Treatment of multiple sclerosis using long-acting glatiramer and adipose derived stem cells
阅读说明：本技术 使用长效格拉替雷和脂肪源性干细胞治疗多发性硬化症 (Treatment of multiple sclerosis using long-acting glatiramer and adipose derived stem cells ) 是由 埃胡德·马罗姆 纳达夫·布莱希·基梅尔曼 弗里达·格里恩斯潘 于 2017-05-15 设计创作，主要内容包括：提供一种治疗多发性硬化症的方法,包括在肠胃外以缓释库形式给予醋酸格拉替雷,并将脂肪源性干细胞给予中枢神经系统。根据一些方面,联合疗法提供协同效应。特别地,联合疗法对进展型多发性硬化症提供益处。(A method for treating multiple sclerosis is provided comprising parenterally administering glatiramer acetate in a sustained release depot form and administering adipose derived stem cells to the central nervous system. According to some aspects, the combination therapy provides a synergistic effect. In particular, combination therapy provides benefits for progressive multiple sclerosis.)
1. A method of treating multiple sclerosis comprising parenterally administering to a subject in need thereof a pharmaceutical composition comprising glatiramer acetate in a sustained release depot form, and administering human adipose derived stem cells (hADSCs) to the Central Nervous System (CNS) of the subject.
2. The method of claim 1, wherein the pharmaceutical composition comprising glatiramer acetate and the hADSCs are administered on the same day.
3. The method of claim 1, wherein the pharmaceutical composition comprising glatiramer acetate and the hADSCs are administered on different days.
4. The method of claim 3, wherein the period of time between administration of the pharmaceutical composition comprising glatiramer acetate and administration of the hADSCs is in the range of 1 to 14 days.
5. The method of claim 1, wherein the pharmaceutical composition comprising glatiramer acetate is administered once every 1 to 6 weeks.
6. The method of claim 1, wherein the pharmaceutical composition comprising glatiramer acetate is administered once every 4 weeks.
7. The method of claim 1, wherein the hADSCs are administered once.
8. The method of claim 1, wherein the hADSCs are administered more than once.
9. The method of claim 8, wherein the hADSCs are administered every 2 to 8 months.
10. The method of claim 1, wherein the pharmaceutical composition comprising glatiramer acetate is administered prior to the hADSCs.
11. The method of claim 1, wherein the pharmaceutical composition comprising glatiramer acetate is administered after the hADSCs.
12. The method of claim 1, wherein the pharmaceutical composition comprising glatiramer acetate is configured for intramuscular administration and the administration is by intramuscular injection.
13. The method of claim 1, wherein the pharmaceutical composition comprising glatiramer acetate is configured for subcutaneous implantation and the administering is by subcutaneous injection.
14. The method of claim 1, wherein the hADSCs are administered intrathecally.
15. The method of claim 1, wherein the hADSCs are administered intracerebroventricularly or Intracerebroventricularly (ICV).
16. The method of claim 1, wherein the hADSCs are obtained from human subcutaneous fat by liposuction aspiration.
17. The method of claim 1, wherein the hADSCs are autologous.
18. The method of claim 1, wherein the hADSCs are allogeneic.
19. The method of claim 1, wherein said hADSCs are characterized by positive expression of CD44, CD73, and CD90 in at least 95% of the cells, positive expression of CD105 in at least 90% of the cells, and negative expression of CD45, CD19, CD11B, and HLADR in at least 95% of the cells.
20. The method of claim 19, wherein said hADSCs are further characterized by positive expression of CD34 in 0.1 to 10% of the cells.
21. The method of claim 1, wherein administering the hADSCs comprises administering about 105-3x108Cells were administered each time.
22. The method of claim 1, wherein said glatiramer acetate comprises acetate salts of L-alanine, L-glutamic acid, L-lysine, and L-tyrosine in a molar ratio of about 0.14 glutamic acid, about 0.43 alanine, about 0.10 tyrosine, and about 0.33 lysine.
23. The method of claim 1, wherein the glatiramer acetate comprises about 15 to about 100 amino acids.
24. The method of claim 1, wherein the pharmaceutical composition comprising glatiramer acetate comprises a pharmaceutically acceptable carrier of biodegradable or non-biodegradable glatiramer acetate.
25. The method of claim 24, wherein the carrier is selected from the group consisting of poly (D, L-lactide-co-glycolide) (PLGA), poly (D, L-lactide) (PLA), Polyglycolide (PGA), polycaprolactone, polyhydroxybutyrate, polyorthoesters, polyalkanoanhydrides, gelatin, collagen, oxidized cellulose, and polyphosphazene.
26. The method of claim 1, wherein the pharmaceutical composition comprising glatiramer acetate is in the form of microparticles prepared by a water-in-oil-in-water double emulsification process.
27. The method of claim 26, wherein the microparticles comprise an internal aqueous phase comprising a therapeutically effective amount of glatiramer acetate, a water immiscible polymer phase comprising a carrier selected from biodegradable and non-biodegradable polymers, and an external aqueous phase.
28. The method of claim 27, wherein the water-immiscible polymer phase comprises a biodegradable polymer selected from PLA and PLGA.
29. The method of claim 27, wherein the external aqueous phase comprises a surfactant selected from polyvinyl alcohol (PVA), polysorbates, polyethylene oxide-polypropylene oxide block copolymers, and cellulose esters.
30. The method of claim 1, wherein the pharmaceutical composition comprising glatiramer acetate comprises about 20-500mg of glatiramer acetate.
31. The method of claim 1, wherein the subject has progressive multiple sclerosis.
32. A pharmaceutical composition comprising glatiramer acetate in the form of a sustained release depot for parenteral administration for use in the treatment of multiple sclerosis in combination with human adipose derived stem cells (hADSCs) administered to the Central Nervous System (CNS).
Technical Field
The present invention relates to a treatment regimen for multiple sclerosis (multiple sclerosis) comprising the administration of a pharmaceutically acceptable salt of glatiramer in a long acting dosage form (long acting nasal forms) and the administration of adipose-derived stem cells. In particular, the invention relates to a combination therapy comprising intramuscular administration or subcutaneous administration of glatiramer acetate in extended release form and intracerebroventricular (intra-ventricular) or intrathecal (intra-administration) administration of adipose-derived stem cells.
Background
Multiple Sclerosis (MS) is a chronic inflammatory disease of the Central Nervous System (CNS) that usually occurs in young people, with women being more common than men. MS affects the ability of nerve cells in the brain and spinal cord to communicate with each other and control bodily functions. Clinical disability is associated with inflammation of myelin (myelin), a protective sheath around CNS axons, damaged by autoimmune attack and neurodegenerative processes. As a result, white matter (whitematemater) of the brain and spinal cord can scar (scarred) due to focal lesions (plaques), leading to neurological dysfunction. There are a number of symptoms of MS. Most patients experience a relapsing-remitting (RRMS) course in the initial phase, characterized by unpredictable relapses, followed by periods of partial or complete recovery (remission), sometimes becoming Progressive (PMS). This progressive MS is classified as secondary progressive MS (secondary progressive MS) (spms). Some patients undergo a progressive course from symptom onset, and this disease pattern is classified as primary progressive ms (primary progressive ms) ((ppms)).
Patients with relapsing remitting MS are often treated with corticosteroids (corticosterioids) during the acute attack (relapses) and with immunomodulatory or immunosuppressive drugs to prevent new relapses and disability progression. In more severe cases, these drugs include interferon beta
All MS treatment options are only partially effective.
Glatiramer Acetate (Glatiramer Acetate):
copolymer-1 (also known as Glatiramer Acetate (GA) and is available under the trade name Gelatirex Acetate (GA)
The mechanism of action of glatiramer acetate is not clear, although some important immunological properties of the copolymer have emerged. Administration of glatiramer acetate can shift the T cell population from pro-inflammatory Th1 cells to regulatory Th2 cells that suppress the inflammatory response (FDA)
Library system for glatiramer acetate (Depot systems):
US8,377,885 discloses a long acting parenteral pharmaceutical composition comprising a therapeutically effective amount of glatiramer, and in particular, a composition comprising a therapeutically effective amount of glatiramer acetate in library form suitable for subcutaneous or intramuscular implantation or injection in the treatment of multiple sclerosis.
US8,796,226 discloses a library composition comprising glatiramer acetate and at least one other drug.
Mesenchymal stem cells (mesnchymal) for the treatment of autoimmune and/or neurodegenerative diseases stem cells)(MSCs)：
MSCs are a source of multipotent self-renewing cells (multipotent self-renewing cells) originally found in adult bone marrow. Naturally, they differentiate to give rise to osteocytes (osteoclasts), chondrocytes (chondrocytes) and adipocytes (adipocytes). MSCs provide a useful source of pluripotent stem cells to replace Embryonic Stem (ES) cells. MSCs potentially circumvent the need for immunosuppression in cell therapy because they are available from autologous sources and because they are characterized by immune-privileged properties that facilitate allogeneic use.
MSCs-based therapies have been shown to be effective in preclinical studies for a number of indications, including graft versus host disease (stroke), myocardial infarction (myocardial infarcation), pulmonary fibrosis, and autoimmune disease. MSCs have also been extensively studied as a therapeutic tool for neurodegenerative diseases such as Alzheimer's Disease (AD), Parkinson's Disease (PD), Amyotrophic Lateral Sclerosis (ALS), Huntington's Disease (HD), and Multiple Sclerosis (MS). In the context of neurodegenerative diseases, MSCs are discussed herein in two respects: its ability to transform into neural cells under specific conditions and its neuroprotective and immunomodulatory effects. When transplanted into the brain, MSCs produce neurotrophic factors and growth factors, thereby protecting and inducing regeneration of damaged tissues. In addition, MSCs have also been explored as gene delivery vectors, e.g., genetically engineered to overexpress glial-derived or brain-derived neurotrophic factor in the brain. Clinical trials involving MSCs are currently underway with MS, ALS, cerebral trauma (traumatic brain injures), spinal cord injury and stroke.
Adipose-derived stem cells (ADSCs):
research over the past decades has shown that adipose tissue is a rich source of multipotent stromal cells in addition to its primary function as an energy reservoir (Zuk et al, Mol Biol Cell 2002; 13: 4279-.
WO 2010/045645 discloses a method for recovering adipose stem cells from adipose tissue.
US8,021,882 discloses a method of producing a stem cell conditioned medium for use in the treatment of nerve injury by providing an adipose stem cell culture and collecting the supernatant.
Constantin et al (2009) Stem cells, 27(10):2624-35 study intravenous administration of adipose-derived mesenchymal Stem cells to mice in chronic Experimental Autoimmune Encephalomyelitis (EAE).
Stepien et al (2016) Mediators of Inflammation, vol.2016 report a one-year follow-up (one-year follow-up) of MS patients with RRMS or SPMS treated by intrathecal injection of autologous adipose stem cells.
WO 2006/057003 discloses inter alia methods of stem cell therapy using bone marrow derived stem cells in combination with glatiramer.
Aharoi et al (2009) J neurommunol, 215(1-2):73-83 reported treatment of EAE-induced mice by intracerebroventricular or intraperitoneal transplantation of Muscle Progenitor Cells (MPCs) in combination with glatiramer acetate.
There is a need in the art for improved methods of treating multiple sclerosis, particularly in patients with progressive disease.
Disclosure of Invention
According to some aspects, the present invention provides combination therapy for Multiple Sclerosis (MS) using a long acting pharmaceutical composition comprising a therapeutically effective amount of a pharmaceutically acceptable salt of glatiramer (e.g., glatiramer acetate) in combination with Adipose Derived Stem Cells (ADSCs). In a specific embodiment, the invention provides a method of treating multiple sclerosis comprising parenterally administering glatiramer acetate in a sustained release depot form and administering adipose derived stem cells to the central nervous system.
The present invention is based in part on the synergistic effect of a combination of glatiramer acetate in a repertoire form and adipose derived stem cells on clinical scoring in an animal model of multiple sclerosis. It has been found that this combination is particularly effective in reducing disease score and delaying disease onset, but also significantly slows disease progression. Unexpectedly, it was found that low doses of glatiramer acetate formulations were more effective when combined with cells than high doses. Thus, according to some embodiments, the methods and compositions provided herein allow for a reduction in the dose of glatiramer acetate administered to a patient as compared to the dose required when glatiramer acetate is administered alone.
According to one aspect, the present invention provides a method of treating multiple sclerosis, comprising parenterally administering to a subject in need thereof a pharmaceutical composition comprising glatiramer acetate, the pharmaceutical composition being in a sustained release depot form, and administering human adipose derived stem cells (hADSCs) to the Central Nervous System (CNS) of the subject.
According to another aspect, the present invention provides a pharmaceutical composition comprising glatiramer acetate in the form of a sustained release depot for parenteral administration for use in combination with human adipose derived stem cells (hADSCs) administered to the Central Nervous System (CNS) for the treatment of multiple sclerosis.
According to some embodiments, the pharmaceutical composition comprising glatiramer acetate and the hADSCs are administered on the same day.
According to other embodiments, the pharmaceutical composition comprising glatiramer acetate and the hADSCs are administered on different days.
According to some embodiments, the time period between administration of the pharmaceutical composition comprising glatiramer acetate and administration of the hADSCs varies between 1 to 14 days. According to other embodiments, the time period between administration of the pharmaceutical composition comprising glatiramer acetate and administration of the hADSCs varies between 1 and 2 weeks.
According to some embodiments, the pharmaceutical composition comprising glatiramer acetate is administered once every 1 to 15 weeks. According to other embodiments, the pharmaceutical composition comprising glatiramer acetate is administered once every 1 to 10 weeks. According to yet other embodiments, the pharmaceutical composition comprising glatiramer acetate is administered once every 2 to 6 weeks. According to some embodiments, the pharmaceutical composition comprising glatiramer acetate is administered once every 4 weeks.
According to some embodiments, the hADSCs are administered once.
According to other embodiments, the hADSCs are administered more than once, e.g., 2 times, 3 times, 4 times, etc. Each possibility represents a separate embodiment of the invention. According to some embodiments, the hADSCs are administered every 2 to 8 months. According to other embodiments, the hADSCs are administered every 3 to 12 months.
According to some embodiments, the pharmaceutical composition comprising glatiramer acetate is administered first prior to administration of the hADSCs.
According to other embodiments, the pharmaceutical composition comprising glatiramer acetate is administered a second time after the administration of the hADSCs.
According to other embodiments, the pharmaceutical composition comprising glatiramer acetate is configured for subcutaneous implantation and the administration is by subcutaneous injection.
According to some embodiments, the administering of the hADSCs is by intrathecal administration
According to other embodiments, the hADSCs are administered by intraventricular or Intracerebroventricular (ICV), i.e., into the ventricles of the brain (brain ventricles).
According to some embodiments, the hADSCs are derived from human subcutaneous fat obtained by liposuction aspiration (lipofection).
According to some embodiments, the hADSCs are autologous.
According to other embodiments, the hADSCs are xenogenic.
According to some embodiments, the hADSCs are characterized by positive expression of CD44, CD73, and CD90 in at least 95% of the cells, positive expression of CD105 in at least 90% of the cells, and negative expression of CD45, CD19, CD11B, and HLADR in at least 95% of the cells. According to some embodiments, the hADSCs are further characterized by positive expression of CD34 in 0.1-10% of the cells.
According to some embodiments, administering the hADSCs comprises administering about 10 at each administration5–3x108And (4) cells.
According to some embodiments, glatiramer acetate comprises acetate salts of L-alanine, L-glutamic acid, L-lysine, and L-tyrosine in a molar ratio of about 0.14 glutamic acid, about 0.43 alanine, about 0.10 tyrosine, and about 0.33 lysine.
According to other embodiments, glatiramer acetate comprises from about 15 to about 100 amino acids.
According to some embodiments, the pharmaceutical composition comprising glatiramer acetate comprises 20 to 500mg glatiramer acetate. According to other embodiments, the pharmaceutical composition comprising glatiramer acetate comprises 20 to 250mg glatiramer acetate. According to yet other embodiments, the pharmaceutical composition comprising glatiramer acetate comprises 20 to 100mg glatiramer acetate.
According to some embodiments, the pharmaceutical composition comprising glatiramer acetate comprises a pharmaceutically acceptable biodegradable or non-biodegradable carrier.
According to some embodiments, the carrier is selected from the group consisting of poly (D, L-lactide-co-glycolide) (PLGA), poly (D, L-lactide-co-glycolide) (PLA), Polyglycolide (PGA), polycaprolactone, polyhydroxybutyrate, polyorthoester, polyanhydride (polykananeanhydride), gelatin, collagen, oxidized cellulose, and polyphosphazene. Each possibility represents a separate embodiment of the invention.
According to some embodiments, the pharmaceutical composition comprising glatiramer acetate is in the form of microparticles prepared by a water-in oil-in water (water-in oil-in water) double emulsification process.
According to some embodiments, the microparticles comprise an internal aqueous phase comprising a therapeutically effective amount of glatiramer acetate, a water immiscible polymer phase comprising a carrier selected from biodegradable and non-biodegradable polymers, and an external aqueous phase. In some embodiments, the water-immiscible polymer phase comprises a biodegradable polymer selected from PLA and PLGA. Each possibility represents a separate embodiment of the invention.
In other embodiments, the external aqueous phase comprises a surfactant selected from polyvinyl alcohol (PVA), polysorbates, polyethylene oxide-polypropylene oxide block polymers, and cellulose esters. Each possibility represents a separate embodiment of the invention.
The subject to be treated as described herein is typically a human. According to some embodiments, the methods and compositions of the invention are used for the treatment of progressive Multiple Sclerosis (MS). Thus, according to some embodiments, the subject is a subject with progressive MS. In some embodiments, the progressive MS is a secondary progressive MS. In other specific embodiments, the progressive MS is a primary progressive MS.
These and other aspects and features of the invention will become apparent from the detailed description, examples and claims that follow.
Drawings
FIG. 1: the effect of GA Depot and ADSCs on EAE was determined by AUC clinical score analysis until
FIG. 2: the effect of GA Depot and ADSCs on EAE was determined by mean peak analysis until
FIG. 3: the effect of GA Depot and ADSCs on EAE was determined by mean day of onset analysis until
FIG. 4: the effect of GA Depot and ADSCs on EAE was determined by mean clinical score analysis until
FIG. 5: effects of GA Depot and ADSCs on body weight of MOG-induced EAE mice until
FIG. 6: the effect of GA Depot in combination with indicated doses of ADSCs on EAE was determined by AUC clinical score analysis until
FIG. 7: the effect of GA Depot in combination with indicated doses of ADSCs on EAE was determined by mean peak analysis until
FIG. 8: the effect of the combination of GA Depot and indicated doses of ADSCs on EAE was determined by mean day of onset analysis until
FIG. 9: the effect of GA Depot in combination with indicated doses of ADSCs on EAE was determined by mean clinical score analysis until
FIG. 10: effect of GA Depot in combination with indicated doses of ADSCs on body weight of MOG-induced EAE mice. Body weight was followed up to
FIG. 11: cell surface marker expression of ADSCs as a function of passage number. Cells were isolated from adipose tissue, cultured as described in the examples section, and analyzed for expression of the indicated markers. The results are the mean of nine (9) samples +/-standard deviation.
Detailed Description
According to some aspects, the present invention provides compositions and methods for treating multiple sclerosis using glatiramer acetate (or any other pharmaceutically acceptable salt of glatiramer) in long acting injectable formulations in combination with adipose-derived stem cells administered to the central nervous system.
Glatiramer acetate depot formulations (GA depot) have been previously described and evaluated in vitro for analysis of glatiramer acetate release profiles and in vivo in mice using Experimental Autoimmune Encephalomyelitis (EAE) induced by Myelin Oligodendrocyte Glycoprotein (MOG). In those studies, GA depot was shown to be involved in ameliorating EAE symptoms
The inventors of the present invention have used the MOG-induced EAE model for assessing the effect of a combination treatment of GA depot with adipose-derived mesenchymal stem cells on disease outcome. MOG is a glycoprotein that is believed to be important in the process of demyelination (myelinization) of the Central Nervous System (CNS). Immunization with the MOG 35-55 peptide was commonly used for induction of chronic EAE in C57BL/6 mice. ADSCs were injected into the ventricles of the brain (ICV) of mice that induced EAE with MOG. Mice were treated with stem cells alone, GA depot alone or in combination with ADSCs and
The present invention discloses for the first time surprising observations demonstrating the synergistic effect of the combination of GA depot and human ADSCs on EAE, including clinical score, mean day of onset, maximum mean disease score and disease duration.
Accordingly, the present invention provides therapeutic methods and regimens for the administration of pharmaceutical formulations comprising pharmaceutically acceptable glatiramer salts, particularly glatiramer acetate, for sustained release administration by parenteral administration, and for the administration of human mesenchymal adipose-derived stem cells. These combination therapies are superior to treatment with GA or stem cells alone in their efficacy against multiple sclerosis. These combination treatments improve and prolong the efficacy as measured by different clinical scores.
There are currently no methods and protocols for treating multiple sclerosis that involve the administration of glatiramer acetate and adipose derived stem cells in a long acting dosage form. Such combination therapy would be beneficial to many patients, particularly those with advanced disease with neurological symptoms or physical disabilities. In particular, such treatment would be beneficial to patients with progressive multiple sclerosis.
The term "treatment" as used herein refers to the inhibition or alleviation of symptoms after the onset of multiple sclerosis. "treating" also encompasses reducing the rate of progression of the disease, or at least one symptom thereof. Common symptoms after the onset of multiple sclerosis include, but are not limited to, vision loss or loss, teeter, speech confusion, and frequency and urinary incontinence. In addition, multiple sclerosis can cause mood changes and depression, muscle spasms and severe paralysis. In particular, the disease is characterized by symptoms such as weakness, numbness, tremors, loss of vision, pain, paralysis, loss of balance, bladder and bowel dysfunction, and cognitive changes (primary symptoms); recurrent urinary tract infections, disuse weakness (disuse weakness), postural adjustments and trunk maladjustment, muscle imbalance, decreased bone density, weak breathing (breathing), dyspnea and bedsores (secondary symptoms); depression (tertiary symptoms). In some embodiments, the treatment comprises: (i) inhibiting the disease condition, i.e., arresting its development; or (ii) remission, i.e., resolution of the disease. Each possibility represents a separate embodiment of the invention. In some embodiments, treating multiple sclerosis according to the present invention comprises slowing disease progression, i.e., relieving the progression of disability.
The "subject" to which the medicament is administered is a mammal, preferably but not limited to a human. The subject is suffering from multiple sclerosis, i.e., is diagnosed with multiple sclerosis.
The term "multiple sclerosis" as used herein refers to an autoimmune disease of the central nervous system, accompanied by one or more of the symptoms described above. In some embodiments, the MS is relapsing-remitting MS. In other embodiments, the MS is a progressive MS. In some embodiments, the progressive MS is a secondary progressive MS. In other embodiments, the progressive MS is a primary progressive MS. In other embodiments, the progressive MS is progressive relapsing MS (progressive delaminating MS).
According to some embodiments, the glatiramer acetate composition is administered every 2 to 6 weeks. According to some embodiments, the glatiramer acetate composition is administered every 4 weeks.
According to some embodiments, the ADSCs are administered once. According to some embodiments, the ADSCs are administered multiple times, e.g., every 2 to 8 months, every 3 to 12 months, or less frequently.
According to some embodiments, the ADSCs are administered once, and the glatiramer acetate composition is administered once every 2 to 6 weeks, e.g., once every 4 weeks.
According to other embodiments, the ADSCs are administered once every 3 to 12 months, such as once every 3,4, 5, 6, 7,8, 9, 10, 11, 12 months, and the glatiramer acetate composition is administered once every 2 to 6 weeks, such as once every 4 weeks.
According to other embodiments, the ADSCs and glatiramer acetate composition are administered according to an alternating schedule.
Adipose-derived stem cells
The present invention uses adipose derived mesenchymal stem cells. As used herein, the term "adipose-derived mesenchymal stem cells" or "adipose-derived stem cells", abbreviated as "ADSCs" or "hADSCs" (i.e., human adipose-derived stem cells), refers to plastic-adherent, pluripotent cell populations obtained from adipose tissue. The cell population is characterized by positive expression of CD44, CD73, and CD90 in at least 95% of the cells, positive expression of CD105 in at least 90% of the cells, and negative expression of CD45, CD19, CD11B, and HLADR in at least 95% of the cells.
In some embodiments, the population of cells is characterized by positive expression of CD44, CD73, and CD90 in at least 98% of the cells, positive expression of CD105 in at least 90% of the cells, and negative expression of CD45, CD19, CD11B, and HLADR in at least 98% of the cells.
The cell population is also characterized by positive expression of CD34 in up to 10% to 20% of the cells. In some embodiments, the population of cells is characterized by positive expression of CD34 in up to 5%, 6%, 7%, 8%, 9%, or 10% of the cells. Each possibility represents a separate embodiment of the invention.
In some embodiments, at least 50% of the cells are positive for CD105, CD73, CD44, and CD90, and negative for CD45, CD19, CD11B, and HLADR.
According to some embodiments, 90 to 100% of the human ADSCs are positive for markers CD44, CD73, and CD 90. According to other embodiments, at least 95% of the human ADSCs are positive for markers CD44, CD73, and CD 90. According to yet other embodiments, at least 98% of the human ADSCs are positive for markers CD44, CD73, and CD 90.
According to some embodiments, 65 to 100% of the human ADSCs are positive for CD 105. According to other embodiments, 80 to 100% of the hADSCs are positive for CD 105. According to yet other embodiments, 90 to 100% of the hADSCs are positive for CD 105. According to yet other embodiments, 80 to 95% of the hADSCs are positive for CD 105.
According to some embodiments, 0.1 to 20% of the human ADSCs express the marker CD 34. According to other embodiments, 0.1 to 10% of the human ADSCs express the marker CD 34. According to yet other embodiments, 0.1 to 5% of the human ADSCs express the marker CD 34. According to yet other embodiments, 0.5 to 2% of the human ADSCs express the marker CD 34.
According to other embodiments, 2 to 10% of the hADSCs are positive for the marker CD 34. According to other embodiments, 2 to 5% of the hADSCs are positive for the marker CD 34. According to some embodiments, at least 90% of the cells, e.g., at least 95% of the cells, are negative for the marker CD 34.
According to some embodiments, at least 90% of the administered human ADSCs are negative for the markers CD45, CD19, CD11B, and HLADR. According to other embodiments, at least 95% of the administered human ADSCs are negative for the markers CD45, CD19, CD11B, and HLADR. According to yet other embodiments, at least 98% of the administered human ADSCs are negative for the markers CD45, CD19, CD11B, and HLADR.
According to some embodiments, at least 50% of the injected human ADSCs are positive for CD105, CD73, CD44, and CD90, and negative for CD45, CD19, CD11B, and HLADR. According to other embodiments, at least 60%, 70%, 80%, or 90% of the injected human ADSCs are positive for CD105, CD73, CD44, and CD90, and negative for CD45, CD19, CD11B, and HLADR. Each possibility represents a separate embodiment of the invention.
Characterization of cell surface marker expression can be performed by methods known in the art, for example, using Fluorescence Activated Cell Sorting (FACS). FACS Protocols are outlined in, for example, Flow Cytometry Protocols, Methods in molecular biology Volume 6992011, Editors: Teresa S.Hawley, Robert G.Hawley Humana Press. Exemplary steps are described below.
Adipose tissue as a source of pluripotent stromal/stem Cells has several advantages over other sources (Baer PC, Geiger h.stem Cells Int 2012; 2012: 812693). For example, subcutaneous fat is ubiquitous in the human body and is readily available in large quantities by liposuction suction. Liposuction (lipofection) is a well-tolerated procedure that produces large volumes of tissue aspirates. Liposuction devices (liposuction) are typically discarded as medical waste, making them a good starting material for adipose-derived matrix/stem cell (ASC) separations. The tissue contains a large number of pluripotent cells, which can be isolated and propagated in culture.
According to some embodiments, the ADSCs are derived from human subcutaneous fat. According to a specific embodiment, the cells are derived from human subcutaneous fat obtained by liposuction aspiration. ADSCs can be harvested by liposuction surgery in various parts of the body including the stomach, hips, thighs, arms, neck and buttocks. Any liposuction procedure may be used to obtain ADSCs in accordance with the present invention, including but not limited to laser, ultrasound, and fat removal by abdominoplasty (abdominopelastic), as is known in the art.
Adipose tissue is treated to isolate adipose-derived stem cells, for example, according to the procedure described in example 1 below. The preparation method generally comprises using buffer (such as PBS and saline) and/or growth medium (such as DMEM, StemMACS)TMOr Plasma-Lyte) and treating the tissue with a tissue dissociation enzyme (such as collagenase) and/or subjecting the tissue to a mechanical agitation/disruption step. It is also possible to use a combination of dispase and collagenase for the digestion of the sample. Colloidal bodies (liposomes) are typically pooled and separated from free stromal cells (including stem cells) and other cells (such as red blood cells, endothelial cells, and fibroblasts) by centrifugation. The red blood cells can be lysed from the suspension particles using a suitable lysis buffer, and the remaining cells filtered or centrifuged.
Alternatively, the isolation may be performed by cell sorting or immunohistochemistry. Bunnell et al, (2008) methods, 45(2) 115-120, review methods for isolation of ADSCs.
In some preferred embodiments, the ADSCs are cultured prior to being provided to a subject in need thereof (or prior to storage for subsequent use). Preferably, the cells are cultured in xeno-free medium. In some embodiments, the ADSCs are grown to 80 to 100% confluence, e.g., about 80% confluence, and subcultured (sub-pooled) to a passage number of 3 to 10, preferably between 3 to 5, or 3 to 4, prior to being administered to the subject. Thus, in some embodiments, the number of passages of the administered cells is between 3 and 5. In some embodiments, the ADSCs are subcultured to
Prior to administration, cells are counted and prepared for injection in a pharmaceutically acceptable diluent/carrier. Typically, the cells are concentrated prior to administration to a subject. The concentration range is usually 1.6X104From ml to 100x106Between/ml.
In some embodiments, a stem cell composition for a single administration according to the methods of the invention comprises 105To 3x108Personal ADSCs. According to some embodiments, the composition comprises 105To 108Personal ADSCs. According to other embodiments,
According to some embodiments, the ADSC compositions of the invention are for systemic administration. Typically, administration is into the Central Nervous System (CNS) of the subject. Such administration may be intended to bypass the blood brain barrier. According to yet other embodiments, the ADSCs are administered directly to a specific region of the brain.
According to some embodiments, the composition is administered to the CNS, e.g. by intraspinal administration. According to some embodiments, the composition is administered intrathecally. According to other embodiments, the composition is administered by the intracerebroventricular or lateral ventricular (ICV) route, i.e., into the ventricles.
Intraventricular drug delivery is drug delivery in the cerebrospinal fluid of the brain cistern (cister) (C1-2 vertebra) and the intracranial ventricles (intracranial ventricular). By directly administering the drug, less drug is required and fewer side effects are experienced than with oral administration. As is known in the art, drugs are typically delivered through an implanted catheter connected to a pump. The pump may be programmable and may be implanted or external.
Intrathecal administration is a route of administration by injecting a drug into the vertebral canal (spinal canal), more specifically into the subarachnoid space (subarachnoid space), to reach the cerebrospinal fluid (CSF), and is useful for spinal anesthesia, chemotherapy, or pain management applications. This approach has also been used to introduce drugs against certain infections, particularly after neurosurgical procedures. The drug needs to be administered in such a way as to avoid the blood brain barrier. Intrathecal or epidural drug delivery involves an intraspinal route of administration. Each route delivers drugs to the Cerebral Spinal Fluid (CSF). Intrathecal delivery involves injecting drugs directly into the CSF of the spinal intrathecal space, whereas drugs injected in the epidural space must pass through the dura to reach the CSF. Thus, epidural administration of drugs may also achieve systemic circulation, while intrathecal administration of drugs is limited to CSF circulating in the spine and ventricles.
Glatiramer formulations
The term "glatiramer acetate" as used herein refers to a compound previously referred to as
The term "parenterally" as used herein refers to a route selected from Subcutaneous (SC), Intravenous (IV), Intramuscular (IM), Intradermal (ID), Intraperitoneal (IP), and the like. Each possibility represents a separate embodiment of the invention.
In some embodiments, the glatiramer formulation is administered by intramuscular, subcutaneous, transdermal, intravenous, or inhalation administration. Each possibility represents a separate embodiment of the invention. According to certain embodiments, the glatiramer formulation is for subcutaneous or intramuscular implantation.
The term "therapeutically effective amount" as used herein is intended to define an amount of glatiramer to achieve the purpose of alleviating the symptoms of multiple sclerosis or at least one of the symptoms of the disease. Suitable dosages for each dosage form include, but are not limited to, 20 to 750 mg. It will be understood, however, that the amount of glatiramer administered will be determined by a physician in light of various parameters, including the chosen route of administration, age, weight, and severity of the patient's symptoms. According to various embodiments of the invention, the therapeutically effective amount of glatiramer varies from about 1 mg/day to about 500 mg/day. Alternatively, this therapeutically effective amount of glatiramer is from about 20mg to about 100mg per day.
The term "long-acting" as used herein refers to a composition that provides long-term, sustained or prolonged release of glatiramer to the general systemic circulation of a subject or to the site of local action of a subject. The term also refers to compositions that provide a long-term, sustained or extended duration of action (pharmacokinetics) of glatiramer in a subject. These compositions are also referred to herein as "sustained release depot forms". In a specific embodiment, the long acting pharmaceutical composition of the present invention provides a dosing regimen ranging from once a week to once every 6 months. It releases a therapeutically effective amount of a pharmaceutically acceptable salt of glatiramer over a period of about one week to about 6 months. According to a more presently preferred embodiment, the administration regimen ranges from once a week, twice a month (approximately once every 2 weeks) to once a month. Based on the desired duration of action, depot preparations (depot formulations) usually contain about 20 to 750mg of active ingredient, which are designed to be released over a period of several weeks to several months.
In certain embodiments, the glatiramer formulation is administered at a concentration of 20 to 30mg GA per 1ml of carrier. In certain embodiments, the carrier is water for injection (WFI). The term "water for injection" or "WFI" as used herein generally refers to sterile, pure water, meeting regulatory standards, e.g., particulate matter, dissolved solids, organic matter, inorganic matter, microorganisms, and endotoxin contaminants. In certain embodiments, the glatiramer formulation is administered in WFI or a buffer comprising a suspending agent (e.g., carboxymethylcellulose, CMC), a buffer (e.g., citrate), and/or a tonicity agent (e.g., NaCl).
In certain embodiments, the glatiramer formulation comprises 10% to 40% solids. In other embodiments, the glatiramer formulation comprises 20% to 30% solids. In certain embodiments, the glatiramer formulation comprises a poly (lactide-co-glycolide) (PLGA) copolymer. In certain embodiments, the PLGA copolymer is a poly (D, L-lactide-co-glycolide) (50:50) copolymer. In some embodiments, the glatiramer formulation comprises 150 to 1500mg of PLGA copolymer per 40mg GA. In some embodiments, the glatiramer formulation comprises 550mg PLGA copolymer per 40mg GA. In certain embodiments, the PLGA copolymer at least partially encapsulates the GA. In certain embodiments, the PLGA copolymer encapsulates GA.
In certain embodiments, less than 30% of the GA is released from the library formulation (depot formulation) within 7 days under continuous stirring in PBS at 37 ℃. In certain embodiments, more than 20% of the GA is released from the library preparation within 75 days in PBS at 37 ℃ under continuous stirring. In certain embodiments, less than 45% of the GA is released from the library preparation within 14 days in PBS at 37 ℃ under continuous stirring. In certain embodiments, more than 90% of the GA is released from the library preparation within 28 days in PBS at 37 ℃ under continuous stirring.
In some embodiments, the library preparations used in the methods of the present invention include, but are not limited to, suspensions of glatiramer or a pharmaceutically acceptable salt thereof in an aqueous phase, an oil phase, or a wax phase; a poorly soluble electrolyte complex of glatiramer or a pharmaceutically acceptable salt thereof; an "in situ" gel-forming matrix based on the association of a water-soluble solvent with glatiramer or a pharmaceutically acceptable salt thereof; and biodegradable polymeric microparticles incorporating glatiramer or a pharmaceutically acceptable salt thereof. Each possibility represents a separate embodiment of the invention. In a specific embodiment, the composition of the invention is in the form of injectable microparticles, wherein glatiramer or a pharmaceutically acceptable salt thereof is entrapped in a biodegradable or non-biodegradable carrier. The microparticle compositions of the present invention may comprise a water-in-oil-in-water double emulsion. Within the scope of the present invention is a particulate composition comprising an inner aqueous phase comprising glatiramer or any pharmaceutically acceptable salt thereof, an oil or water immiscible phase comprising a biodegradable or non-biodegradable polymer, and an outer aqueous phase. The external aqueous phase may also comprise a surfactant, preferably polyvinyl alcohol (PVA), polysorbate, polyethylene oxide-polypropylene oxide block copolymer or cellulose ester. The terms "oil phase" and "water immiscible phase" are used interchangeably herein.
According to a specific embodiment, the long acting pharmaceutical composition of the present invention is in the form of microparticles prepared by a water-in-oil-in-water double emulsification process. In a presently preferred embodiment, the long acting pharmaceutical composition of the invention comprises an inner aqueous phase comprising a therapeutically effective amount of a pharmaceutically acceptable salt of glatiramer, a water immiscible polymer phase comprising a carrier selected from biodegradable or non-biodegradable polymers, and an outer aqueous phase. In other presently preferred embodiments, the water immiscible polymer phase comprises a biodegradable polymer selected from PLA and PLGA. Each possibility represents a separate embodiment of the invention. In other embodiments, the external aqueous phase comprises a surfactant selected from polyvinyl alcohol (PVA), polysorbates, polyethylene oxide-polypropylene oxide block copolymers, and cellulose esters. Each possibility represents a separate embodiment of the invention.
In some embodiments, the composition may comprise any other pharmaceutically acceptable salt of glatiramer, including, but not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydrochloride, hydrobromide, hydroiodide, acetate, nitrate, propionate, decanoate, octanoate, acrylate, formate, isobutyrate, hexanoate, heptanoate, propionate, oxalate, malonate, succinate, tocopheryl succinate, suberate, sebacate, fumarate, maleate, butyne-1, 4-dioate, hexyne-1, 6-dioate, benzoate, chlorobenzoate, methyl benzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methoxinolate, dihydrogenate, dihydrogensulfate, dihydrogenphosphate, pyrophosphate, hydrochloride, hydrobromide, hydroiodide, acetate, nitrate, propionate, caprylate, capryl, Phthalates, terephthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, beta-hydroxybutyrates, glycolates, tartrates, methanesulfonates, propanesulfonates, naphthalene-2-sulfonates, p-toluenesulfonates, mandelates and the like. Each possibility represents a separate embodiment of the invention.
The copolymer can be prepared by any process available to those skilled in the art. For example, the copolymers can be prepared under condensation conditions using the desired molar ratio of amino acids in solution or by solid phase synthesis. The condensation conditions include suitable temperature, pH and solvent conditions for the condensation of the carboxyl group of one amino acid with the amino group of another amino acid to form a peptide bond. A condensing agent, such as dicyclohexylcarbodiimide, may be used to promote the formation of peptide bonds.
Blocking groups may be used to protect functional groups, such as side chain moieties and some amino or carboxyl groups, from undesirable side reactions. The process disclosed in U.S. patent No. 3,849,550, the contents of which are incorporated herein by reference in their entirety, can be used to prepare the copolymers of the present invention. For example, N-carboxyanhydrides of tyrosine, alanine, gamma-benzylglutamic acid and N, epsilon-trifluoroacetyllysine are polymerized in anhydrous dioxamine at ambient temperature using diethylamine as an initiator. The gamma-carboxyl group of glutamic acid can be used as a bromination hydrogen desorption (deblocking) in glacial acetic acid. The trifluoroacetyl group was removed from lysine using one mole of piperidine. It will be readily understood by those skilled in the art that the process can be adapted to produce peptides and polypeptides comprising the desired amino acid (i.e., three of the four amino acids in copolymer 1) by selectively eliminating reactions associated with any of glutamic acid, alanine, tyrosine, or lysine. U.S. patent nos. 6,620,847, 6,362,161, 6,342,476, 6,054,430, 6,048,898 and 5,981,589, the contents of which are hereby incorporated by reference in their entirety, disclose improved processes for preparing glatiramer acetate (
For example, long acting and depot formulations of GA are disclosed in US8,377,885. In a non-limiting example, GA Depot is a sterile lyophilized powder comprising microparticles of polylactic acid (polyglactin) loaded with GA. The formulation is suspended in water for injection and is intended for intramuscular administration, e.g. once every 4 weeks.
The molecular weight of the copolymer can be adjusted during polypeptide synthesis or after polymer preparation. In order to adjust the molecular weight during polypeptide synthesis, the synthesis conditions or the amount of amino acids are adjusted such that synthesis is stopped when the polypeptide reaches approximately the desired length. After synthesis, polypeptides having the desired molecular weight can be obtained by any available size selection method, such as chromatographic analysis of the polypeptide on a molecular weight exclusion column or gel, and collection of the desired range of molecular weights. The polypeptides of the invention may also be partially hydrolyzed to remove high molecular weight materials, such as by acid or enzyme hydrolysis, and then purified to remove the acid or enzyme.
In one embodiment, copolymers having a desired molecular weight can be prepared by a process comprising reacting a protected polypeptide with hydrobromic acid to form a trifluoroacetyl-polypeptide having a desired molecular weight profile. The reaction is carried out at a time and temperature predetermined by one or more test reactions. In the test reaction, the time and temperature are varied and the molecular weight range of a given batch of test polypeptide is determined. Test conditions that provide the optimal molecular weight range for the batch of polypeptide are used for the batch. Thus, trifluoroacetyl-polypeptides having a desired molecular weight profile can be prepared by a process comprising reacting a protected polypeptide with hydrobromic acid at a time and temperature predetermined by a test reaction. The trifluoroacetyl-polypeptide having the desired molecular weight profile is then further treated with an aqueous piperidine solution to form a deprotected polypeptide having the desired molecular weight.
In a presently preferred embodiment, a test sample from a given batch of the protected polypeptide is reacted with hydrobromic acid at a temperature of about 20 to 28 ℃ for about 10 to 50 hours. The optimum conditions for the batch were determined by performing a number of test reactions. For example, in one embodiment, the protected polypeptide is reacted with hydrobromic acid at a temperature of about 26 ℃ for about 17 hours.
In certain embodiments, the dosage form includes, but is not limited to, biodegradable injectable depot systems, such as PLGA-based injectable depot systems; non-PLGA based injectable depot systems and injectable biodegradable gels or dispersions. Each possibility represents a separate embodiment of the invention. The term "biodegradable" as used herein refers to a component that erodes or degrades (at least in part) at its surface over time in contact with substances found in the surrounding tissue fluid or by cellular action. In particular, the biodegradable component is a polymer, such as, but not limited to, a lactic acid-based polymer, for example a polylactide (such as poly (D, L-lactide)), i.e. PLA; glycolic acid based polymers, e.g. Polyglycolide (PLA) (such as from Durect)
In some embodiments, the compositions of the present invention comprise biodegradable copolymers selected from, but not limited to, PLGA, PLA, PGA, polycaprolactones, polyhydroxybutyrates, polyorthoesters, polyalkanhydrides, gelatin, collagen, oxidized cellulose, polyphosphazenes, and the like. Each possibility represents a separate embodiment.
The presently preferred biodegradable polymers are lactic acid based copolymers, more preferably polylactide or poly (D, L-lactide-co-glycolide), i.e. PLGA. Preferably, the biodegradable polymer is present in an amount of about 10% to about 98% w/w of the composition. The lactic acid-based polymer has a monomer ratio of lactic acid to glycolic acid in the range of 100:0 to about 0:100, preferably 100:0 to about 10:90, and an average molar weight of about 1,000 to 200,000 daltons. However, it is understood that the amount of biodegradable polymer is determined by parameters such as duration of use.
The compositions of the present invention may further comprise one or more pharmaceutically acceptable excipients selected from, but not limited to, co-surfactants, solvents/co-solvents, water-immiscible solvents, water-miscible solvents, oily components, hydrophilic solvents, emulsifiers, preservatives, antioxidants, antifoaming agents, stabilizers, buffers, pH adjusting agents, osmotic agents, channeling agents, osmotic agents, or any other excipients known in the art. Suitable co-surfactants include, but are not limited to, polyethylene glycol, polyoxyethylene-polyoxypropylene block copolymers known as "poloxamers", polyglycerol fatty acid esters, such as decaglycerol monolaurate and decaglycerol monocrotonate, sorbitan fatty acid esters, such as sorbitan monostearate, polyoxyethylene sorbitan fatty acid esters, such as polyoxyethylene sorbitan monooleate
The compositions of the present invention may be prepared by any means known in the art. It is currently preferred to incorporate glatiramer or salt copolymers thereof into colloidal delivery systems, such as biodegradable microparticles, to allow for delayed release by diffusion through the polymer walls of the microparticles and by polymer degradation in aqueous or biological fluids in vivo (releaseterredardation). The compositions of the invention may be prepared in the form of injectable microparticles by a process known as "double emulsification". Briefly, a concentrated solution of a water-soluble copolymer is dispersed in a solution of a biodegradable or non-biodegradable polymer in a water-immiscible volatile organic solvent (such as methylene chloride, chloroform, etc.). The "oil-in-water" (w/o) emulsion thus obtained is then dispersed in a continuous external aqueous phase containing a surfactant (e.g., polyvinyl alcohol-PVA, polysorbates, polyethylene oxide-polypropylene oxide block copolymers, cellulose esters, etc.) to form "water-in-oil-in-water (w/o/w) double emulsion" droplets. After evaporation of the organic solvent, the microparticles solidify and are collected by filtration or centrifugation. The collected Microparticles (MPs) were washed with purified water to remove most of the surfactant and unbound peptide, and centrifuged again. The washed MPs were collected without additives or with the addition of cryoprotectants (mannitol) and lyophilized to facilitate their subsequent reconstitution.
The particle size of the "water-in-oil-in-water (w/o/w) double emulsion" can be determined by various parameters including, but not limited to, the amount of force applied at this step, the mixing speed, the surfactant type and concentration, and the like. Suitable particle sizes range from about 1 to 100 μm.
The library system of the present invention comprises any form known to those skilled in the art. Suitable forms include, but are not limited to, biodegradable or non-biodegradable microspheres, implants of any suitable geometry, including implantable rods, implantable capsules, and implantable rings. Each possibility represents a separate embodiment of the invention. Further contemplated are extended release gel reservoirs and erodible matrices. Each possibility represents a separate embodiment of the invention. For example, a suitable implantable system is described in US 2008/0063687, the contents of which are incorporated herein in their entirety. Implantable rods can be prepared using a suitable micro-extruder, as is known in the art.
In accordance with the principles of the present invention, the long acting pharmaceutical compositions of the present invention provide the same or better therapeutic effect as commercially available daily injectable dosage forms, reducing the incidence of and severity of side effects at a local and/or systemic level. In some embodiments, the compositions of the invention provide for extended release or prolonged action of glatiramer in a subject as compared to a substantially similar dose of an immediate release formulation of glatiramer acetate.
The present invention encompasses combination therapies of glatiramer acetate or any other pharmaceutically acceptable salt of glatiramer with adipose derived stem cells and optionally at least one other active agent. Active agents within the scope of the present invention include, but are not limited to, interferons, such as pegylated or non-pegylated alpha-interferon, or beta-interferon, such as interferon beta-1 a or interferon beta-1 b, or interferon tau; having an alternative antiproliferative/antitumor activity(iii) a sexual immunosuppressive agent, such as mitoxantrone (mitoxantrone), methotrexate (methotrexate), azathioprine (azathioprine), cyclophosphamide (cyclophosphamide), or a steroid, such as methylprednisolone (methylprednisone), prednisone (prednisone), or dexamethasone (dexamethasone), or a steroid secreting agent, such as ACTH; adenosine deaminase inhibitors, such as cladribine (cladribine); IV immunoglobulin G (e.g., as disclosed in Neurology, 1998, May 50(5): 1273-81), monoclonal antibodies to various T cell surface markers, such as natalizumab
The invention includes the use of glatiramer acetate or any other pharmaceutically acceptable salt of glatiramer for administration with ADSCs in a depot form suitable for implantation into an individual in need thereof.
The invention encompasses pharmaceutical compositions comprising glatiramer acetate or any other pharmaceutically acceptable salt of glatiramer in the form of a slow release depot for use with hADSCs in the treatment of multiple sclerosis.
The invention also encompasses the combination of glatiramer acetate and ADSCs, with at least one additional drug, preferably an immunosuppressive agent, in particular fingolimod.
The invention also includes the use of glatiramer acetate in the form of a sustained release depot suitable for administration or implantation to a subject in need thereof, together with hADSCs, for the manufacture of a medicament for the treatment of multiple sclerosis.
The following examples are presented to more fully illustrate certain embodiments of the invention. However, they should in no way be construed as limiting the broad scope of the invention. Numerous variations and modifications of the principles disclosed herein may be readily devised by those skilled in the art without departing from the scope of the invention. Examples