSC population of interest.

[0131] FIG. 1B, 1C, 1D, 1E, 1F show the evaluation of the expression of the marker of interest by the population of feline NSCs by analysis of the fluorescence for the corresponding fluorochrome. The graph shows the results of the surface markers (in light grey, the specific marker of interest; in dark grey the corresponding isotype). The positivity threshold is placed using the cells marked by the corresponding isotype. The table indicates the number of events analysed, as well as the median of fluorescence of the population of NSCs.

[0132] FIG. 1B shows the positivity of the expression of the surface marker CD44.

[0133] FIG. 1C shows the positivity of the expression of the surface marker CD8.

[0134] FIG. 1D shows the positivity of the expression of the surface marker CMH-II.

[0135] FIG. 1E shows the positivity of the expression of the surface marker CD29.

[0136] FIG. 1F shows the positivity of the expression of the surface marker CMH-I.

[0137] FIG. 1G shows the analysis of the PBMCs by flow cytometry according to the parameters FSC-A/FSC-H allowing to select the unique cells. An analysis according to the parameters FSC-A/SSC-A allows to then select lymphocytes.

[0138] FIG. 1H and 1I show the evaluation of the expression of the marker of interest by the lymphocytes, by analysis of the fluorescence for the corresponding fluorochrome. The graph shows the results of the surface markers (in light grey, the specific marker of interest; in dark grey the corresponding isotype). The positivity threshold is placed using the cells marked by the corresponding isotype. The table indicates the number of events analysed, as well as the median of fluorescence of the lymphocytes.

[0139] FIG. 1H shows the validation of the specificity of the marker CMH-II on cat lymphocytes.

[0140] FIG. 1I shows the validation of the specificity of the marker CMH-I on cat lymphocytes.

[0141] FIG. 2 shows the proliferative activity illustrated by the accumulation of the number of cell doublings during the passages.

[0142] FIG. 3 shows a photograph representative of the MSCs cultivated 2 weeks in an osteogenic differentiation medium and marked with Alizarin Red. The presence of calcium deposits is highlighted by the red colouring.

[0143] FIG. 4 shows a monitoring form completed by the owners of the animals included in the study.

[0144] FIG. 5 shows a photograph of the mouth of a cat having undergone dental avulsion and treated with the feline NSCs. This photograph shows the reduction in the oral lesions visible before (photograph on the left) and after treatment (photograph on the right).

[0145] FIG. 6 shows the change in the SDAI score of the cats having undergone dental avulsion and treated by the feline NSCs over time (days after perfusion). The median is represented by a horizontal line at the centre of the box plots.

[0146] FIG. 7 shows the change in the SDAI score over time presented for each cat included and monitored having undergone dental avulsion and treated by the feline NSCs.

[0147] FIG. 8 shows the clinical recovery rate calculated at each monitoring visit (D60, D90, D180) for the 7 cats having undergone dental avulsion and treated by the feline NSCs of the study.

[0148] FIG. 9 shows the owner score (/12) evaluated over time (injection D0, 15 days, 2 months, 3 months and 6 months) for each cat having undergone dental avulsion and treated by the feline NSCs.

[0149] FIG. 10 shows a photograph of the mouth of a cat having undergone partial dental avulsion and treated by the feline NSCs. This photograph shows the reduction in the visible oral lesions 15 days after treatment (photograph on the left) and 1 month after treatment (photograph on the right).

[0150] FIG. 11 shows the change in the SDAI score of the cats having undergone a partial dental avulsion and treated by the feline NSCs over time (days after perfusion).

[0151] FIG. 12 shows the clinical recovery rate calculated at each monitoring visit (D60, D90, D180) for the 2 cats having undergone a partial dental avulsion and treated by the feline NSCs of the study.

[0152] FIG. 13 shows the owner score (/12) evaluated over time (day of the perfusion, 15 days, 2 months, 3 months and 6 months) for each cat having undergone a partial dental avulsion and treated by the feline NSCs.

EXAMPLES

Example 1: Obtaining and Characterising Feline Neonatal Stromal Cells (NSCs)

A. Method for Obtaining a Population of Feline NSCs

Collecting Feline Embryonic Membranes

[0153] The feline extra-embryonic membranes (placenta, umbilical cord) are removed aseptically during elective ovariohysterectomy carried out on pregnant cats. In the context of an ovariohysterectomy or hysterectomy, the uterus plus or minus the clamped ovarian ducts are deposited in a transport box containing a buffered saline solution for example such as Dulbecco's phosphate (D-PBS) to be transported to the laboratory at a controlled temperature (4-12° C.).

Isolation of the Feline NSCs from Extra-Embryonic Tissue

[0154] The treatment of the extra-embryonic tissue is carried out at most in the 72 h following the removal. All of the steps of treating the tissue are carried out in a controlled environment, in a microbiological safety cabinet (BSC). The tissue is removed aseptically from its transport box and transferred to a Petri dish of 100 cm.sup.2. The amniotic sac containing the foetus is eliminated by dissection. The placenta having a red colour is transferred into a sterile beaker. The tissue is washed 3 to 5 times in successive baths of D-PBS. The placental tissue is dissected into fragments of approximately 10-20 mm.sup.2 then subjected to enzymatic digestion while incubating the tissue fragments in a solution composed of DMEM (Dulbecco's Modified Eagle Medium) containing 0.5-4 mg/ml of type I collagenase, and more specifically a concentration of 1 mg/ml of type I collagenase. The enzymatic digestion is carried out at 37° C. for 45 min but a digestion between 15 min and 16 h can be carried out by reducing the incubation temperature (ambient temperature (18-22° C. or 4° C.). At the end of the digestion, the enzymatic activity is stopped by dilution, by adding DMEM containing at least 10% foetal bovine serum (FBS) in an equivalent quantity to the solution for enzymatic digestion. The solution is then filtered over a 70-100 μm screen. The cells recovered are centrifuged at 800 g for 10 min. The cell pellet containing the neonatal stromal cells is rinsed in DMEM and again centrifuged at 800 g for 10 min. The cell pellet is replaced in culture medium consisting of DMEM, 10% FBS, 2 mM glutamine and from 0 to 20 ng/ml of fibroblast growth factor (FGF). The cells are counted and inoculated in culture dishes at a density of between 10.sup.4 and 2.Math.10.sup.4 cells/cm.sup.2. The cells are then cultivated in the culture medium described above in a controlled atmosphere at 37° C. and containing 5% CO.sub.2. The medium is changed after 48 h then every 2-3 days. The cells are passaged when the confluence reaches 70-80%.

Isolation of the Feline NSCs from Amniotic Fluid

[0155] The Treatment of the Amniotic Fluid

[0156] is carried out at most in the 24 h following the removal. All of the steps of treating the tissue are carried out in a controlled environment, in a microbiological safety cabinet (BSC). The clamped or sutured uterus, to avoid microbiological contaminations, is transferred under BSC to a 100 mm petri dish. The amniotic sac is separated from the uterus and transferred to a new 100 mm petri dish. The integral sac thus still containing the foetus is pierced using a chisel or scalpel to free the amniotic fluid. The amniotic sac and the foetus are transferred into another dish and the liquid is collected. Optionally, the amniotic fluids of several amniotic sacs can be combined in order to increase the volume of liquid to be treated. Culture medium consisting of DMEM, 10% FBS, 2 mM glutamine and from 0 to 20 ng/ml fibroblast growth factor (FGF) is added to the amniotic fluid in a minimum amount of 50% of the volume removed. Optionally, the suspension can be rinsed with DPBS and centrifuged at 800 g for 10 min. The volume of liquid or of cells re-suspended in an equivalent volume in culture medium is disposed directly in a culture dish in an amount of 100 to 3000 per cm.sup.2. The culture dishes are thus cultivated in the culture medium described above in a controlled atmosphere at 37° C. and containing 5% CO.sub.2. The medium is changed after 48 h then every 2-3 days. After 1-2 weeks of culture, the colonies of feline NSCs are passaged to allow cell amplification.

Cell Passage and Amplification

[0157] At sub-confluence, the cells undergo cell passage and optionally an amplification procedure. The feline NSCs are rinsed with D-PBS and treated with 0.05% trypsin-EDTA for 2-5 min at 37° C. This allows to detach the cells and form a population of isolated cells. The cells are then replaced in amplification medium consisting of DMEM, 10% FBS, 2 mM glutamine and from 0 to 20 ng/ml fibroblast growth factor (FGF) and centrifuged between 300-500 g for 5 to 10 min. The feline NSCs are replaced in amplification medium and counted by manual counting (trypan blue and Malassez cells) or using an electronic counter. They are then inoculated at a rate of 1500 to 5000 cells/cm.sup.2 and cultivated on a plastic cell culture substrate in amplification medium and in a controlled atmosphere at 37° C. and containing 5% CO.sub.2. During the process of amplification the cells can undergo between 0 and 15 cell passages.

Freezing and Cryopreservation of the Feline NSCs

[0158] After the first or second cell passage (P1-P2), the cells can be cryopreserved in seed units. To do this, after counting, the feline NSCs are centrifuged between 300-500 g for 5 to 10 min and the cell pellet is replaced in freezing medium composed either of DMEM medium enriched with 5-20% FBS and 5-10% (vol:vol) DMSO or in a commercial cryopreservation medium, containing a fraction of DMSO or not. The cell concentration is between 1.Math.10.sup.6 and 15.Math.10.sup.6 cells per ml of freezing medium. The freezing of the cells is carried out in conditions of controlled temperature decrease, by using for example a CoolCell® Cell Freezing Containers (BioCision) container and by following the freezing procedure as it is described by the manufacturer. The cells are then transferred for storage in negative cold at temperatures such as −196° C. for long-term storage (greater than 1 year).

[0159] The seed units are used to generate cell units for therapeutic purposes. The seed units are thawed to 37° C. for 3-6 min and amplified in vitro. The cells are inoculated in culture medium at the density of 1500-3000 cells/cm.sup.2. The cells are amplified by successive passage in vitro. When a significant number of cells is produced (for example >150.Math.10.sup.6 cells), the cells are frozen according to the protocol described above. The cells are distributed in hermetically sealed sealable flasks in an amount of 1.Math.10.sup.6-1.5.Math.10.sup.7 cells/ml in freezing medium free of product of animal origin (for example such as the cryopreservation medium Recovery™ Cell culture freezing medium (Thermo Fisher), Cryostem™ freezing medium (Biological Industries), Cryostor™ (Biolife Solution). The flasks are decreased in temperature according to a protocol of controlled temperature decrease, at a rate of −1° C./min to −80° C. The flasks are then transferred at −80° C. for storage for a maximum time of 24 months.

B. Characterisation of the Population of Feline NSCs

[0160] In order to evaluate the purity and the functionality of the feline NSCs isolated from feline placentas, tests are carried out on aliquots of cells currently being amplified or after freezing/thawing of a seed unit or of a therapeutic unit.

Cytometric Analysis of the Feline NSCs

[0161] The expression of surface markers of the feline NSCs is carried out by flow cytometry by using the following anti-feline antibodies: CD8 (vpg9; Biorad), CMH2 (vpg3; Biorad); and the antibodies specific to other species and crossing with the cat: CD44 (IM7; Biolegend); CD29 (TS2/16; Biolegend). For the unconjugated antibody anti-CMH2, a secondary mouse anti-immunoglobulin antibody (IgG) coupled with allophycocyanin (APC) (eBiosciences) is used afterwards. Isotypic controls are used to set the background noise for each fluorochrome used: anti-mouse IgG2a (COL2002/COLI205C; Monoclonal Antibody Center); anti-mouse IgG1 coupled with PE (MOPC-21; Biolegend); anti-rat IgG2b coupled with APC (IM7, Biolegend).

[0162] In summary, the feline NSCs in culture are detached from their plastic substrate by trypsinisation and are rinsed with D-PBS. The cells are distributed into tubes at a rate of 10.sup.5−5.Math.10.sup.5 cells/tube. The cells are centrifuged (500 g/5 min) and replaced in 25-1000 μl of marking buffer (D-PBS and from 0.5-1% (v/v) of bovine serum albumin (BSA) or from 0.5-2% (v/v) foetal bovine serum). The primary antibody coupled or not with a fluorochrome and specifically targeting the membrane marker of interest is added. The optimal concentration of antibody used for the marking must be previously determined by a person skilled in the art. The incubation necessary for the marking must also be determined by a person skilled in the art and be between 15 min and 10 h at 4° C. away from light. In particular, the cells are incubated 30 min at 4° C. away from light. Marking with a secondary antibody targeting the primary antibody can be carried out, after washing with D-PBS, in the case in which the primary antibody is not directly coupled with a fluorochrome. After each incubation with an antibody (primary and secondary) or the corresponding isotype, the cells are washed 2 times in 1 ml of D-PBS and centrifuged (500 g/5 min). The cell pellets are replaced in a volume of 100-500 μl of marking buffer for reading with a flow cytometer (Accuri C6, BD Biosciences).

[0163] FIGS. 1A to 1I show an example representative of the results of cytometric analysis of the feline NSCs.

[0164] Thus, the presence of the markers CD44 and CD29 is revealed, and the absence of the markers CMH-II and CD8 on the surface of the feline NSCs according to the invention.

Evaluation of the Proliferative Activity of the Feline NSCs

[0165] The proliferation of the feline NSCs is evaluated during 6 to 15 consecutive cell passages to determine the proliferative activity of the cells. At each cell passage (1 time per week; or every 6 to 8 days), the cells are detached from their culture substrate using trypsin/EDTA 0.5% for 2-3 min. Culture medium is added and the cells are centrifuged 5 min/300 g. The cell pellet is replaced in a defined volume of culture medium and the cells are counted by a trypan blue exclusion technique using an electronic counter. The doubling number at each cell passage is calculated according to the following formula: Nb of doublings=LOG (Nf/Ni)/LOG (2) (Nf: number of final cells and Ni: number of initial cells). The total number of cell doublings is equal to the sum of the number of cumulative doublings at each cell passage.

[0166] FIG. 2 illustrates the number of cumulative doublings over 8 cell passages of a line of feline NSCs.

[0167] Thus, the feline NSCs are capable of 30 consecutive doublings in the space of 7 weeks (at a rate of one passage per week).

Potential for Osteogenic Differentiation of the Feline NSCs

[0168] The feline NSCs are detached from the plastic substrate by trypsinisation and counted. The feline NSCs are inoculated at a density of between 1500 and 5000 cells/cm.sup.2 in a 6-well plate in amplification medium under a controlled atmosphere at 37° C. and containing 5% CO.sub.2. When the cells reach 50-75% confluence, the proliferation medium is removed and replaced with osteogenic differentiation medium composed of DMEM, 10% FBS, 2 mM glutamine, 0.1 μM dexamethasone (Sigma), 50 μM ascorbic acid 2-phosphate (Sigma) and 10 mM (β-glycerophosphate (Sigma). The medium is renewed 2 times/week, for a period of between 10 and 15 days.

[0169] At the end of the differentiation process, the wells are washed with D-PBS and the cells are fastened with for example a solution of 10% neutral buffered formalin for 1 h at minimum. Colouring with a solution of 1% (weight/volume) Alizarin Red is carried out to reveal the presence of calcium deposits. The wells are then rinsed in H.sub.2O and the colouring is analysed under a microscope. The presence of calcium deposits allows to conclude that there is a capacity to differentiate in the osteogenic line.

[0170] FIG. 3 shows an example of feline NSCs cultivated 14 days in osteogenic differentiation medium and coloured with Alizarin Red.

[0171] The presence of calcium deposits demonstrates that the feline NSCs have a capacity for osteogenic differentiation.

Example 2: Evaluation of the Clinical Effect of a Single Injection of Cryopreserved Feline NSCs for the Care for Feline CGS Refractory to the Conventional Treatments and Having Undergone a Total Dental Avulsion or a Partial Dental Avulsion

A. Programme of the Studies

[0172] This pilot clinical study is multicentric, non-randomised. The cats included in these studies (6 for total avulsion and 2 for partial avulsion) are owners' animals suffering from CGS, in a situation of therapeutic failure as specified in the inclusion criteria below. The inclusion criteria are specified in the following paragraph. There is no restriction in terms of sex, breed, weight of the animals.

[0173] Inclusion criteria: 1—Having inflammatory lesions specific to CGS (confirmed by a dentistry specialist); 2—Having tried other treatments such as ciclosporin, NSAIDs, antibiotics, IFN omega unsuccessfully; 3—Showing a persistence of the clinical signs after 2 months of treatment; 4—Dental extraction (partial or total) necessary and dating to more than 3 months; 5—Stopping the treatments with IFN or corticotherapy

[0174] Non-inclusion criteria: 1—Informed consent not signed by the owner; 2—Gestation, evolutive tumoral process, systemic infectious process, intercurrent disease that can interfere with the evaluation of the treatment; 3—Extreme physical weakening risking the life of the animal; 4—Administration of an unauthorised treatment during the study period (IFN or corticotherapy)

[0175] Criteria for exiting the study: 1—Restarting a treatment including ciclosporin, IFN, corticoids, lactoferrin during the study period; 2—Degradation of the general state during the trial leading to an extreme weakening risking the life of the animal; 3—Appearance of other intercurrent diseases that can interfere with the monitoring of the changes: evolutive tumoral process, systemic infectious process

[0176] The animals included in the study are evaluated by a veterinarian who rates the degree of severity of the diseases by using the Stomatitis Disease Activity Index (SDAI). This score allows to attribute a score of 0 (healthy animals) to 24 (severe CGS). Photographs of the mouth of the cat are taken at D0 and at the end of the study (6 months (M)) to verify the changes in the lesions. The owners of the animals included in the study receive a questionnaire to fill out to evaluate the activity, appetite, behaviour, and comfort of their animal. The animals receive the cell treatment the day of inclusion. The monitoring of the animals is planned at 15 days, 2 months, 3 months and 6 months post-inclusion. During the study, the animals are allowed to continue their non-steroidal anti-inflammatory treatment (NSAID) under the condition that the dose is specified in the study documents. The monitoring of the doses and the frequency of administration of the treatments during the study is an analysis criterion.

[0177] Table 1 summarises the calendar of the study and the various analyses carried out at each monitoring.

TABLE-US-00001 TABLE 1 perfusion 1.sup.st Post-treatment monitoring visit perfusion D 15 D 60 D 90 D 180 Owner informed X Informed consent X obtained Inclusion X Perfusion of MSCs X Monitoring X Clinical evaluation X X X X X by the veterinarian investigator = SDAI Weight X X X X X Owner evaluation X X X X X

[0178] The criteria for evaluating effectiveness are:

1. The evaluation of the clinical score (SDAI) during the study (15 d, 2 mo, 3 mo, 6 mo)
2. The evaluation of the owner score during the study (15 d, 2 mo, 3 mo, 6 mo)
3. The change in the medicinal needs of the cats during the study.

B. Evaluation of the treatment

Veterinarian Evaluation

[0179] The clinical evaluation is carried out by a veterinarian specialised in dentistry, who holds a specialist diploma from the European college (European Veterinary Dental College (EVDC)). The monitoring is programmed at 2 weeks, 2 months, 3 months and 6 months post-treatment. The veterinarian fills out a clinical monitoring form including the evaluation criteria described in detail below, the weight, the current treatments.

[0180] The main criterion for evaluating the effectiveness is the improvement of the clinical signs. This evaluation is based on the activity index of the CGS (SDAI) taking into account various parameters: [0181] Oral inflammation (maxillary and mandibular) (0 none, 1 slight, 2 moderate, 3 severe); [0182] Gingival inflammation (maxillary and mandibular) (0 none, 1 slight, 2 moderate, 3 severe); [0183] The inflammation at the palatoglossal arch (0 none, 1 slight, 2 moderate, 3 severe); [0184] The inflammation of the salivary gland (0 none, 1 slight, 2 moderate, 3 severe); [0185] The oropharyngeal inflammation (0 none, 1 slight, 2 moderate, 3 severe); [0186] The lingual or sublingual inflammation (0 none, 1 slight, 2 moderate, 3 severe).

Owner Evaluation

[0187] The owners are also asked to carry out an evaluation of their cat on the criteria of activity, appetite, behaviour and comfort. Each parameter is scored from 0 to 3, resulting in a score of 12.

[0188] FIG. 4 shows the monitoring form filled out by the owners of the animals included in the study.

Preparation of the Cellular Product

[0189] A dose of feline NSCs cryopreserved at −196° C. is thawed to 37° C. for 3-5 minutes in the laboratory the day before the administration of the treatment in conditions guaranteeing the asepsis of the product. An aliquot of the cellular product is sampled to verify the viability and the quantity of cells by the technique of trypan blue exclusion. The specifications of the product post-thawing are the following: viability greater than 80%, and total number of viable cells at least 10.sup.7. The precise number of viable cells counted is presented in table 2.

[0190] Table 2 below illustrates the results of the cell viability tests upon thawing.

TABLE-US-00002 TABLE 2 CAT ID viability number of viable cells total avulsion 1 93.6 10.56 2 92.7 10 3 92 10.47 4 92 10.6 5 87.6 12.5 6 87.6 12.5 average 90.92 11.11 standard 2.64 1.10 deviation partial avulsion 1 91.1 10.59 2 92.3 13.23 average 91.70 11.91

[0191] The cells compliant with the specifications are transferred into a sterile sealable flask. The product packaged in its flask is transferred into a transport box with controlled temperature (4-12° C.). The treatment is delivered to the veterinary clinic for administration to the patient at D+1 or D+2.

Administration of the Feline NSCs

[0192] The animal can be sedated for the control of the correct execution of the perfusion. The veterinarian chooses the most suitable site to fasten the veinous catheter to the animal. A bag of perfusion solution (Ringer's lactate) is used in combination with heparin. The bag is then homogenised by inversion, then the veterinarian connects the intravenous set to the bag of solution.

[0193] The cellular product (between 1.0.Math.10.sup.7 and 1.4.Math.10.sup.7 viable cells for an average of 1.2.Math.10.sup.7.) is taken out of its package 15 min before the injection. The veterinarian homogenises the cellular suspension inside the flask by rotation. A syringe having a suitable volume with a needle of 18 G-20 G (70 mm) is prepared to suck up the cellular suspension inside the flask. The syringe is then connected onto the injection site at the bottom of the nozzle. The veterinarian slowly pushes the piston in order to eject approximately 0.2-0.5 ml of the suspension every 5 min. The totality of the feline NSCs is administered in approximately 20-30 min. The animals are awoken and kept at the clinic for 4 h minimum to verify their physiological parameters.

Analysis of the Data

[0194] The main evaluation criterion is the SDAI clinical score defined by the veterinarian during each monitoring. The significance of the variability of the SDAI over time is compared to the score upon inclusion in the study (D0) using a non-parametric Friedman test.

C. Results of the Study

[0195] Information on the subjects included in the study with complete or partial dental avulsion.

[0196] Eight cats were recruited over a period of 10 months in 3 investigating centres: 6 females and 2 males. The average age of the cats included in the study is 6.8 years (3-11 years). No cat was excluded from the study during the 6 months. Among the cats included in the study, 6 had undergone a total dental avulsion and 2 cats a partial dental avulsion. Four cats did not take any anti-inflammatories (NSAIDs) upon inclusion; 2 cats took NSAIDs on demand; and 2 cats followed a continuous treatment with NSAIDs.

[0197] Table 3 describes in detail the characteristics of the animals included in the study with complete or partial dental avulsion.

TABLE-US-00003 TABLE 3 CAT age Investigating Weight ID breed (year) gender centre (kg) Total avulsion 1 Unkn. 6 female 1 3.6 2 European 3 female 1 5.4 3 Unkn. 5 female 1 2.6 4 European 11 female 1 3 5 European 10 female 1 5.3 6 Unkn. 4.5 female 2 4.15 Partial avulsion 1 European 7 male 3 4.4 2 European 8 male 2 5.8

[0198] During the administration of the product, one cat vomited. An anti-vomiting drug was prescribed to it. No other undesirable event was observed during the perfusions and the monitoring of the animals included. The animals monitored during or after the perfusion did not show modifications of their physiological parameters: heart rate, respiratory rate.

[0199] Table 4 summarises the treatments administered by the owners of the cats during the 6 months of monitoring.

TABLE-US-00004 TABLE 4 inclusion D 15 D 60 D 90 D 180 TOTAL AVULSION 1 none none none none none 2 NSAID NSAID NSAID NSAID NSAID Buprenor- (1/day) (1/day) (¾ dose) (½ dose) phine 3 NSAID NSAID none NSAID none upon demand 4 none none none none none 5 none none none none none 6 none NSAID NSAID none none (1/month) (1/month) PARTIAL AVULSION 1 NSAID unkn. NSAID unkn. Diluted upon demand injection NSAID every 10 1/month days 2 NSAID NSAID 3xmorphine Antibiotics IFN upon demand upon NSAID upon for 1 week gabapentin demand demand IFN NSAID upon gabapentin gabapentin demand IFN gabapentin

[0200] Four of the six cats with total avulsion did not take NSAIDs upon inclusion. Three of these 4 cats did not take any treatment over the 6 months of monitoring (#1, 4 and 5). One cat resorted to 2 NSAID treatments over the first two months, then did not take any more (#6).

[0201] Two cats with partial avulsion and two cats with total avulsion took NSAID treatment upon inclusion. One of the cats with total avulsion markedly reduced the dose and the frequency of administration (#2), one cat with total avulsion did not take NSAIDs any more at 6 months (#3). The cats with partial avulsion either reduced the dose and the frequency of NSAIDs (#1) or stopped taking NSAIDs at 6 months (#2).

[0202] Change in the veterinarian clinical score of the cats included with complete dental avulsion.

[0203] Of the 6 cats having undergone a complete dental avulsion, 100% showed a favourable clinical change after the administration of feline NSCs. The clinical observations showed a reduction in the oral lesions as illustrated in FIG. 5.

[0204] The reduction of the SDAI clinical score confirms the favourable change in the animals treated. FIG. 6 illustrates the change in the SDAI score over time. A non-significant reduction of the SDAI was observed 15 days post-treatment (median score at D15: 9.5 [4;14] vs median score at D0: 11 [9;13]). Two months post-treatment, a significant reduction in the score is observed (median score at M2: 5 [0;13] vs median score at D0: 11 [9;13]; p=0.0325). At three months post-treatment, a significant reduction in the score is observed (median score at M3: 6.5 [2; 9] vs median score at D0: 11 [9; 13]; p=0.0325). At 6 months, the signifcantness disappears (p=0.143) but the effect of the treatment seems to continue.

[0205] FIG. 7 illustrates the change in the SDAI per cat during the study. A complete resolution of the clinical signs 2 months post-administration in one cat (#4) is observed. In this cat, a slight oropharyngeal inflammation is observed which reappears at 3 and 6 months (score 2/24). At the monitoring at 3 months, a slight degradation in one cat (#3, score goes from 5 to 9) is observed, then the score decreases again to reach 7 at 6 months. An improvement in the state of the other cats is also observed.

[0206] FIG. 8 represents the rate of clinical recovery of each animal having undergone a total avulsion at the various monitoring times. The recovery rate corresponds to the difference (in percentage) between the SDAI score evaluated at a monitoring time (2 months, 3 months, 6 months) and the SDAI score upon inclusion of the same animal, divided by the SDAI score upon inclusion. This ratio takes into account the improvement that occurred, if there is one. It is observed that a single cat (#6) slightly degraded at 2 months, then improved by 30% with respect to the inclusion starting at the monitoring at 3 months. One cat (#4) showed a complete recovery (clinical remission) at the monitoring at 2 months.

[0207] Owner monitoring of the cats included with complete dental avulsion.

[0208] The owner evaluation is based on 4 main criteria: appetite, activity, comfort and behaviour. Each parameter is graded out of /3, and thus allows to obtain a score of 12. The more the score decreases, the more the behaviour of the cat improves. The change is shown in FIG. 9. Between the inclusion and the monitoring at 6 months, an improvement of the score of all the cats included is observed except for cat #5, who only shows a transient improvement. Three cats are considered to be healthy starting at 15 days (#4), starting at 2 months (#1) or starting at 6 months (#6). One cat (#6) included with a score of 2 (few clinical signs) degraded at D15 and at 2 months to finally reach a healthy-cat score at 6 months (score=0). Finally, as observed with the SDAI scores, cat #3 degraded at 3 months to come back to its inclusion score then improved again between 3 and 6 months.

[0209] Change in the veterinarian clinical score of the cats included with partial dental avulsion.

[0210] The 2 cats having undergone a partial dental avulsion also showed a favourable clinical change after the administration of feline NSCs. FIG. 10 shows a reduction in the oral lesions at 1 month after the injection.

[0211] FIG. 11, showing the change in the SDAI for the two cats having undergone a partial dental avulsion shows an almost complete resolution of the clinical signs at 6 months post-administration with a score of 3. While cat #1 showed a fast clinical improvement starting at two weeks, cat #2 showed a recurrence of the symptoms at 2 months that greatly decreased afterwards.

[0212] FIG. 12 shows the rate of clinical recovery of each animal having undergone a partial avulsion at the various monitoring times. For both cats, an improvement in the recovery rate can be observed starting at 90 days.

[0213] Owner monitoring of the cats included with partial dental avulsion.

[0214] FIG. 13 shows the change in the owner score for the two cats with partial dental avulsion. Here again, cat #2 showed a transient reduction in the symptoms to be later considered as healthy at 6 months after injection. The state of health of cat #1 quickly improved after two weeks to reach an owner score of 2 at 6 months.

Conclusion

[0215] All of this data shows that a single perfusion of feline NSCs is safe for the animal and leads to a clinical improvement in all the cats diagnosed with chronic gingivostomatitis and not having responded to the various conventional treatments. Moreover, this clinical improvement can be observed starting at 2 months and the clinical benefit is observed over a period of at least 6 months.

LIST OF THE CITED DOCUMENTS

Patent Documents

[0216] For all intents and purposes, the following patent document(s) are cited: [0217] US20160199414A1 (publication number); and [0218] WO2017062475A1 (publication number).

Non-Patent Literature

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