A NEW TREATMENT FOR MEIBOMIAN GLAND DYSFUNCTION
20220347167 · 2022-11-03
Inventors
Cpc classification
International classification
Abstract
The invention provides compositions and methods for reducing oxygen concentration in the environment of or promoting the expression or function of at least one hypoxia inducible factor (HIF) in one or more dysfunctional meibomian glands (MGs), utilizing a low oxygen mimetic agent capable of promoting the expression or function of at least one HIF and mimicking a low oxygen environment, or a combination thereof, to improve MG functions.
Claims
1. A method of reducing the severity or treating an optical disease or disorder associated with reduced meibomian gland (MG) function in a subject by locally administering to an eye tissue of the subject a composition comprising a low oxygen mimetic compound, wherein reduced severity or treatment of said disease or disorder comprises increased tear secretion, increased tear volume, prolonged tear film break up time, improved tear osmolarity, improved gland expressibility, improved gland expressed oil quality and volume, increased thickness of tear film lipid layer, decreased ocular surface staining, decreased inflammation on the ocular surface, or alleviated ocular discomfort feeling of patients.
2. The method of claim 1, wherein said compound comprises a hypoxia inducible factor (HIF) prolyl hydroxlase inhibitor (HIF-PHI).
3. The method of claim 1, wherein said low oxygen mimetic compound comprises Roxadustat, Daprodustat, Molidustat, Vadadustat, or Desitustat.
4. The method of claim 1, wherein said composition in the form of an eye drop or eye ointment.
5. The method of claim 1, wherein said composition is administered at a concentration of 0.01-100 mg/ml.
6. The method of claim 1, wherein said compound is administered at a concentration of 1-50 μg/ml.
7. The method of claim 1, wherein said compound is administered to an eye tissue by injection.
8. The method of claim 1, wherein said compound is administered by subconjunctival injection, subdermal injection around an eyelid, or periorbital injection.
9. The method of claim 6, wherein said compound is administered at a dose of 1-300 μg per injection.
10. A method of reducing the severity or treating an optical disease or disorder associated with reduced meibomian gland (MG) function in a subject by systemically administering to the subject a composition comprising a low oxygen mimetic compound, wherein reduced severity or treatment of said disease or disorder comprises increased tear secretion, increased tear volume, prolonged tear film break up time, improved tear osmolarity, improved gland expressibility, improved gland expressed oil quality and volume, increased thickness of tear film lipid layer, decreased ocular surface staining, decreased inflammation on the ocular surface, or alleviated ocular discomfort feeling of patients.
11. The method of claim 10, wherein said compound is administered orally at a dose of 50-70 mg per oral administration or 1-10 mg/kg of body weight per injection.
12. The method of claim 10, wherein the oral dose does not exceed 3.0 mg/kg of body weight.
13. The method of claim 1 or 10, wherein local oxygen concentration at the MG in the subject is reduced to no more than 1%.
14. The method of claim 1 or 10, wherein the agent in iii) comprises Roxadustat (Roxa), dimethyloxalyglycine, desferrioxamine, cobalt (II) chloride (CoCl.sub.2), of combinations thereof.
15. A method of promoting differentiation of a meibomian gland epithelial cell (MGEC) by i) reducing local oxygen concentration of the MGEC to no more than 1%; and/or ii) contacting the MGEC with an HIP-PHI.
16. The method of claim 15, wherein the agent comprises roxadustat (Roxa), dimethyloxalyglycine, desferrioxamine, cobalt (II) chloride (CoCl.sub.2), FG-2216, daprodustat/GSK1278863, vadadustat/AKB-6548, molidustat/BAY 85-3934, desidustat/ZYAN1), Dimethyloxalylglycine (DMOG), or combinations thereof.
17. A method of treating obesity and/or reducing weight gain of a subject by administering to the subject a pharmaceutically effective dosage of an HIF-PHI agent capable of reducing oxygen concentration, wherein said subject comprises obesity with high serum cholesterol level.
18. The method of claim 17, wherein the agent comprises Roxadustat (Roxa), dimethyloxalyglycine, desferrioxamine, cobalt (II) chloride (CoCl.sub.2), of combinations thereof.
19. A dosage formulation for administration to a subject, wherein the formulation comprises Roxadustat (Roxa) i) in a therapeutically effective amount between about 0.1-100 mg/ml for administration as an eye drop for treatment of reduced meibomian gland (MG) function; or ii) in a therapeutically effective amount between about 1-300 μg for administration as an injection for treatment of reduced meibomian gland (MG) function.
20. A dosage formulation for administration to a subject, wherein the formulation comprises Roxadustat (Roxa) in a therapeutically effective amount between about 50-500 mg for treatment of obesity and/or elevated serum cholesterol, wherein said effective amount is administered 1-5 times per week.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0116] Low O.sub.2 is usually considered to have adverse effects on human health. However, under certain conditions, low O.sub.2 may be actually beneficial for diseased tissues, according to the latest discoveries. In fact, low O.sub.2 therapy (also called as hypoxia therapy) has been used in both animal models and phase III clinical trials to treat various conditions in recent years, such as mitochondrial disease, wound healing, retinopathy of prematurity, corneal endothelial cell protection, anemia and tissue regeneration. Low O.sub.2 therapy includes reducing the O.sub.2 concentration, either systemically or locally in a tissue, or inducing a cellular response by using hypoxia mimetic drugs.
[0117] The instant disclosure includes, at least, a method for using low O.sub.2 therapy as a new treatment for meibomian gland dysfunction (MGD) and other related diseases or disorders. In fact, MGD is the major cause of dry eye disease (DED), which afflicts countless people throughout the world (i.e., greater than 40 million in the USA), and is one of the most frequent causes of patient visits to eye care practitioners. Prevalence of this disease ranges from approximately 5% up to 75% in different populations, with more affected women than men. Prevalence is also higher in Asian and aging populations, though younger people are increasingly exhibiting symptoms. As the world population is expected to increase from 7.2 billion in 2012 to between 8.3-10.9 billion by 2050, the incidence of DED is expected to rise. According to The Lancet Series on Aging, published in 2014, 2 billion people will be aged 60 years or older by the year 2050. Assuming a prevalence of 25%, 500 million people will have dry eye disease in this demographic alone. The current burden of DED for the USA healthcare system is estimated to be over $3.8 billion, and, because of diminished productivity, $55.4 billion for the USA overall. It has been estimated that manufacturers' global revenues in the dry eye treatments market will climb from $4.6 billion in 2018 to $6.2 billion in 2023. The DED market has been divided into three broad segments: procedures, over-the-counter (OTC) lubricants, and prescription pharmaceuticals. Revenues from OTC lubricants and prescription pharmaceuticals are forecast to grow moderately—in the range of 4.3 percent to 4.5 percent a year. A much higher cumulative annual growth rate of 21.4 percent is expected in procedure revenues.
[0118] Patients with DED continuously experience dryness, stinging, burning, chronic pain, and blurred vision. Moderate to severe DED is associated with significant pain, role limitations, low vitality and poor general health. These symptoms significantly impair quality of life. Additionally, patients with DED frequently report significant disturbances in their psychiatric state, showing symptoms of anxiety and depression. A previous diagnosis of DED, or frequent experiences of dry eye symptoms, has been associated with depression and suicidal ideation. There is currently no global cure for MGD or DED.
[0119] Treatments for MGD include the LipiFlow thermal pulsation system, warm compression, lid cleansing, and the off-label use of antibiotics or intense pulsed light therapy. In addition to these MGD-specific treatments, patients are often prescribed one of the three FDA-approved drugs for DED. All of these are immunosuppressants, which target inflammatory pathways and are designed to alleviate the signs and symptoms of DED. Two of these drugs share the same major ingredient, but with differences in dose and formation. However, the pathophysiology of MGD and DED, and the mechanisms of current treatments are still unclear, and the long-term effects of these treatments are unclear. Although MGD can induce inflammation at the ocular surface, the role of inflammation in the etiology of MGD is controversial and uncertain. In addition, the vast majority of interventional clinical trials of anti-inflammatory and immunomodulatory agents have failed to meet their primary endpoints. It appears that inflammation is not the ideal target for MGD and DED treatment.
[0120] Compared to current treatments for MGD, the instant disclosure provides, at least, a method to target a completely different pathway. It was recently found for the first time that healthy human MGs exist in a physiologically hypoxic environment, and that a low O.sub.2 environment significantly promotes the function of human MG epithelial cells (HMGECs). Low oxygen mimetic drugs can duplicate the function-promoting effect of a low O.sub.2 environment in HMGECs. This discovery reveals a new aspect of the biology of the MG and pathophysiology of MGD, and also exposes the potential for developing new treatments. The instant disclosure provides, at least, a method to use low O.sub.2 therapy to promote the function of diseased MGs by correcting the disease upstream, suggesting that by inducing the physiologically hypoxic environment for MG tissue, normal MG function may be restored.
[0121] The instant disclosure provides, at least, a method to treat patients with MGD and DED with a new therapeutic modality—a low O.sub.2 therapy. Low O.sub.2 therapy includes reducing the O.sub.2 concentration, either systemically or locally in a tissue, or inducing a cellular response by using hypoxia mimetic drugs, such as Roxadustat (Roxa). Even though it may seem counterintuitive, the instant disclosure provides that low O.sub.2 promotes the function of MGs, and Roxa can duplicate the effect of low O.sub.2 on MGs. In order to translate the treatment into the clinical setting, Roxa may be applied locally around the MGs. Because the MG is a superficial tissue located in the eyelids, it is not difficult to apply Roxa topically and induce a local low O.sub.2 reaction in the MGs. In addition, because MGD and DED do not directly impact other organs, Roxa, or other molecules disclosed herein, can be localized at the ocular surface to avoid systemic effects. The effectiveness of systemic use of Roxa, or other molecules disclosed herein, may be tested in treating MGD, and if successful, Roxa, or other molecules disclosed herein, may be further developed into an eye drop or eye ointment. If the project is successful, patients could buy the drug with prescription and use it at home.
[0122] It was discovered, for the first time, that human MGs exist in a physiologically hypoxic environment, and low O.sub.2 environment significantly promotes the function of HMGECs. This discovery significantly advances the current understanding of the pathophysiology of MGD. Several low oxygen mimetic drugs, such as Roxa, can duplicate the positive effect of low O.sub.2 environment in HMGEC function. Roxa showed a stronger lipid-inducing effect in HMGECs than other low oxygen mimetic drugs. Roxa is now marketed for the treatment of anemia with chronic kidney disease in China. Thus, Roxa has passed the toxicity tests for both clinical trial and animal studies.
Agents
[0123] This specification describes at least one agent that may be administered to the subject in a pharmaceutically effective amount, a pharmaceutical composition, and/or a dosage formulation. In vitro, such agent may mimic a low oxygen (hypoxia) concentration in the environment of a meibomian gland epithelial cell (MGEC) (i.e., as low oxygen mimetic agents, such as roxadustat/Roxa), promote the differentiation of the MGEC (thus decreasing the MGEC number), increases levels of at least one of hypoxia-inducible factors (HIFs), such as HIF1α and/or prevents degradation of at least one of hypoxia-inducible factors (HIFs), such as HIF1α, in the MGEC, increase the size of the MG, increase the levels of neutral lipid droplets and/or nonpolar lipid acids in the MGEC, or combinations thereof. The agent may have the similar in vivo functions in a subject. For example, such agent may increase the MG function in a subject having reduced MG functions and/or a disease or disorder related to reduced MG functions, increase tear volume, increase the size of the MG, decrease the serum cholesterol level, decrease the percentage of weight gain, or combinations thereof. In general, the agents described herein is capable of mimicking low oxygen concentration at the MG in a subject, increasing MG functions in a subject, decreasing serum cholesterol levels, and/or decreasing the percentage of weight gain in the subject.
[0124] The agent described herein may comprise, for example, roxadustat (Roxa), dimethyloxalyglycine, desferrioxamine, cobalt (II) chloride (CoCl.sub.2), FG-2216, daprodustat/GSK1278863, vadadustat/AKB-6548, molidustat/BAY 85-3934, desidustat/ZYAN1, Dimethyloxalylglycine (DMOG), or combinations thereof. In some embodiments, the agent comprises an HIF prolyl hydroxylases inhibitor (HIF-PHI). In some embodiments, the HIF-PIH comprises roxadustat (Roxa), daprodustat/GSK1278863, molidustat/BAY 85-3934, vadadustat/AKB-6548, desidustat/ZYAN1.
[0125] A pharmaceutically effective amount of such agent may comprise about 0.1 to about 100 mg/kg, about 0.1 to about 50 mg/kg, about 0.1 to about 20 mg/kg, about 0.1 to about 10 mg/kg, about 0.1 to about 5 mg/kg, about 0.5 to about 100 mg/kg, about 0.5 to about 50 mg/kg, about 0.5 to about 20 mg/kg, about 0.5 to about 10 mg/kg, about 0.5 to about 5 mg/kg, about 1 to about 100 mg/kg, about 1 to about 50 mg/kg, about 1 to about 20 mg/kg, about 1 to about 10 mg/kg, about 1 to about 5 mg/kg, about 5 to about 10 mg/kg, about 5 to about 20 mg/kg, about 5 to about 50 mg/kg, about 5 to about 100 mg/kg, or other amount of the agent.
[0126] In some embodiments, the agents described herein comprises roxadustat (Roxa, a.k.a., FG-4592, ASP1517, AZD9941). Roxa has a structure as Formula I below:
##STR00001##
[0127] In some embodiments, the agents described herein comprises roxadustat (Roxa) comprising a structure of Formula I, or a pharmaceutically acceptable salt or prodrug thereof.
[0128] In some embodiments, the agent may have a structure as Formula II below.
##STR00002##
[0129] In Formula II:
[0130] each X.sup.1 and X.sup.2 is independently O or S;
[0131] each L.sup.1 and L.sup.2 is —O—, —NR.sup.a—, or R.sup.c-substituted or unsubstituted alkylene (e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkylene, or C.sub.1-C.sub.4 alkylene);
[0132] R.sup.1 is —OR.sup.a, NR.sup.aR.sup.b, R.sup.c-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl), or R.sup.c-substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl);
[0133] each R.sup.2, R.sup.3, R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is independently hydrogen, halogen, —OR.sup.a, NR.sup.aR.sup.b, R.sup.c-substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl), or R.sup.c-substituted or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl);
[0134] each R.sup.a and R.sup.b is independently hydrogen, or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl);
[0135] R.sup.c is halogen, oxo, —N.sub.3, —CN, —N.sub.3, —OH, —NH.sub.2, —COOH, —CONH.sub.2, —NO.sub.2, —SH, —SO.sub.2H, —SO.sub.3H, or unsubstituted alkyl; and n is an integer of 0 to 5.
[0136] In some embodiments, X.sup.1 is O. In some embodiments, X.sup.2 is O. In some embodiments, X.sup.1 is S. In some embodiments, X.sup.2 is S.
[0137] In some embodiments, L.sup.1 is —O—, —NH—, or unsubstituted C.sub.1-C.sub.4 alkylene. In some embodiments, L.sup.2 is —O—, —NH—, or unsubstituted C.sub.1-C.sub.4 alkylene. In some embodiments, L.sup.1 is —O—. In some embodiments, L.sup.2 is methylene or ethylene.
[0138] In some embodiments, le is —OH, —OCH.sub.3, —NH.sub.2, —NHCH.sub.3, NR.sup.aR.sup.b, or unsubstituted C.sub.1-C.sub.4 alkyl. In some embodiments, R.sup.1 is —OH or —OCH.sub.3.
[0139] In some embodiments, R.sup.2 is hydrogen, —OR.sup.a, —NR.sup.aR.sup.b, or unsubstituted C.sub.1-C.sub.4 alkyl. In some embodiments, R.sup.3 is hydrogen, —OR.sup.a, —NR.sup.aR.sup.b, or unsubstituted C.sub.1-C.sub.4 alkyl. In some embodiments, each R.sup.a and R.sup.b is independently hydrogen or unsubstituted C.sub.1-C.sub.4 alkyl. In some embodiments, R.sup.2 is —OH, —OCH.sub.3, methyl, or ethyl. In some embodiments, R.sup.3 is —OH, —OCH.sub.3, methyl, or ethyl. In some embodiments, R.sup.2 is —OH. In some embodiments, R.sup.3 is methyl.
[0140] In embodiments, each R.sup.4, R.sup.5, R.sup.6, and R.sup.7 is independently hydrogen, —OH, or unsubstituted C.sub.1-C.sub.4 alkyl. In embodiments, each R.sup.4, R.sup.5, R.sup.6, and R.sup.7 are hydrogen.
[0141] In some embodiments, the agent may have a structure as Formula III below.
##STR00003##
R.sup.1, R.sup.2, R.sup.3, and L.sup.2 are as described herein.
[0142] In some embodiments, R.sup.1 is —OH. In some embodiments, L.sup.2 is unsubstituted methylene or ethylene. In some embodiments, each of R.sup.2 and R.sup.3 is independently —OH or methyl. In some embodiments, R.sup.2 is —OH and R.sup.3 is methyl.
[0143] In some embodiments, the agent described herein comprises a structure selected from the group consisting of Formula I, Formula II, Formula III, and a pharmaceutically acceptable salt or prodrug thereof.
[0144] In some embodiments, the agents described herein comprises daprodustat (a.k.a., GSK1278863). Daprodustat has a structure as Formula IV below:
##STR00004##
[0145] In some embodiments, the agents described herein comprises daprodustat/GSK1278863 comprising a structure of Formula IV, or a pharmaceutically acceptable salt or prodrug thereof.
[0146] In some embodiments, the agents described herein comprises molidustat (a.k.a., Bay 85-3934). Molidustat has a structure as Formula V below:
##STR00005##
[0147] In some embodiments, the agents described herein comprises molidustat/Bay 85-3934 comprising a structure of Formula V, or a pharmaceutically acceptable salt or prodrug thereof.
[0148] In some embodiments, the agents described herein comprises vadadustat (a.k.a., AKB-6548, PG-1016548). Vadadustat has a structure as Formula VI below:
##STR00006##
[0149] In some embodiments, the agents described herein comprises vadadustat comprising a structure of Formula VI, or a pharmaceutically acceptable salt or prodrug thereof.
[0150] In some embodiments, the agents described herein comprises desidustat (a.k.a., ZYAN1). Desidustat has a structure as Formula VII below:
##STR00007##
[0151] In some embodiments, the agents described herein comprises desidustat comprising a structure of Formula VII, or a pharmaceutically acceptable salt or prodrug thereof.
Low Oxygen Concentration
[0152] The instant disclosure provides a method to increase MG function and/or treat a disease or disorder related to reduced MG function in a subject by application of low oxygen environment to the MG or the subject. There are different definitions for hypoxia; the terms physiological, modest, moderate and severe hypoxia and anoxia have been used to designate 10-14, 2.5, 0.5, 0.1 and 0% O.sub.2, respectively [Evans et al. (2006) The Journal of Investigative Dermatology 126:2596-2606]. In the instant disclosure, the terms “hypoxia” or “hypoxic environment” refer to an O.sub.2 concentration of less than 5%, unless expressly taught otherwise. This usage is consistent with that of other studies [McKeown (2014) Br J Radiol 87:20130676]. In some embodiments, an oxygen concentration lower than the physiological oxygen concentration in the mice MG (i.e., 1.3%) is used, such as less than 1.3%, 1%, 0.5%, 0.1%, or less oxygen. A low O.sub.2 environment (generally under 5% O.sub.2) triggers a hypoxic response pathway in mammalian cells, which is centered on the regulated expression of hypoxia-inducible factor (HIF). HIF, which consists of α and β subunits, is the central player that regulates hypoxic responses. To date, three HIFα isoforms (HIF1α, HIF2α and HIF3α) have been identified. Among these isoforms, HIF1α is the primary regulator of cellular responses to O.sub.2 levels. HIF1α is an O.sub.2 sensitive subunit, and its accumulation and degradation are highly sensitive to changing O.sub.2 levels. When the O.sub.2 level increases, HIF prolyl hydroxylases (HIF-PH) are activated and HIF1α is rapidly degraded. In a low O.sub.2 environment, HIF-PH activity is suppressed, thereby increasing the steady-state levels of HIF1α. Then HIF1α can translocate to the nucleus, form a heterodimer with HIF1β, and activate the transcription of numerous genes and pathways involved in cell proliferation, cell differentiation, tissue regeneration and lipid metabolism. HIF2α share similarities with HIF1α, but also has its distinct function in modulating cell function. The function of HIF3α is currently unclear. Roxadustat belongs to the family of HIF prolyl hydroxylases inhibitors (HIF-PHI), which mimics the effect of low O.sub.2 by increasing the levels of hypoxia-inducible factors. The information for human HIF1α (including isoform sequences) may be found at the World Wide Web site at uniprot.org/uniprot/Q16665. For example, the human HIF1α, isoform 1 comprises an amino acid sequence of
TABLE-US-00001 MEGAGGANDKKKISSERRKEKSRDAARSRRSKESEVFYELAHQLPLPHN VSSHLDKASVMRLTISYLRVRKLLDAGDLDIEDDMKAQMNCFYLKALDG FVMVLTDDGDMIYISDNVNKYMGLTQFELTGHSVFDFTHPCDHEEMREM LTHRNGLVKKGKEQNTQRSFFLRMKCTLTSRGRTMNIKSATWKVLHCTG HIHVYDTNSNQPQCGYKKPPMTCLVLICEPIPHPSNIEIPLDSKTFLSR HSLDMKFSYCDERITELMGYEPEELLGRSIYEYYHALDSDHLTKTHHDM FTKGQVTTGQYRMLAKRGGYVWVETQATVIYNTKNSQPQCIVCVNYVVS GIIQHDLIFSLQQTECVLKPVESSDMKMTQLFTKVESEDTSSLFDKLKK EPDALTLLAPAAGDTIISLDFGSNDTETDDQQLEEVPLYNDVMLPSPNE KLQNINLAMSPLPTAETPKPLRSSADPALNQEVALKLEPNPESLELSFT MPQIQDQTPSPSDGSTRQSSPEPNSPSEYCFYVDSDMVNEFKLELVEKL FAEDTEAKNPFSTQDTDLDLEMLAPYIPMDDDFQLRSFDQLSPLESSSA SPESASPQSTVTVFQQTQIQEPTANATTTTATTDELKTVTKDRMEDIKI LIASPSPTHIHKETTSATSSPYRDTQSRTASPNRAGKGVIEQTEKSHPR SPNVLSVALSQRTTVPEEELNPKILALQNAQRKRKMEHDGSLFQAVGIG TLLQQPDDHAATTSLSWKRVKGCKSSEQNGMEQKTIILIPSDLACRLLG QSMDESGLPQLTSYDCEVNAPIQGSRNLLQGEELLRALDQVN (SEQ ID NO: 1; NCBI REFERENCE SEQUENCE: NP_001521.1)
[0153] The information for human HIF2α (a.k.a., Endothelial PAS domain-containing protein 1, or EPAS1) may be found at the World Wide Web site at uniprot.org/uniprot/Q99814. For example, the human HIF2α comprises an amino acid sequence of
TABLE-US-00002 MTADKEKKRSSSERRKEKSRDAARCRRSKETEVFYELAHELPLPHSVSS HLDKASIMRLAISFLRTHKLLSSVCSENESEAEADQQMDNLYLKALEGF IAVVTQDGDMIFLSENISKFMGLTQVELTGHSIFDFTHPCDHEEIRENL SLKNGSGFGKKSKDMSTERDFFMRMKCTVTNRGRTVNLKSATWKVLHCT GQVKVYNNCPPHNSLCGYKEPLLSCLIIMCEPIQHPSHMDIPLDSKTFL SRHSMDMKFTYCDDRITELIGYHPEELLGRSAYEFYHALDSENMTKSHQ NLCTKGQVVSGQYRMLAKHGGYVWLETQGTVIYNPRNLQPQCIMCVNYV LSEIEKNDVVFSMDQTESLFKPHLMAMNSIFDSSGKGAVSEKSNFLFTK LKEEPEELAQLAPTPGDAIISLDFGNQNFEESSAYGKAILPPSQPWATE LRSHSTQSEAGSLPAFTVPQAAAPGSTTPSATSSSSSCSTPNSPEDYYT SLDNDLKIEVIEKLFAMDTEAKDQCSTQTDFNELDLETLAPYIPMDGED FQLSPICPEERLLAENPQSTPQHCFSAMTNIFQPLAPVAPHSPFLLDKF QQQLESKKTEPEHRPMSSIFFDAGSKASLPPCCGQASTPLSSMGGRSNT QWPPDPPLHFGPTKWAVGDQRTEFLGAAPLGPPVSPPHVSTFKTRSAKG FGARGPDVLSPAMVALSNKLKLKRQLEYEEQAFQDLSGGDPPGGSTSHL MWKRMKNLRGGSCPLMPDKPLSANVPNDKFTQNPMRGLGHPLRHLPLPQ PPSAISPGENSKSRFPPQCYATQYQDYSLSSAHKVSGMASRLLGPSFES YLLPELTRYDCEVNVPVLGSSTLLQGGDLLRALDQAT (SEQ ID NO: 2; NCBI Reference Sequence: NP_001421.2)
[0154] The information for human HIF3α (including isoform sequences) may be found at the World Wide Web site at uniprot.org/uniprot/Q9Y2N7. For example, the human HIF3α, isoform 1 comprises an amino acid sequence of
TABLE-US-00003 MALGLQRARSTTELRKEKSRDAARSRRSQETEVLYQLAHTLPFARGVSA HLDKASIMRLTISYLRMHRLCAAGEWNQVGAGGEPLDACYLKALEGFVM VLTAEGDMAYLSENVSKHLGLSQLELIGHSIFDFIHPCDQEELQDALTP QQTLSRRKVEAPTERCFSLRMKSTLTSRGRTLNLKAATWKVLNCSGHMR AYKPPAQTSPAGSPDSEPPLQCLVLICEAIPHPGSLEPPLGRGAFLSRH SLDMKFTYCDDRIAEVAGYSPDDLIGCSAYEYIHALDSDAVSKSIHTLL SKGQAVTGQYRFLARSGGYLWTQTQATVVSGGRGPQSESIVCVHFLISQ VEETGVVLSLEQTEQHSRRPIQRGAPSQKDTPNPGDSLDTPGPRILAFL HPPSLSEAALAADPRRFCSPDLRRLLGPILDGASVAATPSTPLATRHPQ SPLSADLPDELPVGTENVHRLFTSGKDTEAVETDLDIAQDADALDLEML APYISMDDDFQLNASEQLPRAYHRPLGAVPRPRARSFHGLSPPALEPSL LPRWGSDPRLSCSSPSRGDPSASSPMAGARKRTLAQSSEDEDEGVELLG VRPPKRSPSPEHENFLLFPLSLSFLLTGGPAPGSLQDPSTPLLNLNEPL GLGPSLLSPYSDEDTTQPGGPFQPRAGSAQAD (SEQ ID NO: 3; NCBI Reference Sequence: NP_690008.2)
[0155] Multiple methods may be used to lower oxygen concentration in a subject, e.g., in the environment of the MG, such as near the eyes and/or eyelids area. For example, restricting of the blood flow can be accomplished in a number of ways, including by contracting or closing one or more blood vessels around the one or more dysfunctional MGs. For example, restricting the blood flow can be achieved, among other approaches, using one or more of a 532-nm potassium titanyl phosphate (KTP) laser, a 532-nm neodymium yttrium-aluminum-garnet (Nd:YAG) laser, a 578-nm copper vapor laser, 585-600-nm pulsed dye laser (PDL), a dual 595-nm PDL, a long-pulse alexandrite (755 nm), a 800-983-nm diode laser, a 1,064-nm Nd:YAG laser, indocyanine green augmented laser therapy, PDL treatment combined with rapamycin, intense pulsed light (IPL), carbon dioxide (CO.sub.2) laser, cryotherapy, vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR) inhibitors or antagonists, systemic and/or local beta-blockers, anti-angiogenic molecules and mixtures thereof. Any of these devices can be configured specifically for this treatment. The hypoxic status can be induced in one or more of the following ways: pharmaceutically, surgically, using a laser, using an intense-pulsed light, with a device and/or using hypoxia chamber goggles.
[0156] The effects of reduced oxygen concentration can also be mimicked by the systemic or local use of the agents described herein that induce the generation of HIFs in the dysfunctional MGs. These low oxygen mimetic agents include such drugs as one or more of prolyl hydroxylases inhibitors, i.e. FG-4592/roxadustat (Roxa), FG-2216, daprodustat/GSK1278863, vadadustat/AKB-6548, molidustat/BAY 85-3934, desidustat/ZYAN1), Dimethyloxalylglycine (DMOG), desferrioxamine (DFX) and cobalt chloride (CoCl.sub.2), etc.
[0157] In some embodiments, restricting of the blood flow is accomplished by contracting or closing one or more blood vessels around the one or more dysfunctional meibomian glands. In some embodiments, restricting the blood flow is achieved, among other approaches, using one or more of a 532-nm potassium titanyl phosphate (KTP) laser, a 532-nm neodymium yttrium-aluminum-garnet (Nd:YAG) laser, a 578-nm copper vapor laser, 585-600-nm pulsed dye laser (PDL), a dual 595-nm PDL, a long-pulse alexandrite (755 nm), a 800-983-nm diode laser, a 1,064-nm Nd:YAG laser, indocyanine green augmented laser therapy, PDL treatment combined with rapamycin, intense pulsed light (IPL), carbon dioxide (CO.sub.2) laser, cryotherapy, vascular endothelial growth factor (VEGF)/vascular endothelial growth factor receptor (VEGFR) inhibitors or antagonists, systemic and/or local beta-blockers, anti-angiogenic molecules and mixtures thereof. In some embodiments, the hypoxic status is induced pharmaceutically. In some embodiments, the hypoxic status is induced surgically. In some embodiments, the hypoxic status is induced using a laser. In some embodiments, the hypoxic status is induced using an intense-pulsed light. In some embodiments, the hypoxic status is induced with a device, such as hypoxia chamber goggles.
[0158] It was recently found that the oxygen-sensitive protein HIF1α plays an essential role in the regulation of human meibomian gland epithelial cells (HMGECs). To be specific, HIF1α activation promotes HMGEC differentiation. Although exposure to a low O.sub.2 environment is a widely used method to stimulate HIF1α, systemic and prolonged hypoxia can lead to a range of severe pathophysiological reactions and compensations in vivo and will hinder its translation into clinical treatment in the future. In addition to a hypoxic environment, HIF1α expression also can be induced by low oxygen mimetic agents and growth factors. In order to mimic a local hypoxic environment around the MG, but also to minimize the compensatory effects of systemic low oxygen exposure, agents capable of pharmacological activation of the HIF1α pathway under normoxic (21% O.sub.2) conditions were studied. It was found that the hypoxia mimetic Roxadustat (Roxa; also called FG-4592) can duplicate the differentiation-promoting effect of low oxygen by promoting HIF1α in immortalized HMGECs under normoxic condition. Roxa has been used tested for safety in animal studies and has passed the phase III clinical trial for the treatment of anemia in patients with chronic kidney disease.
Combinational Therapy
[0159] In some embodiments, combinations of the low oxygen mimetic agents and the method to induce low oxygen concentration in the environment of MG, both described herein, are used. As described in the Examples, synergy for such combinational therapy was discovered. The agents and the method may be used simultaneously or sequentially.
The Ability to Increase MG Function
[0160] The mechanisms underlying the low oxygen mimetic agents and/or the method/therapy of lowering oxygen concentration described herein were studied in the MGECs and an in vivo animal model. With no intention to be limiting, the agents described herein, such as Roxa, and/or a low oxygen concentration, such as no more than 1% O.sub.2, increases expression levels and/or stability of at least one hypoxia-inducible factors (HIFs), such as HIF1α, in the MGECs. Other functions of the agents and the low oxygen method/therapy are described in the above sections.
Decrease of Cholesterol Levels
[0161] The mechanisms underlying the low oxygen mimetic agents and/or the method/therapy of lowering oxygen concentration described herein were studied in the MGECs and an in vivo animal model. With no intention to be limiting, the agents described herein, such as Roxa, and/or a low oxygen concentration, such as no more than 1% O.sub.2, reduces serum cholesterol levels in the treated subject. For example, the low oxygen mimetic agents and/or the method/therapy of lowering oxygen concentration described herein reduces cholesterol levels (e.g., serum cholesterol levels) in the treated subject to about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less, compared to a subject with a same disease or disorder without the treatment, or a healthy subject. The cholesterol levels in a subject may be measured using standard blood test or other methods known in the art, and may include indexes including at least one of total cholesterol amount [a measure of the total amount of cholesterol in blood, including both low-density lipoprotein (LDL) cholesterol and high-density lipoprotein (HDL) cholesterol], LDL cholesterol amount (the main source of cholesterol buildup and blockage in the arteries), HDL cholesterol amount (HDL helps remove cholesterol from arteries), Non-HDL amount [the total cholesterol minus HDL, including only LDL and other types of cholesterol such as VLDL (very-low-density lipoprotein)], triglycerides amount, etc. In some embodiments, the low oxygen mimetic agents and/or the method/therapy of lowering oxygen concentration described herein reduces the levels of at least one of the total cholesterol amount, the LDL cholesterol amount, the Non-HDL amount, and the triglycerides amount. In some embodiments, the hypoxia-mimetic agents and/or the method/therapy of lowering oxygen concentration described herein reduces the levels of at least one of the total cholesterol amount, the LDL cholesterol amount, the Non-HDL amount, and the triglycerides amount in the treated subject to about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less, compared to a subject with a same disease or disorder without the treatment, or a healthy subject. In some embodiments, the low oxygen mimetic agents and/or the method/therapy of lowering oxygen concentration described herein increases the levels of the HDL cholesterol amount in the treated subject to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 500%, 1000%, or more, than the levels in a subject with a same disease or disorder without the treatment, or a healthy subject.
[0162] The terms “normal cholesterol levels” and “normal serum cholesterol levels” refers to levels in a common healthy subject or a subject without a specific disease or disorder described herein, recognized by a doctor or a medical personnel. For example, healthy serum cholesterol may be less than 200 mg/dL, healthy LDL cholesterol may be less than 130 mg/dL, healthy HDL cholesterol may be higher than 55 mg/dL for women and 45 mg/dL for men, and healthy triglycerides may be less than 150 mg/dL. Normal cholesterol levels may refer to a condition when any of these levels are in the healthy levels. High cholesterol levels may refer to a condition when any of these levels are out of the healthy levels. For example, high cholesterol levels may refer to at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 500%, 1000%, or more, levels than healthy levels for serum cholesterol, LDL, and/or triglycerides, and/or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or less, levels than healthy levels for HDL. For example, normal cholesterol levels may refer to at most 30%, 25%, 20%, 15%, 10%, 5%, or less, more than healthy levels for serum cholesterol, LDL, and/or triglycerides, and at most 5%, 10%, 15%, 20%, 25%, 30%, or more, less than healthy levels for HDL.
Diseases and Disorders
[0163] The diseases and disorders related to reduced MG functions, as a target for the agents and/or the low oxygen treatment described herein, comprises at least one selected from the group consisting of: MG dysfunction (MGD), dry eye disease (DED), Sjogren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, etc.
[0164] According to a summary of from the Tear Film and Ocular Surface Society (see Nichols et al. Invest Ophthalmol Vis Sci. 2011 52:1922-1929), Meibomian gland dysfunction (MGD) is a chronic, diffuse abnormality of the meibomian glands, commonly characterized by terminal duct obstruction and/or qualitative/quantitative changes in the glandular secretion. It may result in alteration of the tear film, symptoms of eye irritation, clinically apparent inflammation, and ocular surface disease. The major risk factors of MGD include aging, androgen deficiency, stem cell imbalance, and retinoic acid (RA) use. These factors lead to disrupted differentiation and renewal of meibocytes, obstruction of meibomian orifices, stasis of meibum, acinar atrophy, gland dropout and eventually to MGD. Meibomian gland disease is used to describe a broader range of meibomian gland disorders, including neoplasia and congenital disease. Other terms such as meibomitis or meibomianitis describe a subset of disorders of MGD associated with inflammation of the meibomian glands. Although inflammation may be important in the classification and in the therapy of MGD, these terms are not sufficiently general, as inflammation is not always present. There is no global cure for MGD. Improvement in the patient's symptoms is the major goal in the treatment of MGD.
[0165] A classification of MGD into two major categories based on meibomian gland secretion is proposed: low-delivery states and high-delivery states. Low-delivery states are further classified as hyposecretory or obstructive, with cicatricial and noncicatricial subcategories. Hyposecretory MGD describes the condition of decreased meibum delivery due to abnormalities in meibomian glands without remarkable obstruction. Obstructive MGD is due to terminal duct obstruction. In the cicatricial form, the duct orifices are dragged posteriorly into the mucosa, whereas these orifices remain in their normal positions in noncicatricial MGD. High-delivery, hypersecretory MGD is characterized by the release of a large volume of lipid at the lid margin that becomes visible on application of pressure onto the tarsus during examination. Each MGD category also has primary causes, referring to conditions for which there are no discernible underlying causes or etiology.
[0166] In some embodiments, a subject to be treated has a diseases or disorder related to increased serum cholesterols, and/or increased weight gain. For example, the subject may have obesity, diabetes, overweight or other related diseases or disorders.
[0167] Diagnostic methods for the diseases or disorders to be treated by the low oxygen mimetic agents and/or the method/therapy to reduce local oxygen concentration in MG, as described herein, are well known in the art. For example, Sjogren's syndrome is a complex and currently incurable autoimmune disorder, which is characterized by dry eyes and a dry mouth. This condition often accompanies other immune system disorders, such systemic lupus erythematosus or rheumatoid arthritis. These diseases cause decreased aqueous tear production, leading to aqueous-deficient dry eye disease (DED). They are also associated with meibomian gland dysfunction (MGD) and evaporative DED.
[0168] Exemplary diagnosis methods for MGD are known in the art. See Rolando and Vagge (2014) Current Ophthalmology Reports volume 2, pages 65-74 or Nichols et al. (2011) Invest Ophthalmol Vis Sci. 52:1922-1929. A summary of specialized and nonspecialized tests for MGD and MGD-related disease is given below, adapted from Nichols et al. (2011).
TABLE-US-00004 Specialized and Nonspecialized Tests for MGD and MGD-Related Disease Testing Specific Tests for a Tests for a Category Test(s) General Clinic Specialized Unit Symptoms Questionnaires McMonnies; McMonnies; Schein; Schein; ODSI; ODSI; DEQ; OCI; DEQ; OCI; SPEED; and others SPEED; and others Signs Meibomian Lid Slit lamp Slit lamp microscopy; function morphology microscopy confocal microscopy Meibomian Meibography gland mass Gland Slit lamp Slit lamp microscopy expressibility; microscopy expressed oil quality and volume Lid margin Meibometry reservoir Tear film lipid Interferometry, Interferometry; slit layer; slit lamp lamp; video thickness, interferometry spread time, spread rate Evaporation Evaporimetry Evaporimetry Tears Osmolarity Osmolarity TearLab device, TearLab device, other other Stability Tear film TFBUT; Ocular TFBUT; Ocular protection index protection index Tear film lipid Spread time Interferometry; spread layer rate; pattern Indices of Tear secretion Schimer 1 Fluorophotometry/ volume and fluorescetin secretion clearance rate Tear volume Not available Volume by fluorophotometry Tear volume Meniscus height Meniscus radius of curvature; meniscometry Tear clearance Tear film index Tear film index Oscular Oscular surface Oxford scheme; Oxford scheme; surface staining NEI/industry NEI/industry scheme Inflammation Biomarkers scheme Flow cytometry; bead arrays; microarrays; mass spectrometry; cyrokines and other mediators; interleukins; matrix metalloproteinases Tests of glandular functions are presented first followed by those for related disorders such as dry eye. OSDI, Oscular Surface Disease Inders; DEQ, Dry Eye Questionaire; OCI, Oscular Comfort Inders; SPEED, Standard Patient Evaluation of Eye Dryness.
[0169] There is no practicable way to measure O.sub.2 level or the activity of HIF proteins in human meibomian gland directly. However, other techniques known in the art, e.g., positron emission tomography (PET) may be used to measure the aerobic glycolysis, as an indirect indicator.
[0170] Exemplary outcome measurements for the improvement of MGD and DED by the low oxygen mimetic agents and/or the method/therapy to reduce local oxygen concentration may include: increased tear secretion and tear volume, prolonged tear film break up time, improved tear osmolarity, improved gland expressibility and expressed oil quality and volume, increased thickness of tear film lipid layer, decreased ocular surface staining, decreased inflammation on the ocular surface, and alleviated discomfort feeling of patients. For example, Meibum quality is assessed in each of eight glands of the central third of the lower lid on a scale of 0 to 3 for each gland: 0, clear; 1, cloudy; 2, cloudy with debris (granular); and 3, thick, like toothpaste (total score range, 0-24). Expressibility is assessed on a scale of 0 to 3 in five glands in the lower or upper lid, according to the number of glands expressible: 0, all glands; 1, three to four glands; 2, one to two glands; and 3, no glands. Staining scores are obtained by summing the scores of the exposed cornea and conjunctiva. Oxford staining score range, 1-15; DEWS staining score range, 0-33. In some embodiments, the therapy and/or the low oxygen mimetic agents, described herein, improve the Meibum quality in at least one of measurable indexes described herein.
[0171] Tear volume and tear secretion may be measured by fluorophotometric examinations. For example, a 0.1% fluorescein solution (1 μl) may be applied to the lateral upper bulbar conjunctiva and mixed with the tear film. Ten seconds after application, the clearance of tear film fluorescein may be determined by measuring the decrease in tear film fluorescein five times with 1 minute intervals, using a Fluorotron Master (Coherent Radiation Inc). A regression line for the clearance of the tear film fluorescein and the tear film turnover rate may be automatically calculated (Eter and Göbbels, Br J Ophthalmol. 2002 86:616-619). Similar tests include Schirmer's test, which determines whether the eye produces enough tears to keep it moist. In some embodiments, the therapy and/or the low oxygen mimetic agents, described herein, improve the tear volume and/or tear secretion of the treated subject to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 500%, 1000%, or more.
[0172] Tear breakup time (TBUT) is a clinical test used to assess for evaporative dry eye disease. To measure TBUT, fluorescein is instilled into the patient's tear film and the patient is asked not to blink while the tear film is observed under a broad beam of cobalt blue illumination. The TBUT is recorded as the number of seconds that elapse between the last blink and the appearance of the first dry spot in the tear film, as seen in this progression of these slit lamps photos over time. A TBUT under 10 seconds is considered abnormal. This patient also has punctate epithelial erosions (PEE) that stain positively with fluorescein, another sign of ocular surface dryness. TBUT measurement is an easy and fast method used to assess the stability of tear film. In some embodiments, the therapy and/or the low oxygen mimetic agents, described herein, improve the tear breakup time (TBUT) of the treated subject to about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 500%, 1000%, or more.
[0173] One of the principal indicators of tear dysfunction is elevated tear film osmolarity (hyperosmolarity), predominantly due to elevated sodium ion concentration. Elevated osmolarity is considered the central mechanism of ocular surface damage and may be the single best marker for dry eye disease. The diagnostic cut-off is in excess of 316 mOsmol/L. In some embodiments, the therapy and/or the low oxygen mimetic agents, described herein, decreases the tear osmolarity of the treated subject to about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less 10%. It was reported that an average tear osmolarity is of 302±9.7 in normal subjects (815) and is of 326.9±22.1 in subjects with keratoconjunctivitis sicca (621). In some embodiments, the therapy and/or the low oxygen mimetic agents, described herein, decreases the tear osmolarity of the treated subject to about 315, 314, 313, 312, 311, 310, 309, 308, 307, 306, 305, 304, 303, 302, 301, 300, 299, 298, 297, 296, 295, 294, 293, 292, 291, 290 mOsmol/L, or less.
Obesity
[0174] Obesity is a medical condition in which excess body fat has accumulated to an extent that it may have a negative effect on health. People are generally considered obese when their body mass index (BMI), a measurement obtained by dividing a person's weight by the square of the person's height, is over, e.g., 30 kg/m.sup.2; the range 25-30 kg/m.sup.2 is usually defined as overweight. In adults, obesity is defined as having a BMI of 30.0 or more, according to the Centers for Disease Control and Prevention (CDC). The relationship between adult BMI and obesity class is: BMI of 18.5 or under is considered underweight, 18.5 to <25.0 is considered “normal” weight, 25.0 to <30.0 is considered overweight, 30.0 to <35.0 is considered class 1 obesity, 35.0 to <40.0 is considered class 2 obesity, 40.0 or over is considered class 3 obesity (also known as morbid, extreme, or severe obesity). For a doctor to diagnose a child over 2 years old or a teen with obesity, their BMI has to be in the 95th percentile for people of their same age and biological sex as below: >5% percentile of BMI is considered underweight, 5% to <85% is considered “normal” weight, 85% to <95% is considered overweight, 95% or over is considered obesity.
[0175] Some East Asian countries use lower values. Obesity increases the likelihood of various diseases and conditions, particularly cardiovascular diseases, type 2 diabetes, obstructive sleep apnea, certain types of cancer, osteoarthritis, and depression.
[0176] The symptoms of obesity may include: Above average body weight, Trouble sleeping, Sleep apnea—a condition in which breathing is irregular and periodically stops during sleep, Varicose veins, Skin problems caused by moisture that accumulates in the folds of your skin, Gallstones, Osteoarthritis in weight-bearing joints, especially the knees.
[0177] BMI is a rough calculation of a person's weight in relation to their height. Other more accurate measures of body fat and body fat distribution include: skinfold thickness tests, waist-to-hip comparisons, screening tests, such as ultrasounds, CT scans, and MRI scans. Other tests to help diagnose obesity-related health risks may include: blood tests to examine cholesterol and glucose levels, liver function tests, a diabetes screening, thyroid tests, heart tests, such as an electrocardiogram (ECG or EKG).
Ocular Surface Disease Index (OSDI)
[0178] The Ocular Surface Disease Index (OSDI) is a 12-item questionnaire that provides a rapid assessment of the symptoms of ocular irritation consistent with ocular surface disease, including posterior blepharitis and dry eye disease, and their impact on vision-related functioning. The 12 items of the OSDI questionnaire are graded on a scale of 0 to 4, where 0 indicates none of the time; 1, some of the time; 2, half of the time; 3, most of the time; and 4, all of the time. The total OSDI score is then calculated on the basis of the following formula: OSDI=[(sum of scores for all questions answered)×100]/[(total number of questions answered)×4]. Thus, the OSDI is scored on a scale of 0 to 100, with higher scores representing greater disability. A negative change from baseline indicates an improvement in vision-related function and the ocular inflammatory disorders described herein. For the therapeutic method described herein, treatment is considered more effective than control (vehicle) as indicated by a mean change (decrease) from baseline for the OSDI of >10 units compared to control.
[0179] Therapeutic treatment is considered more effective than the vehicle as indicated by a mean change from baseline of average score (0-100) for the Ocular Surface Disease Index (OSDI) of >10 units better than vehicle.
Tear Film Break-Up Time
[0180] The standard TBUT measurement is performed by moistening a fluorescein strip with sterile non-preserved saline and applying it to the inferior tarsal conjunctiva. After several blinks, the tear film is examined using a broad beam of the slit lamp with a blue filter. The time lapse between the last blink and the appearance of the first randomly distributed dark discontinuity in the fluorescein stained tear film is measured three times and the mean value of the measurements is calculated. The tear break-up time is evaluated prior to the instillation of any eye drops and before the eyelids are manipulated in any way. Break-up times less than 10 seconds are considered abnormal. A positive change from baseline indicates improvement in symptoms of the ocular inflammatory disorders described herein. The treatment described herein, leads to an improvement in TBUT significantly greater than that observed from treatment with vehicle alone.
Corneal and Conjunctival Staining
[0181] Corneal staining is a measure of epithelial disease, or break in the epithelial barrier of the ocular surface, typically seen with ocular surface disorders such as posterior blepharitis and dry eye, among others. Importantly, corneal staining can exist even without clinically evident dry eye, if there is significant lid disease, such as posterior blepharitis. Corneal staining is highly correlated with ocular discomfort in many, though not all patients; in general corneal staining is associated with high scores in the OSDI, as described above. For corneal fluorescein staining, saline-moistened fluorescein strips or 1% sodium fluorescein solution are used to stain the tear film. The entire cornea is then examined using slit-lamp evaluation with a yellow barrier filter (#12 Wratten) and cobalt blue illumination (staining is more intense when it is observed with a yellow filter). Staining is graded according to the Oxford Schema.
[0182] Conjunctival staining is a measure of epithelial disease or break in the epithelial barrier of the ocular surface, typically seen with ocular surface disorders such as posterior blepharitis and dry eye, among others. Importantly, conjunctival staining, similar to corneal staining, can exist even without clinically evident dry eye, if there is significant lid disease, such as posterior blepharitis. Conjunctival staining can also correlate with symptoms of ocular irritation and high OSDI scores as described above. Conjunctival staining is performed under the slit-lamp using lissamine green. Saline-moistened strip or 1% lissamine green solution is used to stain the tear film, and interpalpebral conjunctival staining is evaluated more than 30 seconds, but less than 2 minutes, later. Using white light of moderate intensity, only the interpalpebral region of the nasal and temporal conjunctival staining is graded using the Oxford Schema (above). The treatment described herein leads to decreases in ocular staining scores beyond what is observed with the vehicle alone.
[0183] Therapeutic treatment is considered more effective than vehicle as indicated by a mean change from baseline in average score (0-5 scale) for corneal and conjunctival staining of >1 unit better than vehicle, e.g. as detected using the Oxford Schema.
Schirmer Test
[0184] The Schirmer test is performed in the presence and in the absence of anesthesia by placing a narrow filter-paper strip (5×3 5 mm strip of Whatman #41 filter paper) in the inferior cul-de-sac. This test is conducted in a dimly lit room. The patient gently closes his/her eyes until five minutes have elapsed and the strips are removed. Because the tear front will continue advancing a few millimeters after it has been removed from the eyes, the tear front is marked with a ball-point pen at precisely five minutes. Aqueous tear production is measured by the length in millimeters that the strip wets during 5 minutes. Results of 10 mm or less for the Schirmer test without anesthesia and 5 mm or less for the Schirmer test with anesthesia are considered abnormal. A positive change from baseline indicates improvement of one or more symptoms of an ocular inflammatory disorder described herein.
Meibomian Gland Evaluation
[0185] In the center of the lower lid, 10 adjacent central glands are located on both sides and the glands are expressed by applying a firm digital pressure at the base of the glands. The number of glands expressed for each eye is documented. The quality of secretion is described as follows: [0186] Clear excreta or clear with small particles (0) [0187] Opaque excreta with normal viscosity (1) [0188] Opaque excreta with increased viscosity (2) [0189] Secretions retain shape after expression (3)
[0190] Posterior blepharitis is associated with lid inflammation and alterations in the quantity and/or quality of the meibomian gland secretions, with severe disease associated with quality grades 2-3, as described above. The treatment described herein leads to improvement in meibomian secretion characterized by a decrease in this score; for example, from 3 to 2, or from 2 to 1. An improvement is indicated by a mean change from baseline (0-3 scale) for meibomian gland secretion quality of >1 unit better than vehicle.
Lid and Lid Margin Erythema
[0191] Lid margin vascular injection (erythema) is defined as a red discoloration, compared to the surrounding eyelid skin and is graded as follows:
TABLE-US-00005 None (0): none Mild (1): redness localized to a small region of the lid margin(s) or skin Moderate (2): redness of most of the lid margin(s) Severe (3): redness of most or all the lid margin(s) and skin Very Severe (4): marked diffuse redness of both lid margins and skin
[0192] The presence or absence of tarsal telangiectasis is also noted. Lid telangiectasia is defined as the presence of at least two blood vessels along the eyelid margin.
Conjunctiva Hyperemia
[0193] Bulbar conjunctival hyperemia is graded as follows:
TABLE-US-00006 None (0): none Mild (1): slight localized injection Moderate (2): pink color, confined to palpebral or bulbar conjunctiva Severe (3): red color of the palpebral and/or bulbar conjunctiva Very Severe (4): marked dark redness of the palpebral and/or bulbar conjunctiva
[0194] The presence or absence of tarsal papillary hypertrophy is also noted.
Formulations and Dosage for Administration
[0195] Formulations of the present invention include those suitable for oral, nasal, topical, transdermal, buccal, sublingual, intramuscular, intracardiac, intraperotineal, intrathecal, intracranial, rectal, vaginal and/or other parenteral (e.g., intravenous) administration of the agents described herein, e.g., Roxa. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient, e.g., Roxa, which can be combined with a carrier material to produce a single dosage form, will generally be that amount of the compound that produces a therapeutic effect.
[0196] Pharmaceutically available and/or effective administration routes may be used to deliver the agents (such as Roxa) described herein to a subject. With no intention to be limiting, the agents may be administered locally or systemically to a subject, including administering to, e.g., the circulatory system and/or the GI tract (the digestive system) of the subject, via, e.g., inhalation, pulmonary lavage, oral ingestion, anal administration, infusion, and/or injection, or to the local area near the eyes or the eyelids (e.g., through topical administration). Administration routes also include, but not limited to, administering to a subject intravenously, intradermally, intraperitoneally, intrapleurally, intratracheally, intramuscularly, subcutaneously, by injection, and by infusion. In some embodiments, the administration routes include oral and/or intravenous administration. In some embodiments, the administration routes comprise local injection, such as subconjunctiva injection, subdermal injection around the eyelids, or periorbital injection.
[0197] Dosages and dosing regimens may be determined by a doctor or a medical personnel accordingly to each patient's individual situation. Generally, the agents (such as Roxa) can be given orally at about 1-20 g/day to improve MG functions and/or to treat a disease or disorder described herein. Oral dosage formulations may be administered either in tablet or powder form, or conveniently dissolved in a little water or another beverage, or included in foodstuffs or in food or medical supplements. The preferred adult dose of the agents (such as Roxa) is generally equivalent to about 10 g per day, but anywhere between about 0.1 g per day up to about 20 g or about 30 g per day may be taken. For example, potential dosages may include about 0.01 g to about 30 g per day, about 0.01 g to about 30 g per day, about 0.1 g to about 30 g per day, about 1 g to about 30 g per day, about 1 g to about 5 g per day, about 5 g to about 10 g per day, or any dosages found effective to the subject. Specifically, potential dosages may include about 0.01 to about 200 mg/kg body weight, about 0.01 to about 100 mg/kg body weight, about 0.05 to about 200 mg/kg body weight, about 0.05 to about 100 mg/kg body, weight, about 0.05 to about 50 mg/kg body weight, about 0.1 to about 200 mg/kg body weight, about 0.01 to about 100 mg/kg body weight, about 0.1 to about 50 mg/kg body weight, about 0.1 to about 20 mg/kg body weight, about 0.1 to about 10 mg/kg body weight, about 0.1 to about 5 mg/kg body weight, about 0.5 to about 200 mg/kg body weight, about 0.5 to about 100 mg/kg body weight, about 0.5 to about 50 mg/kg body weight, about 0.5 to about 20 mg/kg body weight, about 0.5 to about 10 mg/kg body weight, about 0.5 to about 5 mg/kg body weight, about 1 to about 200 mg/kg body weight, about 1 to about 100 mg/kg body weight, about 1 to about 50 mg/kg body weight, about 1 to about 30 mg/kg body weight, about 1 to about 20 mg/kg body weight, about 1 to about 10 mg/kg body weight, about 1 to about 5 mg/kg body weight, etc. In some embodiments, the agents (such as Roxa) is administered to the subject in a dosage regimen of about 0.1 to about 10 mg/kg body weight. The oral dosing of Roxa was 50-70 mg three times per week to treat anemia. Unless more proofs are available otherwise, similar doses should be used for treating MGD and DED (and other diseases or disorders described herein). In some embodiments, the dose for treating obesity is about 50-500 mg per time, for example, about 50-300 mg per time, about 50-200 mg per time, about 50-100 mg per time, about 100-500 mg per time, about 100-300 mg per time, about 100-200 mg per time, about 200-500 mg per time, about 200-300 mg per time, about 300-500 mg per time, etc. In some embodiments, the dose is about 1-300 μg per injection, for example, about 1-200 μg per injection, about 1-100 μg per injection, about 1-50 per injection, about 1-20 μg per injection, about 1-10 μg per injection, about 20-300 per injection, about 20-200 μg per injection, about 20-100 μg per injection, about 50-300 per injection, about 50-200 μg per injection, about 50-100 μg per injection, about 100-200 per injection, about 100-300 μg per injection, about 200-300 μg per injection, etc. In some embodiments, the concentration of the agents (e.g., Roxa) in eye drops is about 0.01-100 mg/ml, for example, about 0.01 mg/ml to about 50 mg/ml, about 0.1 mg/ml to about 50 mg/ml, about 0.5 mg/ml to about 50 mg/ml, about 1 mg/ml to about 50 mg/ml, about 1 mg/ml to about 30 mg/ml, about 1 mg/ml to about 20 mg/ml, about 1 mg/ml to about 10 mg/ml, about 0.01 mg/ml to about 30 mg/ml, about 0.01 mg/ml to about 20 mg/ml, about 0.01 mg/ml to about 10 mg/ml, about 0.1 mg/ml to about 50 mg/ml, about 0.1 mg/ml to about 20 mg/ml, about 0.1 mg/ml to about 10 mg/ml, about 50 mg/ml to about 100 mg/ml, about 10 mg/ml to about 100 mg/ml, about 20 mg/ml to about 100 mg/ml, about 30 mg/ml to about 100 mg/ml, etc. With no intention to be limiting, the approved dose for Evrenzo® (roxadustat) Tablets to treat anemia associated with chronic kidney disease in dialysis patients is, for adults, 50 mg (for patients not on erythropoiesis-stimulating agent treatment) or 70 or 100 mg (for patients switching from erythropoiesis-stimulating agents) as the starting dose, orally administered three times weekly, while the thereafter dosages should be adjusted according to the patient's condition with a maximum dose not exceeding 3.0 mg/kg.
[0198] In some cases, the agents (such as Roxa) is administered twice or more, e.g., 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more. For example, the agents (such as Roxa) is administered at least once per week, e.g., at least twice per week, at least three times per week, at least four times per week, at least five times per week, at least six times per week, at least seven times per week. Alternatively, the agents (such as Roxa) is administered at least once per day, e.g., at least twice per day, at least every eight hours, at least every four hours, at least every two hours, or at least every hour. The compositions of the invention (e.g., the dosage formulation of the agents, such as Roxa) are administered for a duration of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, 4 weeks, five weeks, six weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 2 years, 3 years, 4 years, 5 years or more. For example, the composition of the invention are administered one dose every two weeks for 4 to 6 weeks or until the cancer is treated.
[0199] Optionally, the subject is also treated with at least one additional agent and/or therapy (e.g., low oxygen treatment). In some cases, the agents (such as Roxa) described herein is administered simultaneously with the at least one additional agent and/or therapy (e.g., low oxygen treatment). Alternatively, the agents (such as Roxa) described herein and the at least one additional therapy (e.g., low oxygen treatment) are administered sequentially.
EXAMPLES
[0200] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature cited above.
[0201] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.
Example 1
[0202] The Role of Hypoxia-Inducible Factor 1α in the Regulation of Human Meibomian Gland (MG) Epithelial Cells
[0203] This Example describes an exemplary design and experimentation to analyze the HIF1α expression in the human MG under hypoxic environment or upon Roxa treatment. The effect of Roxa on stimulating differentiation in IHMGECs was also analyze.
[0204] Optimal meibomian gland (MG) function is critically important for the health and wellbeing of the ocular surface [Knop et al. (2011) Investigative Ophthalmology & Visual Science 52:1938-1978; Bron et al. (2017) The Ocular Surface 15:438-510]. These glands synthesize and secrete a proteinaceous lipid mixture (i.e. meibum) that enhances the stability and prevents the evaporation of the tear film [Knop et al. (2011); Bron et al. (2017); Green-Church et al. (2011) Investigative Ophthalmology & Visual Science 52:1979-1993]. MG dysfunction (MGD), in turn, leads to a loss of meibum, destabilization and hyperevaporation of the tear film, and dry eye disease (DED) [Knop et al. (2011); Bron et al. (2017); Green-Church et al. (2011); Bron and Tiffany (2004) The Ocular Surface 2:149-165]. Indeed, MGD is the most common cause of DED [Knop et al. (2011); Bron et al. (2017); Green-Church et al. (2011); Bron and Tiffany (2004); Nelson et al. (2011) Investigative Ophthalmology & Visual Science 52:1930-1937; Lemp et al. (2012) Cornea 31:472-478; Nichols et al. (2011) Investigative Ophthalmology & Visual Science 52:1922-1929].
[0205] There is no known cure for MGD. This fact is likely due to the relative lack of information concerning the physiological regulation of the human MG in health and disease. Recently, it was discovered that a hypoxic environment is beneficial for MG function [Liu et al. (2019)]. More specifically, it was found that human and mouse MGs exist in a hypoxic environment in vivo, and that low oxygen (O.sub.2) stimulates differentiation of immortalized human meibomian gland epithelial cells (IHMGECs) in vitro. This hypoxic response involves an increased expression of sterol regulatory element binding protein 1 (SREBP1), an enlargement of lysosomes and a rise in deoxyribonuclease (DNase) II activity [Liu et al (2019)]. SREBP1 is a key controller of lipid synthesis [Horton et al. (2002) The Journal of Clinical Investigation 109:1125-1131], and DNase II is a biomarker for terminal differentiation and holocrine secretion [Zouboulis (2017) The Journal of Investigative Dermatology 137:537-539; Fischer et al. (2017) The Journal of Investigative Dermatology 137:587-594]. In contrast, the local hypoxic environment may be lost in MGD. The results suggest that re-induction of this hypoxic status may be a breath of fresh air for the treatment of MGD in future.
[0206] The mechanism(s) underlying this effect of hypoxia on the MG is unknown, but it is hypothesized that it is due to an increase in the levels of hypoxia-inducible factor1 α (HIF1α). HIF1a is the primary regulator of cellular responses to hypoxia [Wang et al. (1995) Proceedings of the National Academy of Sciences of the United States of America 92:5510-5514; Iyer et al. (1998) Genes & Development 12:149-162]. In other tissues HIF1α expression can be induced by multiple stimuli, including hypoxia, low oxygen mimetic agents, and certain growth factors [Coimbra et al. (2004) Osteoarthritis Cartilage 12:336-345; Stroka et al. (2]001) FASEB Journal 15:2445-2453; Nakada et al. (2017) Nature 541:222-227; Ferrari et al. (2017) Proceedings of the National Academy of Sciences of the United States of America 114:E4241-E4250; Jain et al. (2016) Science 352:54-61; Kim et al. (2018) Leukemia 32:2672-2684; Botusan et al. (2008) Proceedings of the National Academy of Sciences of the United States of America 105:19426-19431]. Typically, HIF1α is rapidly degraded, but exposure to hypoxic conditions [i.e. under 5% O.sub.2; see Bracken et al. (2006) The Journal of biological chemistry 281:22575-22585] prevents degradation and stabilizes this protein. Once stabilized, HIF1a translocates to the nucleus and activates the transcription of numerous genes [Wenger and Gassmann (1997) Biol Chem 378:609-616], such as those promoting cell survival and proliferation, and glucose (e.g. glucose transporter 1) and lipid metabolism (e.g. SREBP1) [Liu et al. (2019); Wang et al. (1995); Stroka et al. (2001); Bensaad et al. (2014) Cell Rep 9:349-365; Lee et al. (2015) Stem Cells 33:2182-2195].
[0207] A common way to induce HIF1α expression, other than low O.sub.2, is through the use of the low oxygen mimetic drugs Roxadustat (Roxa), dimethyloxalylglycine, desferrioxamine and cobalt (II) chloride (CoCl.sub.2) [Jain et al. (2016) Science 352:54-61; Abdel-Rahman et al. (2019) Biomed Pharmacother 109:1688-1697; Ogle et al. (2012) Neurobiol Dis 45:733-742]. These compounds create a pseudohypoxic environment and initiate a HIF1α-mediated hypoxic response, albeit under normoxic conditions, both in vivo and in vitro [Hill et al. (2008) Journal of the American Society of Nephrology 19:39-46; Maybin et al. (2018) Nat Commun 9:295; Hoppe et al. (2016) Proceedings of the National Academy of Sciences of the United States of America 113:E2516-2525; Nguyen et al. (2013) Journal of Cell Science 126:1454-1463; Wang and Semenza (1993) Blood 82:3610-3615; Tian et al. (2011) The Journal of biological chemistry 286:13041-13051; Taheem et al. (2018) Stem Cells 36:1380-1392]. These drugs have been used to facilitate wound healing [Tang et al. (2018) Cellular physiology and biochemistry: international journal of experimental cellular physiology, biochemistry, and pharmacology 46:2460-2470], treat retinopathy of prematurity [Hoppe et al. (2016) Proceedings of the National Academy of Sciences of the United States of America 113:E2516-2525], protect corneal endothelial cells [Bhadange et al. (2018) Cornea 37:501-507], and regenerate tissues [Yu et al. (2018) Biomaterials 165:48-55; Heber-Katz (2017) Trends Mol Med 23:1024-1036].
[0208] This Example evaluated whether HIF1α is present in the human MG and determined whether exposure to 1% O.sub.2 (hypoxia), or to Roxa treatment at 1% or 21% O.sub.2 (normoxia), increases HIF1α expression in IHMGECs. It is further examined whether Roxa can mimic a hypoxic condition by stimulating differentiation in IHMGECs.
Methods and Materials
Human Tissues
[0209] Human eyelid tissues were collected after ectropion surgery from patients without MGD (1 male, 2 females, age range=70 to 82 years), and processed for frozen section preparation as previously described in Liu et al. (2019) The ocular surface 17(2):310-317. All samples were de-identified according to Health Insurance Portability and Accountability Act requirements. The use of human tissues was approved by the Institutional Review Board of the Massachusetts Eye and Ear Infirmary and Schepens Eye Research Institute and adhered to the tenets of the Declaration of Helsinki.
Immunofluorescence Staining
[0210] Frozen sections of human MGs were incubated overnight at 4° C. in a moist chamber with an antibody specific for HIF1α (28b, 1:50, SC-13515, Santa Cruz Biotechnology, Dallas, Tex.). After three times of phosphate buffered saline rinses, the sections were incubated with donkey anti-mouse secondary antibody (1:200, 2492098, EMD Millipore, Temecula, Calif.) for 1 hour at room temperature. The primary antibody was replaced with mouse IgG (Cell Signaling Technology, Danvers, Mass.) for negative controls. All slides were mounted with ProLong Gold antifade containing 4′,6-diamidino-2-phenylindole (DAPI) blue nuclear stain (Invitrogen, Thermo Fisher Scientific, USA).
Cell Culture
[0211] Authenticated IHMGECs were grown and passaged in keratinocyte serum-free medium (KSFM) with 5 ng/mL epidermal growth factor (EGF) and 50 μg/mL bovine pituitary extract (BPE) (Thermo Fisher Scientific, Waltham, Mass.) under 21% O.sub.2 (normoxia) [Liu et al. (2010) Investigative ophthalmology & visual science 51:3993-4005; McDermott et al. (2018) Current eye research 43:1097-1101; Liu et al. (2013) Investigative ophthalmology & visual science 54:2541-2550]. Upon reaching 60% to 70% confluence, culture media were replaced by a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 (DMEM/F12, Corning Life Sciences, Tewksbury, Mass.), supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, Ga.) [Sullivan et al. (2014) Investigative ophthalmology & visual science 55:3866-3877; Liu et al. (2014) JAMA ophthalmology 132:226-228]. Cells were cultured for 4-10 days under 21% O.sub.2 (normoxic) or 1% O.sub.2 (hypoxic) conditions. Culture plates of cells in the hypoxic condition were incubated inside hypoxia chambers filled with a premade 1% O.sub.2, 5% CO.sub.2 and 94% N.sub.2 gas mixture (Linde Gas North America, Hammond, Ind.), according to published protocols in Liu et al. (2019) The ocular surface 17(2):310-317 and Wright and Shay (2006) Nat Protoc 1:2088-2090. Following cell medium changes, the chambers were re-flushed with the gas mixture before returning to the incubator. Cells were cultured in the presence or absence of 50 μM Roxa (Selleck Chemical, Houston, Tex.) under normoxic or hypoxic conditions for 4 to 10 days. Cell culture experiments (n=3 wells/treatment) were repeated at least 3 times.
Western Blot
[0212] After culture in DMEM/F12+10% FBS under normoxic conditions for 7 days, cells were exposed to 21% O.sub.2, 1% O.sub.2 and 10 μM Roxa, alone or in combination, for an additional 6 hours. The cells were lysed in 2× Laemmli sample buffer (Bio-Rad Laboratories, Hercules, Calif.) and sonicated to extract nuclear protein [Andersen et al. (2002) Current biology: CB 12:1-11]. Human hepatocellular carcinoma cell lines (HepG2 cells) (America Type Culture Collection, Manassas, Va.), which express high concentration of HIF1α under CoCl.sub.2 stimulation for 4 hours [Yoo et al. (2014) Journal of cellular biochemistry 115:1147-1158], were used as a positive control for HIF1α antibody. The CoCl.sub.2 was purchased from Sigma-Aldrich Corp (St. Louis, Mo.). Cell lysates were then evaluated for HIF1α (1:1000, BD Biosciences, San Jose, Calif.) or β-actin (1:10,000, Cell Signaling Technology) expression. Secondary antibodies were horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse IgG (both 1:5000, Sigma-Aldrich). Densitometry was performed using ImageJ (at the World Wide Web website of rsb.info.nih.gov/ij).
Lipid Analyses
[0213] After four days of incubation, cells seeded on chamber slides (Corning, Rochester, N.Y.) were stained with 0.3% Oil red 0 (ORO, Fisher Biotec, Wembley, Wash., Australia) according to the published protocol in Liu et al. (2010) Investigative ophthalmology & visual science 51:3993-4005) and Liu et al. (2014) Toxicology 320:1-5. Slides were viewed using a Nikon Eclipse E800 microscope. The size of the stained area was quantified using ImageJ [Deutsch et al. (2014) Anal Biochem 445:87-89].
[0214] Cells seeded in 10-cm polystyrene-coated dishes (Corning, Falcon) were extracted with chloroform and methanol, and the lipid extractions were separated by high-performance thin-layer chromatography (HPTLC), as previously described in Liu et al. Toxicology 2014; 320:1-5, Liu and Ding (2014) Investigative ophthalmology & visual science 55:5596-5601, and Liu et al. (2015) Cornea 34:342-346. In brief, for nonpolar lipid analysis, the plate (Silica Gel 60, Merck, Darmstadt, Germany) was developed in hexane: diethyl ether: acetic acid (80:20:1.5, vol/vol/vol) alone. For phospholipids analysis, the plate was developed sequentially in chloroform:ethanol:water (60:30:5 vol/vol/vol) and then hexane:diethyl ether:acetic acid (80:20:1.5, vol/vol/vol) [Liu and Ding (2014) Investigative ophthalmology & visual science 55:5596-5601]. Bands were visualized according to published protocols [Ponec et al. (1988) Journal of lipid research 29:949-961], and the intensities of bands were quantified using ImageJ [Liu and Ding (2014) Investigative ophthalmology & visual science 55:5596-5601].
Single Radial Enzyme Diffusion Assay (SREDA)
[0215] After being treated with 21% O.sub.2, 1% O.sub.2, 10 μM Roxa or a combination for 10 days, cell numbers were counted with a hemocytometer and the cell lysates and cell culture supernatants were collected, as previously described in Liu et al. (2019) The ocular surface 17(2):310-317. DNase II activity in fresh samples was analyzed by SREDA in agarose gels according to published protocols in Liu et al. (2019) The ocular surface 17(2):310-317 and Oka et al. (2012) Nature 485:251-255. Cell lysates and culture media were used to measure the DNase II activity inside and outside of the cells, respectively [Liu et al. (2019) The ocular surface 17(2):310-317]. After incubation for 3-24 hours at 37° C., the area of the dark circle in the gel was measured under an UV trans-illuminator at 312 nm. For comparison between conditions, values were normalized to cell number.
Statistical Analysis
[0216] The significance of the differences between groups was determined by using ANOVA and Fisher's LSD multiple comparisons test (Prism 5, GraphPad Software, Inc., La Jolla, Calif.). Values with p<0.05 were considered statistically significant.
Results
Expression of HIF1α in the Human MG
[0217] Human eyelid tissues were stained for HIF1α expression to determine whether HIF1α is present in the human MG. As shown in
Influence of Low O.SUB.2 .Levels and Roxa on HIF1α Expression in IHMGECs
[0218] To determine whether HIF1α is also expressed in vitro, and whether different treatments impact the expression of this protein, IHMGECs were exposed to Roxa (50 μM) in both normoxic (21% O.sub.2) and hypoxic (1% O.sub.2) environments for a 6-hour period.
[0219] As shown in
Impact of Low O.SUB.2 .and Roxa on Lipid Accumulation and Composition in IHMGECs
[0220] To examine whether 1% O.sub.2 exposure and/or Roxa administration stimulate the accumulation of neutral lipids within IHMGECs [Liu et al. (2019) Ocul Surf 17:310-317], IHMGECs were treated with Roxa (50 about 20 μg/ml) in either normoxic (21% O.sub.2) or hypoxic (1% O.sub.2) environments conditions for 4 days.
[0221] As shown in
Impact of Low O.SUB.2 .and Roxa on the Terminal Differentiation of IHMGECs
[0222] Low O.sub.2 increases DNase II activity and promotes cell terminal differentiation of IHMGECs (Liu et al. Ocul Surf 2019; 17:310-317). To determine whether Roxa treatment can reproduce this hypoxic effect, IHMGECs were treated with Roxa in both normoxic and 1% O.sub.2 conditions for 10 days, prior to counting cell numbers and analyzing DNase II activity in cell lysates and supernatants.
[0223] As the result, administration of both Roxa and low O.sub.2 significantly increased the DNase II activity in IHMGEC lysates (
DISCUSSION
[0224] It was recently discovered that a hypoxic environment is beneficial for meibomian gland (MG) function. The mechanism(s) underlying this effect was unknown, but may be due to an increase in the levels of hypoxia-inducible factor 1α (HIF1a). In other tissues, HIF1α is the primary regulator of cellular responses to hypoxia and HIF1α expression can be induced by multiple stimuli, including hypoxia and low oxygen mimetic agents. In the current studies described in this Example, human eyelid tissues were stained for HIF1α. Immortalized human MG epithelial cells (IHMGECs) were cultured for varying time periods under normoxic (21% O.sub.2) or hypoxic (1% O.sub.2) conditions, in the presence or absence of the low oxygen mimetic agent Roxadustat (Roxa). IHMGECs were then processed for the analysis of cell number, HIF1α expression, lipid-containing vesicles, neutral and polar lipid content, DNase II activity and intracellular pH. The results show that HIF1α protein is present in human MG acinar epithelial cells in vivo. The findings also demonstrate that exposure to 1% O.sub.2 or to Roxa increases the expression of HIF1α, the number of lipid-containing vesicles, the content of neutral lipids, and the activity of DNase II, and decreases the pH in IHMGECs in vitro. The data support that the beneficial effect of hypoxia on the MG is mediated through an increased expression of HIF1α.
[0225] The exemplary experimentation in this Example illustrate that HIF1α mediates the beneficial effects of hypoxia on HMGEC function. While HIF1α is expressed by human MG acinar epithelial cells in vivo, exposure to 1% O.sub.2 or to Roxa increased the levels of HIF1α, the number of lipid-containing vesicles, the content of neutral lipids, and the activity of DNase II in IHMGECs in vitro. These data support an important role for HIFα in the regulation of MG function.
[0226] HIF1α proteins are primarily located within nuclei of HMGECs, and especially in those cells situated near the central areas of MG acini. This nuclear location is consistent with findings in other sebaceous glands [Rosenberger et al. (2007) The Journal of Investigative Dermatology 127:2445-2452; Revenco et al. (2017) Stem Cells 35:1355-1364] but uncommon in non-sebaceous tissues [Talks et al. (2000) The American Journal of Pathology 157:411-421]. The accumulation of HIF1α in the center of acinar complexes may be because this area is likely the most hypoxic in the MG. The reason is that the O.sub.2 source for the MG is the vasculature located beyond the MG basement membrane [Knop et al. (2011) Investigative Ophthalmology & Visual Science 52:1938-1978]. By Krogh's law, this would establish an O.sub.2 gradient, with the highest O.sub.2 concentration within the blood vessels and the lowest in the central acinar region. Such hypoxic conditions would be expected to prevent HIF1α degradation and promote its translocation to the nucleus [Bracken et al. (2006) The Journal of Biological Chemistry 281:22575-22585]. The nuclear location of HIF1α in HMGECs indicates that this protein is active and able to stimulate gene transcription [Bracken et al. (2006); Wenger and Gassmann (1997) Biol Chem 378:609-616].
[0227] In this Example, exposure to hypoxic conditions, as well as to the low oxygen mimetic Roxa, were found to increase the levels of HIF1α in IHMGECs. The mechanisms underlying these low O.sub.2 and drug responses may be the same. Under normoxic conditions, HIF-prolyl hydroxylase (PH) domain enzymes destabilize HIF proteins by hydroxylating two prolyl residues in the alpha (a) subunit [Yang et al. (2014) Hypoxia (Auckl) 2:127-142]. This HIF1α hydroxylation enables its association with the von Hippel-Lindau tumor suppressor pVHL E3 ligase complex, and leads to the degradation of HIF1α via the ubiquitin-proteasome pathway [Yang et al. (2014)]. However, both low O.sub.2 levels and Roxa inhibit HIF-PH, thereby enhancing the steady-state levels of HIF1α [Bracken et al. (2006)]. Roxa treatment was more effective than hypoxia alone in stimulating the accumulation of HIF1α. This result may be due to a stronger HIF-PH inhibiting effect, or to a delay in achieving the desired O.sub.2 concentration in the culture media during the 6-hour time course of our experiments. O.sub.2 has a relatively low solubility and diffusion rate in aqueous solutions, and its concentration in culture media may not reach equilibrium with the hypoxic chamber air for up to three hours [Allen et al. (2001)Am J Physiol Lung Cell Mol Physiol 281:L1021-1027]. Thus, the IHMGECs may have been exposed to a final 1% O.sub.2 environment, as compared to Roxa, for less time. The combination of both low O.sub.2 and Roxa induced the highest levels of HIF1α.
[0228] The 1% O.sub.2 environment and Roxa administration led to a significant accumulation of neutral lipid-containing droplets in IHMGECs. This effect may have been mediated through the increased content of HIF1α, given that this protein is involved in modulating lipid signaling and synthesis, lipid uptake and transport, FFA and sterol metabolism, and lipid droplet biogenesis [Bensaad et al. (2014) Cell Rep 9:349-365; Mylonis et al. (2012) Journal of Cell Science 125:3485-3493; Liu et al. (2014) Toxicology Letters 226:117-123]. These droplets are the major lipid storage organelle in most eukaryotic cells, and contain variable amounts of neutral lipids, including triglycerides and free and esterified sterols, enclosed by a phospholipid monolayer [Olzmann and Carvalho (2019) Nature Reviews Molecular Cell Biology 20:137-155]. The enhanced lipid droplet content in IHMGECs in response to low O.sub.2 and/or Roxa exposure is consistent with the effect of a hypoxic environment in other cell types [Mylonis et al. (2012); Gordon et al. (1977) The American Journal of Pathology 88:663-678; Taniguchi et al. (2013) Nature Medicine 19:1325-1330].
[0229] A change in the amount of neutral lipids in response to low O.sub.2 in IHMGECs was not found in a previous study [Liu et al. (2019) The Ocular Surface 17(2):310-317]. These inconsistent findings may be due to several factors, including differences in experimental design and staining procedures. In the previous study, IHMGECs were exposed to 3% O.sub.2, different from the 1% O.sub.2 in this Example. One concern for an exposure to a low O.sub.2 environment is that systemic and prolonged hypoxia may lead to a range of severe pathophysiological reactions and compensations in vivo and would hinder its translation into clinical treatment in the future. However, the current Example utilized an even lower O.sub.2 concentration (1% O.sub.2) because mouse MGs were reported to stain positively for pimonidazole [Liu et al. (2019) The Ocular Surface 17(2):310-317], indicating that the oxygen level in MGs is less than 1.3% [Gross et al. (1995) International Journal of Cancer Journal international du cancer 61:567-573]. In addition, in the previous study, LipidTOX reagent was used to identify neutral lipids in IHMGECs, detecting most staining occurred within lysosomes [Sullivan et al. (2014) Investigative ophthalmology & visual science 55:3866-3877; Liu et al. (2014) Toxicology 320:1-5]. These lysosomes may well be lamellar bodies, which contain cholesterol, neutral lipids, phospholipids and various enzymes [Liu et al. (2014) Toxicology 320:1-5; Schmitz and Muller (1991) Journal of Lipid Research 32:1539-1570; Feingold (2007) Journal of Lipid Research 48:2531-2546]. In this Example, ORO and bright-field microscopy permitted the visualization of neutral lipids in lipid droplets [Mehlem et al. (2013) Nat Protoc 8:1149-1154; Fam et al. (2018) Materials (Basel) 11:1768]. Both lamellar bodies and lipid droplets are organelles specialized for lipid storage, and there is evidence that lamellar bodies may be derived from, or transform into, lipid droplets [Menon et al. (2018) Journal of Dermatological Science 92:10-17]. Considering that both lipid droplets and lamellar bodies exist in the human MG [Gorgas and Volkl (1984) The Histochemical Journal 16:1079-1098; Jester et al. (1981) Investigative Ophthalmology & Visual Science 20:537-547], their specific roles in lipid dynamics await clarification. Lastly, different methods were used to quantitate the extent of neutral lipid expression in the previous study versus this Example. For the former the intensity of LipidTOX staining was measured, whereas in this Example the area of ORO staining was calculated. Intensity and area measurements do not necessarily lead to equivalent results.
[0230] The administration of 1% O.sub.2 and/or Roxa promoted the terminal differentiation of IHMGECs, as indicated by the significant increase in DNase II activity in cell lysates and culture supernatants. DNase II is a lysosomal enzyme [Ohkouchi et al. (2013) PloS one 8:e59148], which is typically activated by acidic conditions (pH 4.5-5.5) and then translocates from lysosomes to the nucleus [Evans and Aguilera (2003) Gene 322:1-15]. Within that location DNase II triggers programmed cell death and ultimately holocrine secretion [Fischer et al. (2017) The Journal of Investigative Dermatology 137:587-594; Barry and Eastman (1993) Arch Biochem Biophys 300:440-450]. The heightened levels of HIF1α may have mediated this process. Activation of HIF1α stimulates glycolysis and lactic acid production, which decreases the intracellular pH [Brahimi-Horn and Pouyssegur (2007) Essays Biochem 43:165-178]. HIF1α also reduces the intracellular pH through modulation of carbonic anhydrase (CA) IX [Logsdon et al. (2016) Molecular Cancer Therapeutics 15:2722-2732]. CA IX expression, recently identified in HMGECs in vivo, is increased by HIF1α [Kaluz et al. (2009) Biochimica et biophysica acta 1795:162-172]. The CA enzymes, in turn, could increase intracellular H+ mobility and promote cellular acidification [Boron (2004) Adv Physiol Educ 28:160-179]. These HIF1α effects on pH would destabilize lysosomal membranes, cause the release of lysosomal enzymes, and lead to the activation of DNase II [Tapper and Sundler (1990) The Biochemical Journal 272:407-414; Sundler (1997) Acta Physiol Scand 161:553-556; Torriglia et al. (1998) Molecular and Cellular Biology 18:3612-3619; Eastman (1994) Cell Death and Differentiation 1:7-9]. Hypoxic environments and mimetics also induce such lysosomal disruption in other cell types [Eastman (1994); Smith et al. (1974) The Journal of pharmacology and experimental therapeutics 191:564-574; Abraham et al. (1967) Nature 215:194-196; Loegering et al. (1975) Experimental and Molecular Pathology 22:242-251].
[0231] Overall, an important role for HIF1α in the regulation of MG function is illustrated in this Example. Local administration of hypoxia mimetics may be beneficial for the treatment of MGD.
Example 2
Roxadustat (Roxa) Treatment for Meibomian Gland Dysfunction (MGD) In Vivo
[0232] After the discovery that the oxygen-sensitive protein HIF1α plays an essential role in the regulation of human meibomian gland epithelial cells (HMGECs), alternative treatments for MGD and other related diseases or disorders have been tested, aiming to avoid any pathophysiological reactions and compensations caused by the systemic and prolonged hypoxia treatment. In addition to a hypoxic environment, HIF1α expression also can be induced by low oxygen mimetic agents and growth factors. In order to mimic a local hypoxic environment around the MG, but also to minimize the compensatory effects of systemic low oxygen exposure, pharmacological activation of the HIF1α pathway under normoxic (21% O.sub.2) conditions were tested. The hypoxia mimetic Roxadustat (Roxa; also called FG-4592) duplicated the differentiation-promoting effect of low oxygen by promoting HIF1α in immortalized HMGECs under normoxic condition. Roxa has previously been tested for safety in animal studies and has passed a phase III clinical trial for the treatment of anemia in patients with chronic kidney disease. Thus, Roxa may be a safety drug to use in the current studies.
[0233] Animal studies were used to determine the capacity of Roxa to ameliorate MGD in an model of apolipoprotein E-deficient (ApoE −/−) mice, which have recently been described as a model for MGD. These knockout mice have disrupted lipid regulation (i.e. cholesterol and heart disease) and are widely used as a model of obesity with accelerated atherosclerosis.
[0234] Specifically, two groups of ApoE KO (ApoE −/−) mice, comprising eight (8) male mice for each group, were treated with either vehicle control or Roxa. In addition, four (4) age-matched wild-type C57BL/6J male mice were untreated, as normal meibomian gland controls.
[0235] At the beginning of the study, the 16 ApoE−/− mice and 4 wild-type (WT) C57BL/6J mice, 12 weeks of age, were measured for their weight, tear volume, corneal fluorescein staining and tear breakup time (TBUT). The ApoE −/− mice were randomly divide into 2 groups (n=8 mice/group) and treated with vehicle control (2% DMSO in sterile saline, subcutaneous injection) or Roxa (10 mg/kg, subcutaneous injection). The injections were administrated three times per week (on Monday, Wednesday, and Friday mornings) for 12 weeks. The ApoE −/− mice were weighted weekly (on Mondays) to determine Roxa injection volume for each individual. At the end of the study, the weight, tear volume, corneal fluorescein staining and tear breakup time (TBUT) were measured again. The eyelids of each mouse were then dissected to assess meibomian gland size and morphology.
[0236] As shown in
[0237] As shown in
[0238] Besides the changes in the eye, Roxa treatment also significantly reduced the serum cholesterol level (
[0239] The mice eyelids were collected and dissected after sacrificing. The dissected samples were stained with different antibodies and dyes for lipids. For example, the dissected mice lid samples were stained with Oil Red O and LipidTox™ (Invitrogen) for evaluation of lipid accumulation. The sections were stained for pimonidazole, DNase II, HIF1α, PPARγ, Desmoglein and Desmocollin for the evaluation of cell differentiation and maturation. Roxa treatment promoted expression of the several key markers for MG cell differentiation in mice. These markers include intracellular lipid accumulation, as well as expression of peroxisome proliferator-activated receptor gamma (PPAR-γ), Desmoglein and Desmocollin in MG cells. These results further indicate that Roxa treatment may be used to treat obesity or other diseases or disorders characterized with increased lipid accumulation.
[0240] The results from the studies suggest that Roxa can cure or at least alleviate MGD and DED. A clinical study will be planned for further testing Roxa drug development. Roxa may be developed into a topically applicable eye drop or eye ointment for a phase I clinical trial.
OTHER EMBODIMENTS
[0241] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
[0242] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications and sequence (nucleic acid or protein) citations cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
[0243] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.