Treatment of Placental Chronic Histiocytic Intervillositis Using an Inhibitor of Interleukin-1

Abstract

The present invention concerns the use of an inhibitor of interleukin-(IL-1), in particular of IL-1α and/or IL-1β, for the prevention or treatment of chronic histiocytic intervillositis (CHI) or a symptom associated thereof, eventually in combination with the use of at least one molecule conventionally prescribed to treat CHI and/or an interleukin-18 (IL-18) inhibitor. Said inhibitor of interleukin-1 (IL-1), in particular of IL-1α and/or IL-1β, may also be used for diagnosing in vitro CHI in a subject suspected of suffering from CHI or for monitoring in vitro the effectiveness of a treatment for CHI in a subject in need thereof.

Claims

1. A method of treating chronic histiocytic intervillositus (CHI) or at least one associated symptom, comprising administering an inhibitor of IL-1α and/or IL-1ß, wherein said symptom is at least partly due to CHI.

2. The method according to claim 1, wherein said inhibitor is an anti-IL-1 antibody.

3. The method according to claim 2, wherein said anti-IL-1 antibody is an anti-IL-1ß antibody.

4. The method according to claim 3, wherein said anti-IL-1ß antibody is Canakinumab.

5. The method according to claim 1, wherein said inhibitor is an antagonist of IL-1 receptor.

6. The method according to claim 3, wherein said antagonist of IL-1 receptor is anakinra.

7. The method according to claim 1, wherein the associated symptom is selected from the group consisting of early miscarriage, in utero growth retardation, and in utero fetal death.

8. (canceled)

9. (canceled)

10. The method to claim 1, wherein the inhibitor of IL-1α and/or IL-1ß is administered together, concomitantly or sequentially with a molecule selected from the group consisting of aspirin, low molecular weight heparins, hydroxychloroquine, a corticosteroid, azathioprine, a polyvalent immunoglobulins, and an anti-TNF compounds.

11. The method according to claim 1, wherein the inhibitor of IL-1α and/or IL-1ß is administered together, concomitantly or sequentially with an inhibitor of interleukin-18 (IL-18).

12. The method according to claim 11, wherein the inhibitor of IL-18 is an anti-IL-18 antibody.

13. A method for diagnosing in vitro CHI in a subject comprising the steps of: a. measuring an expression level E1 of IL-1α and/or IL-1ß in a sample subject; and b. comparing of the expression level E1 to a reference expression level E2 of IL-1α and/or IL-1ß, in a sample from a healthy subject, wherein a value E1 greater for IL-1α and/or IL-1ß than a value E2 is the indication of a subject suffering from CHI.

14. A method for monitoring in vitro the effectiveness of a treatment for CHI in a subject, comprising the steps of: a. measuring an expression level E1′ of IL-1α and/or IL-1ß in a sample of a subject suffering from CHI before the subject receiving any treatment for CHI; b. measuring an expression level E2′ of IL-1α and/or IL-1ß in a sample of the subject suffering from CHI after beginning treatment for CHI; and c. comparing expression levels E1′ and E2′, wherein a value E2′ less than a value E1′ indicates effective treatment.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0033] FIG. 1 represents violin plot of differential expression levels of proteins involved in the inflammasome pathway (CASP1, PYCARD, NOD2, NLRP3) of healthy placentas (control, CTRL) versus those with CHI (intervillitis, CHI) (*: p<0.05; **: p<0.01).

[0034] FIG. 2 represents violin plot of differential expression levels of IL-1β and IL-18 mRNA of healthy placentas (control, CTRL) versus those with CHI (intervillitis) (*: p<0.05; **: p<0.01).

[0035] FIG. 3 represents (A) the immunohistochemical labelling of the IL-1β protein in tissue sections of CHI placentas (intervillitis, CHI) compared to samples of healthy placentas (control, CTRL) (B) the number of IL-1β R clusters in tissue sections of CHI placentas (intervillitis, CHI) compared to samples of healthy placentas (control, CTRL).

EXAMPLES

Example 1: Differential Expression of mRNA of the Inflammasome Activation Pathway of CHI Placentas Compared to Healthy Placentas

[0036] In the context of the present invention, RNA from 18 paraffinized samples of CHI placentas (intervillitis) grade II or III and 6 paraffinized samples of healthy placentas (control) was extracted with Qiagen RNeasy FFPE kit. For each patients, two 20 μm placenta tissue sections were used. After the deparaffinization step, tissues were digested with proteinase K. Supernatants were treated with DNAse I. Total RNA were then treated trough RNeasy MinElute column and eluted with 30 μl of RNase-free water.

[0037] The transcriptomic analysis of the 24 patients samples was made according to the manufacturer Nanostring protocol by using the PanCancer Immune Profiling Pattern. The samples were analysed with the nCounter XT Gene Expression Assays leading to the mRNA counts for each sample.

[0038] The statistical analysis was made with the nSolver Advanced analysis software. Four different plug-ins were used to analyse the data: plug-in Overview, plug-in Normalization, plug-in Gene set analysis and plug-in Cell type profiling.

[0039] The results obtained are shown in the table 1 below.

TABLE-US-00001 TABLE 1 Differential p. adjusted rRNAm expression Fold change value NOD2 (NLRC2) yes 6.21 0.00705 NLRC5 yes 4.54 0.0218 NLRP3 yes 3.14 0.0422 PYCARD (ASC) yes 5.68 0.0088

[0040] The results showed a significative overexpression of of the actors of the inflammasome activation pathway: PYCARD (Fold Change=5.58: 0.01), NLRP3 (Fold Change=3.14; p<0.05), NLRC5 (Fold Change=4.54; p<0.05) et NOD2 (Fold Change=6.21; p<0.01) in pathological placentas compared to healthy placentas.

[0041] The significant overexpressions of PYCARD, NOD2 and NLRP3 mRNA were represented in violin plots in FIG. 1.

Example 2: Expression Level of IL-1β and IL-18 mRNA of CHI Placentas Compared to Healthy Placentas

[0042] In the context of the present invention, RNA from 18 paraffinized samples of CHI placentas (intervillitis) grade II or III and 6 paraffinized samples of healthy placentas (control) was extracted with Qiagen RNeasy FFPE kit. For each patients, two 20 μm placenta tissue sections were used. After the deparaffinization step, tissues were digested with proteinase K. Supernatants were treated with DNAse I. Total RNA were then treated trough RNeasy MinElute column and eluted with 30 μl of RNase-free water.

[0043] The transcriptomic analysis of the 24 patients samples was made according to the manufacturer Nanostring protocol by using the PanCancer Immune Profiling Pattern. The samples were analysed with the nCounter XT Gene Expression Assays leading to the mRNA counts for each sample.

[0044] The statistical analysis was made with the nSolver Advanced analysis software. Four different plug-ins were used to analyse the data: plug-in Overview, plug-in Normalization, plug-in Gene set analysis and plug-in Cell type profiling.

[0045] The results obtained are shown in the table 2 below.

TABLE-US-00002 TABLE 2 Differential p. adjusted rRNAm expression Fold change value IL-1β yes 5.66 0.00498 IL-18 yes 3.96 0.0343

[0046] The results showed a significative overexpression of the cytokines produced by the inflammasome actors in pathological placentas: IL-1β (Fold change=5.66; p<0.01) and 11-18 (Fold Change=3.96; p<0.05).

[0047] The significant overexpressions of IL-1β and IL-18 mRNA were represented in violin plots in FIG. 2.

Example 3: Overexpression Level of IL-1β Protein in CHI Placentas Compared to Healthy Placentas

[0048] In the context of the present invention, the IL-1β protein expression level was analysed by immunohistochemistry.

[0049] All samples have been fixed in 10% buffered formalin. Immunohistochemical staining was performed for all samples with anti-IL1β antibody (Cell Signaling®, clone 3A6, dilution 1/100).

[0050] Formalin-fixed and paraffin-embedded tissues were cut (3 μm thickness) and mounted onto slides. Specimens were deparaffinated (OTTIX) for 6 min. After rinsing in distilled water, slides were incubated at 95-100° C. for 20 min with unmasking solution. After rinsing in TBS-Tween (0.1%), endogenous peroxidase activity was blocked by 3% H.sub.2O.sub.2 for 10 min at room temperature. Blocking buffer was added onto the sections of the slides in a humidified chamber at room temperature for 20 min. Then, 100 μl diluted primary antibody were added to the sections on the slides and incubated overnight in a humidified chamber at 4° C. After rinsing, 100 μl appropriately diluted secondary antibody were added to the sections, slides were incubated in a humidified chamber at room temperature for 30 min. After washing, 100 μl DAB substrate solution were applied. Slides were counterstained with hematoxylin and mounted. Immunohistochemically stained slides were reviewed without knowledge of the diagnosis. Numbers of aggregates were evaluated in 50 high power fields for each sample.

[0051] The labelling was made according to the ImmPress manufacturer protocol.

[0052] The antibodies used are described in table 3.

TABLE-US-00003 TABLE 3 Secondary Target Provider Reference Dilution Antibody IL-1β Cell signaling 3A6 1/100 mouse Mouse VectorLab ImmPRESS — — secondary HRP Reagent antibody kit MP-7402

[0053] The labelling was analysed in a blinding way by an anatomopathologist specialized in placenta analysis.

[0054] The labelling results are shown in FIG. 3A. The results showed that the IL-1β protein is localized in the syncytial knots and in the fibrinoid substance deposits.

[0055] The quantitative analysis showed in FIG. 3B that the IL-1β protein is overexpressed in CHI placentas (180 IL-1β clusters counted for fifty high power fields analysed) compared to healthy placentas (81 IL-1β clusters counted for fifty high power fields analysed)

Example 4: Evaluation of the Interest of the Biopharmaceutical Drug Kineret® (Anakinra) in the Prevention of Recurrence of Chronic Histiocytic Intervillitis

[0056] This is a phase I/II pilot clinical trial regarding recurrent CHI. Although this is a rare disease, patients with recurrent CHI are frequently sent to referral centers due to the therapeutic impasse and are seeking new therapeutic strategies.

[0057] Although data on anakinra during pregnancy are limited, no effect of anakinra has been reported in terms of fertility, embryo-fetal, peri- or post-natal development (rats and rabbits) at supra-therapeutic doses. The literature provides reassuring data on the tolerance of this molecule in humans during pregnancy, for conditions for which anakinra cannot be suspended during pregnancy (Still's disease, periodic syndrome associated with cryopyrin).

[0058] The main objective of this study is therefore to demonstrate that ankinra can be a promising therapy for the prevention of recurrent CHI, but secondary purposes are also to evaluate the effect of anakinra during pregnancy in terms of: obstetrical outcome, clinical-biological tolerance of a the molecule during pregnancy and within 12 months postpartum, and impact of the treatment on placental anatomo-pathology.

[0059] The cohort studied comprises 15 patients with a history of recurrent CHI: at least two documented episodes of CHI leading to obstetric complications (spontaneous miscarriage, late fetal loss, intrauterine growth retardation, fetal death in utero, pre-eclampsia). CHI is diagnosed during histological examination of the placenta of the previous pregnancy according to the classification of Rota et al. (J. Gynecol. Obstet. Biol. Reprod., 35(7): 711-719, 2006) [6]. It is to be noted that minor patients, patients unable to give informed consent, patients having an immunosuppressive treatment during pregnancy, patients with immune deficiency and patients with known hypersensitivity to anakinra, are excluded from the study. This inclusion period of patients lasts 24 months.

[0060] Patients randomized to the anakinra group receive anakinra at a dose of 100 mg/day subcutaneously once daily from the beginning of pregnancy (no later than 8 weeks of gestation) and up to 34 weeks of amenorrhea. This participation period lasts in fact 21 months (duration of pregnancy and 12 months of post-partum follow-up).

[0061] The pregnancy outcome is evaluated according to a main judgment criterion based on the live birth rate for these patients at high risk of recurrence of CHI (rate which may exceed 60%), as well as to secondary judgment criteria based on obstetrical complications (spontaneous miscarriages, vasculoplacental unsufficiency, fetal death in utero, prematurity), maternal infectious complications during pregnancy and during post-partum, fetal malformations, pediatric infectious complications in the first year of life, and histogical examination of the placenta and CHI grade evaluation if present.

LIST OF REFERENCES

[0062] 1. Doss et al., Hum. Pathol., 26(11): 1245-1251, 1995 [0063] 2. Labarrere et Mullen, Am. J. Reprod. Immunol. Microbiol., 15(3): 85-91, 1987 [0064] 3. Marchaudon et al., Placenta, 32(2): 140-145, 2011 [0065] 4. Boyd et al., Hum. Pathol., 31(11): 1389-1396, 2000 [0066] 5. Mekinian et al., Autoimmunity, 48(1): 40-45, 2015 [0067] 6. Rota et al., J. Gynecol. Obstet. Biol. Reprod., 35(7): 711-719, 2006