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WO2020009566A1 - Méthodes de traitement de la sarcoïdose - Google Patents

Méthodes de traitement de la sarcoïdose Download PDF

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Publication number
WO2020009566A1
WO2020009566A1 PCT/NL2019/050394 NL2019050394W WO2020009566A1 WO 2020009566 A1 WO2020009566 A1 WO 2020009566A1 NL 2019050394 W NL2019050394 W NL 2019050394W WO 2020009566 A1 WO2020009566 A1 WO 2020009566A1
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Prior art keywords
cells
sarcoidosis
jak1
inhibitor
thl
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Inventor
Mirjam KOOL
Jelle Rindert MIEDEMA
Rudolf Wilhelmus HENDRIKS
Caroline Elizabeth BROOS
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Erasmus University Medical Center
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Erasmus University Medical Center
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Priority claimed from PCT/NL2018/050437 external-priority patent/WO2019132654A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/437—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/541—Non-condensed thiazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the invention is in the field of medical diagnosis and monitoring of disease progression.
  • the present invention pertains to methods of predicting disease progression in non-treated pulmonary sarcoidosis (PS) patients (wherein presence of the prognostic biomarker is indicative of chronic disease), to methods of devising treatment plans for PS patients (comprising measuring the prognostic biomarker and devising treatment based on the outcome).
  • the present invention pertains to methods of identifying patients with PS eligible for treatment with an anti- PS drug comprising testing the patient for the prognostic biomarker, wherein the patient is eligible for treatment if the biomarker is present.
  • the invention further provides for methods of treating PS, comprising testing for the presence of biomarker to confirm presence or stage of disease and administering a therapeutically effective amount of an anti-PS drug to the patient if the sample tests positive.
  • the invention further provides for methods of treating PS comprising administering a specific interleukin 23 blocker, or administering a JAK/STAT inhibitor.
  • PS Pulmonary sarcoidosis
  • Non-necrotizing granulomas are the key pathological feature. These aggregates of differentiated immune cells contain a core with alveolar macrophages, multinucleated giant cells and a shell comprised of mostly T- cells.
  • Diagnosis of PS is estabhshed when clinicoradiological findings are supported by histological evidence of noncaseating epithelioid cell granulomas.
  • the diagnosis of pulmonary sarcoidosis is preferably conform guidelines of the American Thoracic Society (ATS), the European
  • ERS Respiratory Society
  • WASOG World Association of Sarcoidosis and Other Granulomatous Disorders
  • immunological features are depression of cutaneous delayed-type
  • Thl helper T cell type 1
  • corticosteroids notably prednisolone.
  • Additional treatment with non corticosteroids such as methotrexate (MTX) or tumor necrosis factor (TNF)— inhibitor therapy (e.g. infliximab or adalimumab) is available.
  • MTX methotrexate
  • TNF tumor necrosis factor
  • a prognostic biomarker could offer steroid dose-sparing possibilities to patients not at risk of developing chronic disease.
  • the present inventors have found that the number of T helper cells of the Thl7 lineage that produce interferon, and in particular their quantity within the population of all memory CD4 + T cells is predictive of a chronic disease pattern in sarcoidosis.
  • the present invention provides a method for typing a pulmonary sarcoidosis (PS) patient as having an increased risk of developing a chronic, disease pattern, the method comprising the steps of: a) enumerating in a sample obtained from said patient the ThlT. l cells within said population of total CD4 + T cells, optionally including
  • the chronic disease pattern is a progressive disease pattern.
  • the control value can be any group of subjects not suffering from chronic or progressive sarcoidosis.
  • healthy human subjects can provide very suitable control values.
  • samples obtained from individuals who undergo bronchoscopy for non-related illnesses, such as for community-acquired pneumonia or chronic obstructive pulmonary disease may serve as control values in aspects of this invention.
  • the methods may comprise the step of a) enumerating in a sample obtained from said patient the total memory CD4 + T cells and the Thl7.1 cells within said population of total memory CD4 + T cells; and the step of b) calculating the proportion of Thl7.1 cells to the total memory CD4 + T cells. It was found that the quantity of Th 17.1 cells within the
  • ThlT. l cells may also occur within the population of total CD4 + T cells, in particular in samples known to contain high proportions of memory CD4 + T cells as part of the total CD4+ T cell population.
  • embodiments wherein ThlT. l cells are enumerated as part of the total CD4 + T cells are particularly useful in applications utilizing BALF as a sample, since >95% of all CD4 + T cells in BALF is memory CD4 + T cell.
  • the invention provides a method as described above wherein the method further comprises, in addition to enumerating the Thl7.1 cells in step a), also, and preferably as a part of step a), the step of enumerating the Thl cells within said population of total (memory) CD4 + T cells in said sample.
  • a method of the invention as described above comprises the step of a) enumerating in a sample obtained from said patient the total memory CD4+ T cells the Thl 7.1 cells, and the Thl cells within said population of total memory CD4+ T cells.
  • a method of the invention as described above further comprises the step of b) calculating the proportion of Thl 7.1 cells to the total memory CD4+ T cells.
  • a method of the invention as described above further comprises the step of c) calculating the proportion of Thl cells to the total memory CD4+ T cells.
  • a method of the invention as described above further comprises the step of d) calculating the ratio between the proportions calculated in steps b) and c).
  • a method of the invention as described above further comprises the step of e) comparing the ratio obtained in step d) to a control value.
  • a method of the invention as described above further comprises the step of f) typing said patient as having an increased risk of developing a chronic, preferably a chronic progressive, disease pattern if said ratio is increased relative to the control value.
  • the sample in a method as described above is a tissue or tissue aspirate sample, preferably a bronchoalveolar lavage fluid (BALF) or a mediastinal lymph node (MLN) sample, preferably from a non-treated pulmonary sarcoidosis (PS) patient.
  • BALF bronchoalveolar lavage fluid
  • MN mediastinal lymph node
  • control sample is a corresponding (same sample site) sample from a healthy subject , a subject not suffering from chronic and/or progressive sarcoidosis, a sarcoidosis patient experiencing disease resolution, or a pooled sample of either or a mixture of these.
  • the present invention provides the use of Thl7.1-cells or ratio of Thl7.1/Thl populations as predictive/prognostic biomarker in pulmonary sarcoidosis.
  • the predictive biomarker used herein is the BALF IL-12p40 protein level, which level is highest in patients developing chronic disease.
  • the present invention provides a method of treating of treating sarcoidosis in a subject in need thereof, comprising: administering to a subject in need thereof, a therapeutically effective dose of a specific interleukin 23 blocker, preferably selected from tildrakizumab, and guselkumab. This treatment is based on altering Th 17-cell
  • a specific IL-23 blocker only targets IL-23. Such a specific blocker is preferred over dual blockers, which may also block other interleukines.
  • the present invention provides a specific interleukin 23 blocker, preferably selected from tildrakizumab and guselkumab, for use in the treatment of pulmonary sarcoidosis.
  • cytokine receptor signaling such as the IFN-g receptor
  • the Janus kinase (JAK)/Signal Transducer and Activator of Transcription Factor (STAT)-l axis is upregulated in sarcoidosis and IFN-g induces STAT1 expression.
  • This provides a link between Thl7.1 cells and JAK/STAT signaling, and possible implications for suppressive effects of pathway on Thl7-cell differentiation.
  • Altering Thl7-cell differentiation e.g. IL-23 blockade
  • effector cytokine signaling e.g. JAK/STAT
  • non pulmonary sarcoidosis diseases are contemplated for indications in aspects of this invention.
  • pulmonary sarcoidosis may, in aspects of his invention be extended to applications of non pulmonary sarcoidosis.
  • pulmonary sarcoidosis is a preferred embodiment.
  • the present invention provides a method of treating sarcoidosis in a subject in need thereof, comprising: administering to a subject in need thereof, a therapeutically effective dose of a JAK/STAT inhibitor, preferably baricitinib.
  • a JAK/STAT inhibitor preferably baricitinib.
  • Baricitinib is know to interfere in effector cytokine signaling (e.g. the IFN-g signaling pathway, i.e. the effector cytokine produced by Thl7.1 cells). This treatment aspect is therefore based on altering Th 17-cell effector cytokine signaling.
  • the present invention provides a method of treating sarcoidosis, comprising administering to a subject in need thereof, a therapeutically effective dose of a JAK/STAT inhibitor, most preferably a JAK1 inhibitor.
  • the sarcoidosis is chronic sarcoidosis.
  • the JAK/STAT inhibitor interferes with IFN-g receptor signaling.
  • the JAK/STAT inhibitor is a JAK1 inhibitor.
  • the JAK1 inhibitor may, e.g., be a JAK1 -selective inhibitor, or a JAK1-JAK2 inhibitor (i.e. an inhibitor that inhibits both JAK1 and JAK2), or a JAK1-JAK3 inhibitor (i.e. an inhibitor that inhibits both JAK1 and JAK3).
  • the JAK/STAT inhibitor is selected from Baricitinib, Tofacinib, Peficitinib, Filgotinib, and Upadacitinib.
  • the JAK/STAT inhibitor is Baricitinib.
  • Baricitinib is administered at an oral dose of 1-10 mg once daily.
  • the JAK/STAT inhibitor is Filgotinib.
  • Filgotinib is administered at an oral dose of 1-500, preferably 50-200 mg, such as 80-100 mg once daily.
  • the JAK/STAT inhibitor is Tofacitinib.
  • Tofacitinib is administered at an oral dose of 1-10 mg, preferably about 5 mg twice daily, preferably in immediate release form; or about 10 mg once daily, preferably in extended release form.
  • the JAK/STAT inhibitor is Peficitinib.
  • Peficitinib is administered at an oral dose of 100-150 mg once daily.
  • the JAK/STAT inhibitor is Upadacitinib.
  • Upadacitinib is administered at an oral dose of 15-30 mg once daily.
  • the JAK/STAT inhibitor is selected from tofacinib, peficitinib, fdgotinib, and upadacitinib.
  • the JAK/STAT inhibitor in particular the JAK1 inhibitor, is not baricitinib. In certain another preferred embodiment of aspects of this invention, the JAK/STAT inhibitor, in particular the JAK1 inhibitor, is not fdgotinib. In certain another preferred embodiment of aspects of this invention the JAK/STAT inhibitor, in particular the JAK1 inhibitor, is not Ruxolitinib.
  • the JAK2 inhibiting effect of a JAK1-JAK2 inhibitor, or the JAK3 inhibiting effect of a JAK1-JAK3 inhibitor is not relied on for therapeutic efficacy.
  • the JAK1 inhibitor is preferably as selective as possible for inhibiting the JAK1 target.
  • the present invention provides a JAK/STAT inhibitor, most preferably a JAK1 inhibitor, for use in the treatment of sarcoidosis.
  • the sarcoidosis is pulmonary sarcoidosis, most preferably chronic pulmonary sarcoidosis.
  • the JAK/STAT inhibitor interferes with IFN-g receptor signaling.
  • the JAK/STAT inhibitor is a JAK1 inhibitor.
  • JAK1 inhibitors are highly effective in treating PS.
  • JAK1 inhibitor includes reference to a JAK1 -selective inhibitor, a JAK1-JAK2 inhibitor (i.e. an inhibitor that inhibits both JAK1 and JAK2), and a JAK1-JAK3 inhibitor (i.e. an inhibitor that inhibits both JAK1 and JAK3). All of these are able to inhibit JAK1.
  • the JAK/STAT inhibitor is selected from Baricitinib, Tofacinib, Peficitinib, Filgotinib, and Upadacitinib.
  • the JAK/STAT inhibitor is selected from Tofacinib, Peficitinib, Filgotinib, and Upadacitinib.
  • the JAK/STAT inhibitor is Baricitinib.
  • the JAK/STAT inhibitor preferably baricitinib
  • suitable dosages for barcitinib include 4 mg once daily.
  • the JAK/STAT inhibitor is Filgotinib.
  • Filgotinib is administered at an oral dose of 1-500, preferably 50-200 mg, such as 80-100 mg once daily.
  • the JAK/STAT inhibitor is Tofacitinib.
  • Tofacitinib is administered at an oral dose of 1-10 mg, preferably about 5 mg twice daily, preferably in immediate release form; or about 10 mg once daily, preferably in extended release form.
  • the JAK/STAT inhibitor is Peficitinib.
  • Peficitinib is administered at an oral dose of 100-150 mg once daily.
  • the JAK/STAT inhibitor is Upadacitinib.
  • Upadacitinib is administered at an oral dose of 15-30 mg once daily.
  • the JAK/STAT inhibitor is selected from Tofacinib, Peficitinib, Filgotinib, and Upadacitinib. In another preferred embodiment of this aspect, the JAK/STAT inhibitor is not baricitinib.
  • the JAK/STAT inhibitor is not filgotinib.
  • JAK/STAT inhibitor may also be selected from any of the JAK/STAT inhibitors cited hereinabove including the group of JAK1 inhibitors Ruxolitinib, Itacitinib (INCB39110),
  • the JAK/STAT inhibitor is administered in dose that reduces IFN-g production in Thl7.1 cells, preferably in Thl7.1 cells in the BALF of sarcoidosis patients, more preferably pulmonary sarcoidosis patients.
  • the administration of the JAK/STAT inhibitor is for treatment of sarcoidosis, preferably pulmonary sarcoidosis, wherein said treatment comprises the administration of a therapeutically effective dose and regimen of the JAK/STAT inhibitor, preferably by oral administration of the drug.
  • a therapeutically effective dose and/or regimen as defined herein preferably blocks or lowers IFN-g production in Thl7.1 cells (in BALF) of a patient in need of said treatment.
  • a therapeutically effective dose and/or regimen as defined herein refers to a treatment involving at least partial counteraction of the reduced pulmonary function associated with pulmonary sarcoidosis.
  • therapeutically effective dose and/or regimen as defined herein may also, in preferred embodiments of aspects of this invention, be defined as a dose and regimen associated with an increase in at least 5% of Forced Vital Capacity (FVC) as measured by FEV testing, a decrease in 18 F-FDG-PET uptake of at least 10% and/or an improvement of inflammatory activity measured as a decline in biomarker soluble interleukin-2 receptor (sIL-2R) of at least 20- 40%, preferably at least 40% compared to the untreated patient (e.g. baseline levels, prior to treatment).
  • sIL-2R is preferably determined in blood, preferably in a blood sample, preferably about 3-6 months after the start of the treatment. A reduction 40% relative to baseline levels is generally considered clinically relevant also in the case of PET SUV max values.
  • treatment may optionally further comprise subjecting the patient to an FEV test for determining Forced vital capacity (FVC; the total amount of air exhaled during the FEV test).
  • FVC Forced vital capacity
  • treatment may optionally further comprise subjecting the patient to 18 F-fluorodeoxyglucose positron emission tomography ( 18 F-FDG-PET) in order to determine maximum standardized uptake value (SUVmax) as an inflammatory marker.
  • 18 F-FDG-PET maximum standardized uptake value
  • Positive treatment outcome, and hence attainment of therapeutically effective dose and/or regimen, is associated with a reduction in SUVmax of at least 10% in one or both lungs.
  • a therapeutically effective dose and/or regimen in aspects of this invention result in clinically significant improvements in patient health, and may as such be referred to as clinically effective dose or regimen
  • JAK/STAT inhibitor may be administered in the form of a pharmaceutically acceptable salt, solvate, polymorph, or metabolite.
  • the JAK1 inhibitor is not baricitinib.
  • the sarcoidosis is pulmonary sarcoidosis, preferably chronic pulmonary sarcoidosis.
  • treatment involves at least partial counteraction of the reduced pulmonary function associated with pulmonary sarcoidosis.
  • improved pulmonary function is and increase in at least 5% of Forced Vital Capacity (FVC) as measured by FEV testing, compared to baseline levels prior to treatment.
  • FVC Forced Vital Capacity
  • a therapeutically effective dose is associated with a decrease in 18F-FDG-PET uptake of at least 10%, and/or an improvement of
  • inflammatory activity measured as a decline in the soluble interleukin-2 receptor (sIL-2R) of at least 20%, compared to baseline levels prior to treatment.
  • sIL-2R soluble interleukin-2 receptor
  • the above treatment regimens are aimed at reducing the harmful effect of these ThlT. l cells in sarcoidosis. In this way it is aimed to improve the prognosis by inhibiting chronic inflammation in this disease.
  • the present invention provides a method of identifying patients with pulmonary sarcoidosis eligible for treatment with an anti -pulmonary sarcoidosis drug comprising testing the patient for the prognostic biomarker Thl7.1-cells or ratio of Thl7.1/Thl cells, as defined herein above, wherein the patient is eligible for treatment if the biomarker level is increased relative to a control value.
  • control values can be obtained for this aspect as described for other aspects herein.
  • the present invention provides a method of treating pulmonary sarcoidosis, comprising identifying a patient with pulmonary sarcoidosis as eligible for treatment with an anti-pulmonary sarcoidosis drug as described above, and administering to eligible patients a therapeutically effective amount of an anti-pulmonary sarcoidosis drug as described above.
  • Thl, Th2, Thl7, CCR6+ DP, Thl7.1 and unclassified cells which include CCR6-CCR4-CXCR3- cells, CCR6-CCR4+CXCR3+ cells and CCR6+CCR4-CXCR3- cells) of total memory CD4+ T cells in MLN.
  • MLN mediastinal lymph nodes
  • DP double-positive
  • Th T helper
  • SRC sarcoidosis
  • Thl7.1 cells and CCR6+ DP cells are highly proliferative in sarcoidosis MLN Expression of Ki67 was determined ex-vivo in Thl,
  • Proportions Ki67+ total memory T cells (data are from 15 controls and 13 patients).
  • C Representative flow cytometry analysis of Thl7, CCR6+ DP and ThlT. l cells in MLN from one control and one sarcoidosis patient.
  • D Delta (D) MFI of CTLA4 (data are from 17 controls and 13 patients).
  • FIG. 3 Higher Thl7.1 cell proportions in sarcoidosis MLN and BALF than PB Proportions Thl7, CCR6+ DP and Thl7.1 cells were determined in total memory CD4+ T cells from sarcoidosis PB, MLN and BALF. A-C. Proportions of Thl7, CCR6+ DP and Thl7.1 cells of total memory CD4+ T cells. Statistics: Horizontal hnes indicate median values and significance was determined using a Mann-Whitney U test, *** p ⁇ 0.001. Data are from 35 (PB), 17 (MLN) and 36 (BALF) patients.
  • CCR C-C chemokine receptor
  • PB peripheral blood
  • MLN mediastinal lymph nodes
  • BALF broncho-alveolar lavage fluid
  • DP double positive
  • Th T helper.
  • A-B Proportions Thl7.1 and Thl cells of total memory CD4+ T cells at time of diagnosis.
  • C Ratio of the proportions of Thl7.1 versus Thl cells. Statistics: Horizontal lines indicate median values and significance was determined using a Mann-Whitney U test, * p ⁇ 0.05 ** p ⁇ 0.01.
  • Th T helper.
  • CD45RA+FoxP3- naive T cells CD45RA-FoxP3- memory T cells, CD45RA- FoxP3int activated (non-suppressive/-regulatory) T cells, CD45RA+FoxP3int naive regulatory T cells and CD45RAFoxP3high activated Tregs.
  • the total memory T cell pool consists of memory T cells plus activated T cells.
  • the total (non-suppressive/-regulatory) T cell pool consists of naive T cells plus total memory T cells.
  • Th cell subsets can be classified according to chemokine-receptor expression.
  • CCR6- memory T cells subsets including CCR4-CXCR3+ Thl cells and CCR4+CXCR3- Th2 cells; and CCR6+ T cell subsets, which include CCR4+CXCR3- Thl 7 cells, CCR4+CXCR3+ DP Th cells and CCR4-CXCR3+ ThlT. l cells.
  • ThlT. l cells i.e. IFN-y-producing Thl 7 cells
  • CCR6+CCR4+CXCR3+ DP cells are thought to reflect an intermediate Thl7 and Thl7.1 cell population, expressing both RORyt/IL- 17A and T-bet/lFN-g in patients with rheumatoid arthritis (RA).
  • RA rheumatoid arthritis
  • CCR C-C chemokine receptor
  • CXCR CXC chemokine receptor
  • DP double-positive
  • Th T helper
  • Treg regulatory T cells.
  • FIG. 7 Representative flow cytometry analysis of BALF (A) and MLN (B) from one sarcoidosis patient and one control.
  • CCR C-C chemokine receptor
  • BALF broncho-alveolar lavage fluid
  • MLN MLN
  • FIG. 8 Proportions of total CCR6+ and Thl cells were determined in total memory CD4+ T cells from sarcoidosis PB, MLN and BALF.
  • A-B Proportions of total CCR6+ and Thl cells of total memory CD4+ T cells.
  • CCR C-C chemokine receptor
  • PB peripheral blood
  • MLN mediastinal lymph nodes
  • BALF broncho-alveolar lavage fluid
  • DP double positive
  • Th T helper.
  • A-B Proportions of Thl7 and Thl 7.1 cells of total memory CD4+ T cells in PB and MLN of 17 sarcoidosis patients.
  • C-D Proportions of Thl7 and Thl7.1 cells of total memory CD4+ T cells in PB and BALF of 15 sarcoidosis patients.
  • Statistics Significance between median values of paired samples was determined using a Wilcoxon signed rank test, * p ⁇ 0.05 ** p ⁇ 0.01 *** p ⁇ 0.001.
  • BALF bronchoalveolar lavage fluid
  • Th T helper
  • SRC sarcoidosis
  • Figure 11 shows the pulmonary function (FVC percent predicted) and the treatment regimen of the patient described in Example 2.
  • Figure 12 shows the 18F-FDG-PET scan of the patient described in Example 2 at November 2017 (prior to baricitinib treatment) and March of 2018 (following 3 months of baricitinib treatment).
  • Figure 13 shows the targets of Baricitinib in sarcoid granuloma pathogenesis.
  • Figure 14 shows the effect of tofacitinib on the mRNA expression of IFN-g (relative to 18S expression) in Thl7, Thl7.1 and Thl cells as described in Example 3.
  • Figure 15 shows the effects of baricitinib, tofacitinib and filgotinib on STAT1 phosphorylation in IFN-g stimulated monocytes of healthy subjects and sarcoidosis patients (top graph), and the effects of baricitinib, tofacitinib and filgotinib on STAT5 phosphorylation in naive IL-2 stimulated CD4 T-cells of healthy subjects and sarcoidosis patients (bottom graph) as described in Example 4.
  • PS pulmonary sarcoidosis
  • the term“pulmonary sarcoidosis”, abbreviated“PS” is used herein in their art-recognized meaning, and denotes a disease involving abnormal collections of inflammatory cells that form lumps known as granulomas.
  • the cause of PS is unknown.
  • the disease usually begins in the lungs, skin, or lymph nodes, and can manifest itself throughout the body. Multiple complaints may be present with sarcoidosis in general. The most common is fatigue, which can last for a long time even if disease activity is no longer present. Overall malaise, shortness of breath, joint complaints, temperature increase, weight loss and skin complaints may be present.
  • the prognosis is good, and especially the acute form (see below) usually causes few problems; the complaints will gradually decrease automatically.
  • sarcoidosis is present in the heart, kidneys, liver and / or central nervous system, or extensive presence in the lungs, then the outcome is less favorable.
  • staging is a way to indicate the location of granulomas- the lungs, the lymph nodes, or both- and the nature of the disease, and unlike in cancer, staging is no direct indication of severity. Chest radiograph changes are divided into four stages: l.bihilar lymphadenopathy (granulomas in the lymph nodes); 2.bihilar
  • lymphadenopathy and reticulonodular infiltrates granulomas in the lungs
  • 3.bilateral pulmonary infiltrates granulomas in the lungs, but not in the lymph nodes
  • Radiographs often have the chronic, progressive disease. As indicated, the radiograph stages are not a progression. A patient might have stage two sarcoidosis, and then with treatment or time become stage one, or three, or go into remission altogether. Sometimes there is an acute syndrome with fever, general malaise, painful and swollen joints, particularly affecting the ankles, with red to purple patches on the skin, called erythema nodosum. This pattern is also referred to as Lofgren Syndrome. The symptoms can be present for a while, but usually disappear within two years, even without treatment.
  • chronic sarcoidosis refers to a multisystemic disorder of unknown cause that is characterized by the formation of immune
  • aspects of this invention are preferably aimed at the chronic progressive form of PS, also referred to as chronic sarcoidosis herein, which term does not refer to the acute form of PS referred to as Lofgren Syndrome.
  • Diagnosis of PS is estabhshed when clinicoradiological findings are supported by histological evidence of noncaseating epithelioid cell granulomas.
  • the diagnosis of pulmonary sarcoidosis is preferably conform guidelines of the American Thoracic Society (ATS), the European
  • ERS Respiratory Society
  • WASOG Gr anulom atous Disorders
  • sonographic features of mediastinal or hilar lymph nodes are obtained, and presence of granules in lymph nodes on endobronchial ultrasound (EBUS) has a high specificity (99.3%) for the diagnosis of sarcoidosis.
  • Prognosis of PS in aspects of this invention is performed based on the biomarker level (ThlT.l quantity or Thl7.1/Thl ratio) as part of the total population of T cells, preferably of total CD4 + T cells, more preferably of total CD4 + T cells, more preferably as part of BALF or MLN.
  • biomarker level ThlT.l quantity or Thl7.1/Thl ratio
  • total memory CD4+ T cells indicates the memory (e.g. CD45RA-) T cell subset of T helper (CD4+; Th) cells within the T cell (CD3+) fraction of lymphocytes, and in a preferred embodiment, as explained in Figure 5, refers to the combined subpopulations of CD45RA FoxP3 Memory T cells, plus CD45RA FoxP3 int Activated (non- suppressive/non-regulatory) T cells, within the total CD3 + CD4 + T cell population with the exclusion of suppressive or regulatory T (Tregs) cells. Tregs could contaminate the ThlT. l cell evaluation, and are therefore preferably excluded.
  • CD45RA was used to differentiate the naive (CD45RA + ) from memory (CD45RA ) T cell populations, whereas
  • the term“total memory CD4+ T cells” preferably does not include CD45RA + FoxP3 Naive T cells, CD45RA + FoxP3 int Naive Tregs, and CD45RA FoxP3 h, - h Activated Tregs.
  • CD45RO markers for memory T cells
  • Th cell subsets can be classified according to 1) chemokine-receptor expression and/or 2) transcription factor expression. Based on chemokine-receptor expression (including the C-C chemokine receptor (CCR), and CXC chemokine receptor (CXCR), in particular CCR6, CCR4, and CXCR3, the following subsets can be
  • CCR6 memory T cells subsets include CCR4 CXCR3 + Thl cells and CCR4 + CXCR3 Th2 cells; CCR6 + T cell subsets include CCR4 + CXCR3 Thl 7 cells, CCR4 + CXCR3 + double-positive Th cells and CCR4 CXCR3 +
  • Thl cells are identified as T-bet positive, Th2 cells as GAT A3 positive, Thl 7 cells as ROR/t positive and Thl7.1 cells express both T-bet and RORyt.
  • Thl 7.1 cells in a sample in accordance with this invention does not necessarily require marker-specific immunological gating methods, such as those based on flow cytometry, described herein.
  • Alternative methods for enumerating Thl 7.1 cells based on alternate gating methods or markers, even by other means than flow cytometry, are envisioned within the context of the present invention. Such alternate methods could be based on detection of Thl 7.1 -specific gene expression profiles within the cell, production of (a combination of) specific proteins within the cell, or alternative cell-surface markers for either alternative gating or physical separation of cells that are not of interest.
  • T cell is used herein in its art-recognized meaning, and denotes a CD3 + lymphocyte.
  • Lymphocytes T cells, B cells, and NK cells constitute the majority of the peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • the T cell fraction is further subdivided into CD4 + and CD8 + T cells.
  • CD4 + T cells are known as T helper (Th) cells and are further classified into various functional subtypes based on the expression profiles of specific cytokines, surface markers, or transcription factors. These include Thl and Thl7 cells as well as other described subpopulations such as Treg (regulatory T) cells, Th2, Th9, follicular helper, and TR1 types.
  • Thl7.1 cell is used herein in its art-recognized meaning, and denotes a IFN-y-producing Thl 7 cell, also classified as a CCR4 CXCR3 + subset of the CCR6 + memory T cells within the total memory CD4 + T cell (Th) population, or total T cell population in the BALF.
  • CCR6 + T cells include a CCR4 and CXCR3 + subset designated Thl7.1 cells.
  • Thl7.1 cells also different phenotypes of cells can be present: e.g. IL-17A + /IFN-y + (indicated in hterature as
  • Thl 7/Th 1 -cells cells and IFN-g single-positive cells.
  • Thl7.1 cells are thought to reflect an intermediate Thl 7 and Thl 7.1 cell population, expressing both RORyt/IL-17A and T-bet/IFN-g.
  • Thl7.1 cells are not one single cell type, Thl 7-lineage cells are highly plastic and can change phenotype quickly. From benign to pathogenic
  • Interleukin-23 (IL-23) is required for terminal differentiation of
  • Thl7 cells and maintenance of the Thl7 phenotype also induces IFN- g production in Thl7 cells, which then become Thl7.1-cells.
  • IL-23 which is composed of pl9 and p40 subunits, is a proinflammatory cytokine that contributes to the formation and maintenance of Thl7(. l) cells in
  • IL-23/Thl7 pathway is a therapeutic target for the treatment of inflammatory arthritis.
  • IL-23 plays an important role in Thl7 cell signaling pathways, known to be up- regulated in, for instance, psoriasis and also within sarcoidosis.
  • Antibodies that inhibit interleukin -23 can inhibit the expression of molecular and clinical disease characteristics associated with psoriasis.
  • Guselkumab (CNTO 1959, Janssen Research and Development) is a fully human IgGl lambda monoclonal antibody that inhibits interleukin-23-specific
  • An aspect of the present invention pertains to the use of IL-23 inhibitors in the treatment of pulmonary sarcoidosis, through inhibition of the terminal differentiation of Thl7 cells into ThlT. l cells.
  • IL-23 is a heterodimeric cytokine composed of an IL12B (IL-12p40) subunit (that is shared with IL- 12) and the IL23A (IL-23p l9) subunit.
  • IL-12p40 IL12B
  • IL23A IL-23p l9
  • a specific IL- 23 blocker refers to a compound that antagonizes or inhibits the activity of interleukin 23, while preferably not affecting the activity of IL-12.
  • Well known examples of such compounds are tildrakizumab
  • tildrakizumab is usually by subcutaneous injection, at a recommended dose of 100 mg at weeks 0, 4, and every twelve weeks thereafter.
  • TremfyaTM is also administered as a 100 mg subcutaneous injection, with dosing regime of once every eight weeks, following two starter doses at weeks 0 and 4.
  • therapeutically effective dose for the treatment of sarcoidosis can be determined using routine experimentation, and taking into account the pharmacodynamics of the compound.
  • a suitable dose is 100 mg of the specific interleukin 23 blocker in the form of a humanized mAh.
  • specific interleukin 23 blocker includes reference to anti-IL-23(p l9) specific antibodies tildrakizumab and guselkumab and antibodies equivalent thereto.
  • interleukin 23 blockers as used herein do not show this cross reactivity to IL-12, and are therefore referred to as being IL-23-specific.
  • treatment aspects of this invention are preferably not directed to the use of interleukin 23 blockers showing IL23/IL-12 cross-reactivity.
  • specific interleukin 23 blocker in the context of this invention refers to compounds, in particular antibodies, that prevent Th 17-lineage subset differentiation into ThlT. l cells. Methods to determine such blocking or inhibition of differentiation are well within reach of the average skilled artisan, and may include methods as described herein for typing samples of patients suffering from sarcoidosis.
  • Thl7 cells are brought under differentiation conditions in the presence (positive blocking test) and absence (negative control) of the specific interleukin 23 blocker, and wherein blocking of the differentiation is indicative of the suitability of compound for potential use in therapeutic methods as described herein.
  • a further aspect of this invention pertains to treatment aspects comprising the use of a JAK/STAT inhibitor.
  • JAK/STAT inhibitor refers to a Janus kinase inhibitor, also known as JAK inhibitor or jakinib, which compounds function by inhibiting the activity of one or more of the Janus kinase family of enzymes. It is currently estabhshed that the JAK family consists of four members, which mediate signaling of various cytokines and growth factors in humans. Cytokines bind to their respective receptors, thereby activating JAK complexes, formed from combinations of JAK1, JAK2, JAK3, and tyrosine-protein kinase (TYK)2. Activated JAK complexes promote phosphorylation of signal transducers and activators of transcription
  • STATs which regulate the expression of target genes.
  • IFN-g signahng is mediated by JAK1- JAK2 and STAT1 phosphorylation.
  • JAK1 inhibitor refers to a Janus kinase inhibitor or jakinib, i.e. a small-molecule drug that inhibits the activity of its target JAK1, a kinase that binds to the intracellular domain of the Type I/II cytokine receptor, thereby blocking cytokine signaling.
  • the JAK1 inhibitor prevents JAK1 from phosphorylating STATs and other substrates, so that intracellular signals cannot be transduced. Because JAKs are critical for multiple different cytokines, jakinibs block the action of a range of cytokines.
  • JAK inhibitors The selectivity of JAK inhibitors is extensively reviewed by Schwartz et al., 2017 (Nat Rev Drug Discov. 2017 December 28; 17(1): 1-41), to which explicit reference is made in the context of the term JAK1 inhibitor.
  • many first- generation jakinibs that block multiple JAKs and therefore inhibit the actions of a large variety of cytokines, as well as several newer pan-JAK inhibitors are indicated in Table 3 and Table 4 of Schwartz et al., 2017. Express reference is made to these Tables for indicating suitable
  • JAK1 inhibitors for use in this invention include jakinibs that block JAK1, optionally in addition to blocking other kinases.
  • suitable first- generation JAK1 inhibitors include ruxolitinib, tofacitinib, and baricitinib, which block JAK1- JAK2, JAK1-JAK3 and JAK1-JAK2, respectively.
  • a newer pan-jakinib is peficitinib which has IC50 of 3.9, 5.0, 0.71 and 4.8 nmol/L for JAK1, JAK2, JAK3 and TYK2 enzymatic activity respectively.
  • JAK 1 -selective inhibitors A variety of next-gen JAK inhibitors is also indicated in Tables 3 and 4 of Schwartz et al., 2017, including small molecule drugs that are selective for one particular JAK isoform. Filgotinib, Upadacitinib, and Solcitinib are examples thereof and selectively block JAK1, and are referred to herein as JAK 1 -selective inhibitors, all of which are useful in aspects of this invention.
  • JAK 1 inhibitor includes reference to a JAK 1- selective inhibitor, a JAK1-JAK2 inhibitor, a JAK1- JAK3 inhibitor, and a JAK1-TYK2 inhibitor.
  • JAK inhibition could be used in treating chronic granulomatous disease, in particular chronic sarcoidosis. It was found that while such patients can be non- responsive to anti-TNFa therapy, e.g. based on infliximab or adalimumab, JAK1-2 inhibition with haric.it.inih (LY3009104; CAS# 1187594-09-7) proved an effective therapy (see Examples below). Baricitinib blocks IFN-g- signaling.
  • a preferred compound for use in aspects of this invention relating to JAK/STAT inhibitors is baricitinib.
  • Baricitinib is a low-molecular-weight compound that binds to the ATP-binding site of the Janus kinase.
  • Baricitinib is a reversible inhibitor of the Janus kinases JAK1 and JAK2.
  • the JAK/STAT inhibitor is preferably a JAK1 and JAK2 (i.e. a JAK1-2 combined) inhibitor or a JAK1 or JAK2 (i.e. a JAK1/2) inhibitor, more preferably a JAK1 inhibitor.
  • JAK/STAT inhibitors may be equally efficacious in treatment aspects of this invention. Many JAK inhibitors are now in clinical stage or are used for treatment of
  • JAK inhibitors in the treatment of diseases such as RA, psoriasis, and inflammatory bowel disease (IBD) is well established and recognized.
  • IBD inflammatory bowel disease
  • JAK inhibitors include Tofacinib (CP-690550; CAS# 477600-75-2) and Peficitinib (ASP015K, JNJ-54781532; CAS# 944118-01-8), both of which target JAK1 and JAK3. Further well known examples of such JAK inhibitors also include Filgotinib (GLPG0634; CAS#: 1206161-97-8) and Upadacitinib (ABT-494; CAS# 1310726-60-3), both of which target JAK1. Any of these may be used in aspects of this invention, preferably the more specific JAK1 inhibitors. As indicated herein, those that interfere with cytokine receptor signaling of IFN-g (JAK1, JAK2, or JAK1-JAK2) are preferred.
  • treating sarcoidosis in the context of this invention by interfering with IFN- g receptor signaling is aimed at inhibiting or blocking the IFN-g induced STAT1 expression.
  • the term“JAK/STAT inhibitor” in the context of this invention refers to compounds that prevent JAK signahng through IFN- g. Methods to determine whether a compound exhibits such blocking or inhibiting effect are well within reach of the average skilled artisan, and may include methods wherein inhibition of intracellular ST ATI
  • phosphorylation by a test inhibitor in response to stimulation of whole cells ex vivo with IFN-g is measured, e.g., by using flow cytometry as described in Vakkila et al. 2008 Scand J Immunol 67:95-102, and wherein inhibition of intracellular STAT phosphorylation, preferably ST ATI phosphorylation (pSTATl), is indicative of the suitability of the tested JAK/STAT inhibitor as a therapeutic agent in treating sarcoidosis, preferably chronic sarcoidosis as described herein.
  • STAT phosphorylation preferably ST ATI phosphorylation
  • STAT1 controlled genes or proteins preferably CXCL9 and CXCL10
  • expression of STAT1 controlled genes or proteins may be determined, in particular the increase or decrease in such expression, preferably a decrease in response to the administration of the JAK inhibitor, as described in detail in the legend to Figure El in Meesilpavikkai, 2018 J Allergy Clin Immunol, Volume 142(1): pp 328-330. e2.
  • pSTAT3 and pSTAT5 inhibition are signaling intermediates of IL-6 (and IL-23, a.o.) and IL-2 (and IL-7, a.o.) respectively.
  • sample refers to a biological sample encompassing a variety of sample types obtained from an individual and can be used in a diagnostic or monitoring assay.
  • the term encompasses blood and other liquid samples of biological origin, solid tissue samples, such as a biopsy specimen, or aspirate, or tissue cultures or cells derived therefrom and the progeny thereof.
  • the term encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents, solubilization, or enrichment for certain components.
  • the term encompasses a clinical sample, and also includes cells in cell culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples.
  • the term“mediastinal lymph node” abbreviations
  • MLN MLN
  • MLN hilar lymph nodes
  • sampling of lymph nodes of the lungs in aspects of this invention may entail sampling of HLN as well as sampling of MLN.
  • Cell samples of such lymph nodes may be obtained through fine-needle aspiration. Sampling of the MLN and obtaining an MLN aspirate is a preferred embodiment in aspects of this invention.
  • bronchoalveolar lavage a procedure in which a bronchoscope is passed through the mouth or nose into the lungs and fluid is injected into a small part of the lung and then collected for examination.
  • Bronchoalveolar lavage is commonly used in immunological research as a means of sampling T-cell populations. It is not a standard procedure when diagnosing sarcoidosis, as it is not always necessary.
  • Suitable samples for use in methods of the present invention may thus include, but are not limited to samples of T cells obtained from peripheral blood (PB), lungs, HLN or MLN. Samples of T cells in the lungs through procurement of BALF is preferred.
  • typing refers to differentiating or stratifying between individuals according to diagnostic or prognostic disease status.
  • the typing is preferably based on a comparison of (i) a biomarker level in a sample of an individual and (ii) a biomarker control value.
  • the typing differentiates individuals suffering, or suspected of suffering from, PS, in a group having an increased risk of developing a chronic disease pattern and a group not having such an increased risk.
  • the number of Th 17.1 cells in a sample of an individual is measured as the biomarker for determining the diagnostic or prognostic disease status. Such measurements, however, may but do not necessarily need to provide the exact cell number as an outcome.
  • the present inventors have found that it is the proportion of Th 17.1 cells, relative to other T cells in the sample of a subject, that - when compared to the corresponding proportion of Th 17.1 cells in samples of healthy subjects or patients undergoing disease resolution - are elevated at time of diagnosis in patients subsequently developing chronic, in particular progressive, sarcoidosis.
  • Thl7.1 cells are preferably enumerated as a proportion of the total T cell population, even more preferably as a proportion of the total memory CD4+ T cell population as defined herein. Still, more preferably, Thl7.1 cells are enumerated as a proportion of the total Thl and/or Thl7 cell
  • any T cell subset can be used in determining the proportion of Thl7.1 cells in aspects of this invention, including but not limited to:
  • Thl 7.1 cells in a healthy individual It is preferred that the number or proportion of Th 17.1 cells in a sample of an individual is determined by flow cytometry.
  • flow cytometric analysis is that multiple cell (surface) markers can be
  • Flow cytometry data analysis is built upon the principle of gating. Gates and regions are placed around populations of cells with common characteristics, usually forward scatter, side scatter and marker expression, to investigate and to quantify these populations of interest.
  • the proportion of ThlT. l cells in the total T cell population in the BALF, more preferably in the total memory CD4+ T cell or Th population is determined, as a proportion of other T cell populations, preferably wherein the following Th cell subpopulations are enumerated by flow cytometry (cells classified according to chemokine-receptor expression):
  • Thl7.1 CCR6+CCR4-CXCR3+.
  • the skilled person will be able to determine the presence or absence of higher proportions of Thl 7.1 cells in samples of sarcoidosis patients compared to control samples.
  • the skilled person will be able to determine when patients are at risk of developing chronic sarcoidosis, and specifically progressive chronic sarcoidosis with need for treatment, by determining the presence of significantly higher ThlT. l proportions (e.g. Thl7.1/Thl) relative to control values, e.g. in patients that show disease resolution.
  • Thl7.1 cells i.e. IFN-y-pro during Thl7 cells
  • CCR6, CXCR3, RORC and TBX21, but not CCR4 are most informative in diagnosis of disease occurrence and prediction of
  • Thl7.1-cells have a pathogenic phenotype, and Thl7.1-cells are unresponsive to corticosteroid treatment.
  • Thl7.1- cells may constitute a suitable therapeutic target, for instance by using biologicals. It is therefore an aspect of this invention to treat or prevent progressive, chronic forms of sarcoidosis with IL23 inhibitors, such as anti- IL23 antibodies.
  • a suitable biological having such activity includes
  • the present invention considers that the genetic variations within the IL23R gene or other genes involved in the IL-23/Th 17-signaling pathway predispose for (chronic) sarcoidosis. Hence, it is an aspect of this invention to reduce the number of Th 17.1 cells in a patient for the purpose of supporting treatment of (chronic) sarcoidosis, by replacing, compensating, complementing or repairing genetic anomalies in the IL23R gene through gene therapy, Crispr/CAS or RNA interference technology.
  • Example 1 Thl7.1 ratios in BALF and MLN are linked to pulmonary sarcoidosis
  • Exclusion criteria were use of immunomodulatory medication 3 months prior to study inclusion; respiratory tract infection 4 weeks prior to study inclusion; concomitant pulmonary disease (including chronic obstructive pulmonary disorder and asthma), autoimmune diseases, malignancies, human immunodeficiency virus seropositivity, pregnancy, and allergies.
  • concomitant pulmonary disease including chronic obstructive pulmonary disorder and asthma
  • autoimmune diseases including malignancies, human immunodeficiency virus seropositivity, pregnancy, and allergies.
  • sarcoidosis patients donated BALF, MLN- derived fine-needle aspiration (FNA) or peripheral blood (PB). In total 32 of these patients donated material of multiple organs, see figure 6.
  • Control MLN were collected from 22 lung transplantation donors without signs of pulmonary inflammation (routinely assessed by a chest X- ray and bronchoscopy).
  • EUS- or EBUS-FNA endobronchial ultrasound guided (EUS- or EBUS)-FNA from draining MEN were performed with a 22G (or 19G) needle.
  • MLN aspirates were filtered through a 100 pm cell strainer (BD Biosciences) and centrifuged. Cells were stored at -150 °C. Control MLN were collected from lung transplantation donors. Control MLN were processed as lymph node aspirates.
  • BALF, MLN and PB mononuclear cells were stained for intra- and extracellular markers using the antibodies shown in Table 2.
  • Figure 5A were identified on the basis of chemokine-receptor expression ( Figure 5B).
  • Fixable Aqua Dead Cell Stain kit for 405 nm (Invitrogen, Molecular Probes) was used as live-dead marker. At least 100.000 cells per sample were measured on a Flow cytometer LSRII (BD Biosciences), and the mean fluorescent intensity of cytotoxic T-lymphocyte antigen 4 (CTLA4) was standardized to average expression in healthy control PB cells, stated as delta (A) MFI. Ki67 staining was used to assess the proliferative status of cell subsets.
  • CTLA4 cytotoxic T-lymphocyte antigen 4
  • the CCR6+ double positive (DP) cells are thought to reflect an intermediate stage between Thl 7 and Thl 7.1 cells.
  • sarcoidosis MLN contained higher proportions of CCR6+ Th cells than control MLN ( Figure 1C).
  • Figure ID we identified significantly increased proportions of Thl7.1 cells in sarcoidosis MLN compared with control MLN ( Figure ID).
  • sarcoidosis MLN contains increased proportions of Thl 7.1 precursor cells, i.e. Thl 7 and CCR6+ DP cells, compared with controls ( Figure 1D/E).
  • Thl cell proportions were significantly decreased compared with controls, although Thl cells remained (like in controls) the most prominent memory T cell population (Figure 1D/E).
  • Thl7.1 cells and CCR6+ DP cells are highly proliferative in sarcoidosis MLN
  • Thl7.1 cells in sarcoidosis MLN could be caused by enhanced proliferation, therefore we assessed proliferative status of T cell subsets.
  • Proportions of proliferative (Ki-67+) CD4+ memory T cells were significantly increased in sarcoidosis MLN compared with controls ( Figure 2A).
  • ThlT. l cells and CCR6+ DP cells exhibited high fractions of proliferating Ki-67+ cells compared with controls ( Figure 2B/C). This was particularly striking for the Thl7.1 subpopulation, which in controls contained only very few Ki-67+ cells.
  • CCR6+ DP cells contained the highest fraction of proliferating cells (-15%, which was significantly higher than any of the other T cell subsets) in sarcoidosis MLN ( Figure 2B).
  • CCR6+ DP cells also showed decreased CTLA4 expression in sarcoidosis MLN compared with controls ( Figure 2D) as we have previously also shown for Thl7 cells [Broos CE, et al. 2015. Am J Respir Crit Care Med 2015; 192(6):763-65]
  • Thl7.1 cells as a highly proliferative Th subset in MLN of sarcoidosis patients compared with controls.
  • CCR6+ DP cells also proliferate highly compared with other Th cell subsets in sarcoidosis MLN.
  • ThlT.l cells are not a prominent population in the PB of sarcoidosis patients. Rather, patients show an increase in Thl7.1 cells in MLN, which is further enhanced in sarcoidosis lungs (e.g. BALF).
  • Thl7.1 cells are significantly increased in sarcoidosis MLN compared with control MLN, reflecting what we have previously found in sarcoidosis lungs [Ramstein J, et al. 2015. Am J Respir Crit Care Med 2015 doi: 10.1164/rccm.201507-14990C]. Furthermore, Thl7.1 cell proportions are highest in the granulomatous BALF when compared with sarcoidosis-derived MLN and PB. Higher proportions of Thl7.1 cells in the lungs significantly correlated with development of chronic disease. Together these data suggest that Thl7.1 cell proportions in pulmonary sarcoidosis can be evaluated as diagnostic and/or prognostic marker in clinical practice and could serve a new therapeutic target.
  • Thl7 cells display considerable plasticity and can produce IFN-g
  • IFN- g -producing Thl7 cells play a key role in the development of autoimmune diseases.
  • Interferon-y-producing Thl7 cells or so called Thl7.1 cells, are described to be pathogenic in several autoimmune diseases and chronic inflammatory disorders, including Crohn’s disease and arthritis.
  • CCR6+ Thl7.1 cells rather than classical CCR6- Thl cells are the major source for IFN-g production in sarcoidosis BALF.
  • Thl 7 and CCR6+ DP cells can be precursor cells of Thl 7.1 cells, these data suggest that initial Thl 7-lineage subset differentiation occurs within sarcoidosis MLN.
  • Thl7 cells are induced within sarcoidosis MLN, our data suggest that chronically inflamed sites such as sarcoidosis lungs (but possibly also granulomatous parts of the MLN) play a role in accelerating ThlT.l cell differentiation by inducing conversion of Thl7 towards Thl7.1 cells. For example, comparing different involved
  • Thl7.1 cells were augmented in sarcoidosis lungs compared with MLN and PB.
  • Thl7.1 cell fractions were also found increased compared with PB and shared clonal ancestry with Thl7 cells.
  • Thl7.1 cells were abundantly present
  • CCR6+ DP cells which were previously suggested to represent and intermediated stage between Thl7 and Thl7.1 cells, as the subset with the highest fraction of proliferating cells.
  • Sarcoidosis CCR6+ DP cells also showed decreased coinhibitory CTLA4 expression compared with controls, likely contributing to increased proliferative capacity.
  • CTLA4 expression was also observed in sarcoidosis MLN Thl7 cells [Broos CE, et al. 2015.
  • IL-23p l9 expression in sarcoid lungs remains uncertain, high expression of IL-12p40 compared with IL-12p70 may very well indicate elevated levels of IL-23, since IL-12p40 is also a subunit of IL-23.
  • IL-23 transcription was enhanced in sarcoid skin lesion compared with controls, and serum amyloid A (SAA), an antigen that was speculated to contribute to development of chronic (pulmonary) sarcoidosis, has been described to increase IL-23 production by dendritic cells [Ather JL, Ckless K, Martin R, et al. 2011. J Immunol 187(l):64-73].
  • SAA serum amyloid A
  • Thl7.1 cell proportions at time of diagnosis in lungs of patients who develop chronic disease compared to patients who underwent resolution. Together with our previous finding of increased BALF Thl7.1 cells proportions in a population that included progressive patients who are on first-, second- and/or third- fine therapy [Ramstein J, et al. 2015. Am J Respir Crit Care Med doi:
  • IL-23R expression is essential for conversion of Thl7 cells towards pathogenic Thl7.1 cells during chronic inflammation in mice.
  • GW AS genome-wide association study
  • This Example describes the successful treatment of a patient (54- year old male) with refractory sarcoidosis, by the use of baricitinib as the medicament for treatment.
  • This molecule targets cytokine regulation through Janus Kinases (JAK) 1 and 2, the signaling pathway of which has been indicated as a hypothetical target in the treatment of sarcoidosis.
  • JNK Janus Kinases
  • 18 F-fluorodeoxyglucose positron emission tomography 18 F-FDG- PET was used to show diffuse pulmonary uptake and pulmonary function in the patient studied, as measured by Forced Vital Capacity (FVC). Both declined progressively, despite treatment with corticosteroids, methotrexate and tumor necrosis factor alpha blockers. During treatment with baricitinib, 18 F-FDG-PET uptake decreased, FVC increased, and patients’ quality of bfe improved.
  • the present Example illustrates that JAK1-2 inhibition is a viable treatment strategy in therapy-resistant sarcoidosis.
  • the patient was diagnosed with pulmonary sarcoidosis in 1998 based on compatible clinical features and radiology, the histologic
  • CT Chest computed tomography
  • the patient consented to off-label treatment with baricitinib 4mg once daily, by oral administration.
  • the patient subsequently stopped taking maintenance therapy with 5mg prednisone daily on his own initiative.
  • the treatment effect of baricitinib and disease activity were measured by inflammatory markers (18F-FDG-PET uptake and serum sIL2R), pulmonary function parameter (FVC), and patient reported outcome (KSQ).
  • 18F-FDG-PET with measurement of SUVmax and biomarker sIL-2R can be used for evaluating disease activity and treatment response (Treglia et al. 2014. Acad Radiol 21:675-84; Maturu et al. 2016. Sarcoidosis Vase Diffuse Lung Dis 33:372- 80).
  • Pulmonary fibrosis leads to substantial morbidity and mortality.
  • immunomodulatory agents may be added as second-line steroid-sparing treatment in refractory cases.
  • Therapy targeting tumor necrosis factor alpha (TNFa) also shows positive effects on FVC in refractory sarcoidosis.
  • Thl7.1-cells IFN-y-producing T helper 17 cells
  • This questionnaire combines organ specific, medication and general health status modules. In our patient, the general health score improved. Although the minimal important difference for this questionnaire has not yet been determined, we beheve that this increase of 90% in score represents a clinically relevant improvement. After three months, the patient wished to continue treatment with baricitinib.
  • JAK1-2 inhibitors are as effective as baricitinib in inhibiting JAK/STAT pathways in sarcoidosis patients
  • T-cell subsets were isolated from 2 healthy donors: ThlT. l cells (CD3+CD4+CD45RA-CCR6+CXCR3+CCR4-), Thl7 cells
  • CD3+CD4+CD45RA-CCR6-CXCR3+CCR4- were purified and 100.000 cells were placed in culture and were stimulated with anti-CD3/CD28 for 3-4 days with and without tofacitinib (250 uM).
  • IFN-g mRNA levels were examined. IFN-g has been shown to be high in pathogenic Thl7.1 cells (see above).
  • peripheral blood mononuclear cells were isolated from 5 healthy donors and 9 sarcoidosis patients. ⁇ 1*10 L 6 PBMCs were incubated with JAK inhibitors baricitinib (300 nM), tofacitinib (250 uM), or filgotinib (10 uM), or left untreated for 1 hr at 37 degrees Celsius.
  • phospho-STAT response in Th-cells and monocytes of sarcoidosis patients is higher after cytokine stimulation than in healthy subjects.
  • Monocytes of sarcoidosis patients show a higher response to IFN-g stimulation than healthy controls, as evidenced by higher phosphorylated STAT1 levels.
  • Naive CD4 T-cells of sarcoidosis patients show a higher response after IL2 stimulation when compared to healthy controls as evidenced by higher phosphorylated STAT5 levels.
  • JAK inhibitors baricitinib (JAK1/2), tofacitinib (JAK 1/3), filgotinib (JAK1)
  • JAK inhibitors baricitinib (JAK1/2), tofacitinib (JAK 1/3), filgotinib (JAK1)
  • Thl7.1 cells react the strongest to JAK1 inhibition when compared to Thl or Thl7 cells. This supports our earlier finding that the pathogenic Thl7.1 cell is a particularly suitable target for JAK inhibition therapy in sarcoidosis, in particular JAK1 inhibition. Following JAK1 inhibition, the Thl7.1 cells produce less IFN-v and are thereby rendered less pathogenic.

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Abstract

La présente invention concerne un procédé de traitement de la sarcoïdose, comprenant l'administration à un sujet qui en a besoin, d'une dose thérapeutiquement efficace d'un inhibiteur de JAK/STAT. La présente invention concerne également un inhibiteur de JAK/STAT destiné à être utilisé dans le traitement de la sarcoïdose.
PCT/NL2019/050394 2018-07-04 2019-06-26 Méthodes de traitement de la sarcoïdose Ceased WO2020009566A1 (fr)

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