Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
  • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
  • A61K31/706—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom
  • A61K31/7064—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines
  • A61K31/7076—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing six-membered rings with nitrogen as a ring hetero atom containing condensed or non-condensed pyrimidines containing purines, e.g. adenosine, adenylic acid
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/16—Purine radicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/16—Purine radicals
  • C07H19/167—Purine radicals with ribosyl as the saccharide radical

Definitions

• Diabetes is a group of metabolic diseases in which there are high blood sugar levels over a prolonged period.
• Type I diabetes results from the pancreas's failure to produce enough insulin.
• Type II diabetes begins with insulin resistance, a condition in which cells fail to respond to insulin properly, and may cause a lack of insulin as the disease progresses.
• Insulin, or insulin analogs are generally used for treating type I diabetes.
• Metformin is generally recommended as a first line treatment for type II diabetes.
• Compound C Compound D Compound E but not the individual compounds, can significantly attenuate G6pc expression, thereby representing a novel way to reduce hepatic glucose output ⁇ See, WO2015/137983);
• a combination of compounds C, D and E can reduce hepatic glucose output and improve glucose tolerance in an insulin-resistant, diabetic mouse model, so that a combination of compounds C, D and E can be useful in treatment of obesity,
• Compounds C and D may be useful in treating sarcopenia caused by progressive loss of MT function in the kidney or skeletal muscle (See, WO2015/137983);
• Compound C can enhance gluconeogenesis in brain cells which may be beneficial for the survival of brain cells in AD (See, US20160082033);
• Compounds C and D can inhibit Tau hyperphosphorylation in AD brains (See, US20160082033).
• compound C or compound D might be useful alone.
• these compounds are shown not to be useful individually, but to be effective in combination.
• the present disclosure provides new selenium-containing compounds, sharing some structural relationship with compounds of formulas I and II above (and specifically with compounds C and D), that surprisingly show potent activity alone in a variety of contexts.
• the present disclosure demonstrates, among other things, that provided compounds exhibit bioactivity comparable to or better than the CDE combination in reducing hepatic glucose output and/or improving glucose tolerance in insulin-resistant, diabetic subjects.
• the present disclosure also teaches that provided compounds likely have bioactivity comparable to or better than Compounds C and/or D in enhancing gluconeogenesis (e.g., in brain cells), and/or in inhibiting Tau hyperphosphorylation (e.g., in AD brains).
• compositions that contain and/or deliver such compounds (and/or one or more degradants and/or active metabolites thereof), as well as various methods (e.g., of manufacture, characterization, and/or use) and/or materials (e.g., intermediates, degradants, metabolites [in particular, active metabolites], etc) related to such provided compounds.
• provided technologies relate to and/or are particularly useful in modulating glucose metabolism; enhancing AS 160 phosphorylation for translocation of glucose transporter proteins (GLUTs) from cytosolic vesicles to plasma membrane for glucose uptake; and/or enhancing glucose uptake in both liver and skeletal muscles.
• GLUTs glucose transporter proteins
• provided technologies relate to and/or are particularly useful in treatment of hyperinsulinemia, obesity, diabetes, hyperglycemia, polycystic ovary syndrome (PCOS), Alzheimer's disease (AD), and/or sarcopenia.
• provided technologies relate to and/or are particularly useful in treatment of type II diabetes related disorders, such as diabetic retinopathy, nephropathy, neuropathy, and vascular disorders.
• the present disclosure provides a compound of formula
• each of R 2 and R 3 is independently H or -C(0)-R, wherein each R is independently Ci.
• R 2 and R 3 cannot be both H; or R 2 together with R 3 form -(CH2)n-C(0)-(CH 2 )m-, wherein each of n and m is independently 0-3, and n+m ⁇ 3;
• R 5 is -C 1-6 alkyl or -C 1-6 alkyl-CH( H 2 )COOH;
• R 8 is H or halogen
• X is H or halogen
• each of the carbocyclic, heterocyclic, -(CH 2 ) n -, and -(CH 2 ) m - moieties may optionally be substituted 1-3 times by -OH, halogen, H 2 , CN, or
• each C 1-6 alkyl moiety may optionally be substituted 1-3 times by -OH, halogen, NH 2 , or CN.
• the present disclosure provides a compound of formula
• X is H or halogen
• each R 5 ' is independently H or halogen
• each R is independently each of which, independently, may optionally be substituted 1-3 times by halogen.
• the present disclosure provides a compound of formula
• R 8 is H or halogen
• X is H or halogen
• each R' is independently H or halogen.
• the present disclosure provides compositions which comprise or deliver a compound of any one of formulas (l)-(3). In some embodiments, the present disclosure provides compositions comprising a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof. In some embodiments, the present disclosure provides compositions which deliver an active moiety of a compound of any one of formulas (l)-(3).
• the present disclosure provides methods of treating a disease, disorder, or condition by administering a compound or composition as described herein.
• provided methods enhance AS 160 phosphorylation for translocation of glucose transporter proteins (GLUTs) from cytosolic vesicles to plasma membrane for glucose uptake.
• provided methods enhance glucose uptake in both liver and skeletal muscles.
• provided methods attenuate hyperinsulinemia without impairing kidney function and/or resulting in liver damage.
• the present disclosure provides methods for treating an insulin-related disorder, comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for treating insulin resistance disorder comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for treating glucose metabolism disorders, comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• glucose metabolism disorders involve a blood glucose level which is not within the normal range.
• glucose metabolism disorders relate to defective glucose uptake and/or transport.
• glucose metabolism disorders are Diabetes Mellitus, glyceraldehyde-3 -phosphate dehydrogenase deficiency, glycosuria, hyperglycemia, hyperinsulinism, or hypoglycemia.
• the present disclosure provides methods for treating disorders of glucose transport, comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• disorders of glucose transport are glucose-galactose malabsorption, Fanconi-Bickel syndrome, or De Vivo disease (GLUT1 deficiency syndrome (GLUT IDS)).
• the present disclosure provides methods for treating obesity comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for treating diabetes comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for treating hyperglycemia comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for treating polycystic ovary syndrome (PCOS) comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• PCOS polycystic ovary syndrome
• the present disclosure provides methods for treating
• AD Alzheimer's disease comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for treating sarcopenia comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for inhibiting glucose production, comprising administering a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for increasing glucose tolerance, comprising administering a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for activating and/or restoring insulin receptor function and its downstream signaling in a subject in insulin- resistant state, comprising administering a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for treating mitochondria-associated diseases (e.g., caused by dysfunctional mitochondria), comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• mitochondria-associated diseases can be degenerative diseases (e.g., cancer, cardiovascular disease and cardiac failure, type 2 diabetes, Alzheimer's and Parkinson's diseases, fatty liver disease, cataracts, osteoporosis, muscle wasting such as sarcopenia, sleep disorders and inflammatory diseases such as psoriasis, arthritis and colitis).
• the present disclosure provides methods for enhancing mitochondrial function, comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for enhancing gluconeogenesis in the brain, comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• provided methods increase glucose uptake in the brain.
• provided methods are for maintaining or restoring brain functions including memory and learning.
• the present disclosure provides methods for preparing a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for characterizing a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for preparing a composition as described herein.
• the present disclosure provides methods for characterizing a composition as described herein.
• Fig 1 Effects of insulin and pure compounds (listed in Table 1) on glucose production in HepG2 cells. Cells were treated with 0.24% DMSO (the maximal volume of tested compound solvent), insulin or listed compounds in serum-free glucose production media for 48 hr. Data were normalized by cell number as described above, and presented as mean ⁇ SEM of at least 3 samples per group.
• HepG2 and H4IIE cells were treated with 0.24% DMSO (the maximal volume of Compound #43 solvent), insulin, Compound #43 and metformin in serum-free glucose production media for 48 hr, while rat liver cells were treated for 24 hr.
• Glucose levels in culture media were normalized by cell number in each sample. Data are presented as mean ⁇ SEM of between 3 and 8 samples per group. [0034] Fig 3. Differential effects of Compound #C, #50 and #43 on blood glucose levels and serum HbAlc levels in Lepr db/db mice after chronic treatment. Lepr mice were intraperitoneally injected with saline (containing 0.2% the compound solvent DMSO),
• Fig. 4 Effects of Compound #43 and its sulfur analog #68 on blood glucose levels and HbAlc levels in Lepr db/db mice after chronic treatment.
• Lepr db/db mice were intraperitoneally injected with saline(containing 0.2% the compound solvent DMSO),
• blood glucose and HbAlc levels of overnight-fasting mice were determined.
• HbAlc levels in Compound #43 and #68-treated mice were divided by the average HbAlc levels in saline-treated mice to obtain the relative HbAlc levels. Data are presented as mean ⁇ SEM of the indicated number of animals.
• Fig. 5 Differential effects of Compound #43, #69 and #70 on fasting blood glucose and HbAlc levels in Lepr db/db mice after chronic treatment.
• Male 41 -day-old Lepr db/db mice were intraperitoneally injected with saline (containing 0.2% the compound solvent DMSO), Compound #43 (0.136 mg), #69 (0.145 mg) and #70 (0.153 mg) at the dose of 25 ⁇ g selenium of each compound per kilogram body weight daily for 43 days (for blood glucose assay) and 90 days (for HbAlc assay), fasted overnight, and then subjected to blood glucose analysis (using a glucometer) or blood HbAlc assay.
• FIG. 6 Acute treatment of Compound #43 resulted in a decrease in blood glucose level in Lepr db/db male mice. 8-10-week-old Lepr db/db male mice were fasted overnight, and then injected intraperitoneally with saline (containing 0.2% DMSO, the maximal injected volume of Compound #43 stock solvent), 0.0054, 0.054, 0.54 and 5.4 mg Compound #43 /kg body weight.
• saline containing 0.2% DMSO, the maximal injected volume of Compound #43 stock solvent
• Lepr db/db male mice under ad-libitum feeding conditions Blood glucose levels of 6-week-old Lepr db/db maIe mice with free access to food and water were determined before and at 24 hr after an intraperitoneal injection of saline (containing 0.2% the compound solvent DMSO) or Compound #43 at a dose of 5.4 mg/kg body weight.
• the relative blood glucose levels before i. p. injection was normalized by the average glucose level of all five mice within the group, and referred to as 100%). After 24 hr injection, the relative blood glucose level in each mouse was normalized by its glucose level before injection. Different letters represents a statistically significant difference (P ⁇ 0.05) between groups.
• Lepr db/db mice A-B. Male 38-day-old Lepr db/db mice were intraperitoneally injected with (A) saline (containing 0.2%> the compound solvent DMSO), Compound #43, Compound #C and Compound #50 daily for 43 days, or with (B) Compound #68 or Compound #43 for 60 days.
• C Male 41 -day-old Lepr db/db mice were intraperitoneally injected with saline (containing 0.2%> the compound solvent DMSO), Compound #43, Compound #69 and Compound #70 daily for 43 days. The daily injected dose of all listed compounds was 25 ⁇ g selenium or sulfur per tested compound per kilogram body weight.
• mice were fasted overnight, injected with glucose (2 g/kg body weight) and blood glucose levels immediately before glucose injection (referred to as zero time point) and at 0.25 hours, 0.5 hours, 1 hour and 2 hours post- glucose injection were measured using a glucometer with the maximal reading of 600 mg/dL. A glucose level in excess of this limit was recorded as 600 mg/dL. Data are presented as Mean ⁇ SEM of indicated number of animals. * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 when compared to (A, C) saline-treated or (B) Compound #68-treated mice at the same time point.
• FIG. 9 Attenuated G6pc mRNA in the livers of Lepr db/db mice after chronic treatment with Compound #43.
• Lepr db/db mice at postnatal day 38 were intraperitoneally injected with saline (containing 0.2% the compound solvent DMSO), Compound #50 and #43 at the dose of 25 ⁇ g selenium of each compound per kilogram body weight daily for 52 days.
• QRT-PCR was performed on liver RNA isolated from these compound-treated mice.
• G6pc mRNA level in each sample was normalized by Actb mRNA level and data are presented as mean ⁇ SEM of five mice per group. P value is relative to the saline group.
• AML-12 and human HepG2 cells and the cooperative action of both Compound #43 and insulin in the inhibition of G6pc expression in AML-12 cells.
• AML-12 cells were treated without (Control), or with compound CDE combination, Compound #C, Compound #D, Compound #50 and Compound #43 at a dose of 300 parts per billion (ppb) of selenium (equivalent to 3.8 uM of each compound) in serum-free, Insulin-Transferrin-Sodium selenite supplement (ITS) and Dexamethasone (Dex)-free media for 24 hr.
• ppb parts per billion
• AML-12 cells were pretreated with Compound #43 in FBS-containing but ITS/Dex-free media for 24 hours followed by retreatment of Compound #43 in the presence or absence of 10 nM insulin in serum/ITS/Dex-free media for 6 hours.
• G6pc mRNA level in each sample was normalized by Actb mRNA level and data are presented as mean ⁇ SEM of indicated number of sample in each group. P value in panel A-B was compared to the Control group. Different letters in panel C represents a statistical significance (P ⁇ 0.05) between those two groups.
• Fig. 11 Inhibition of G6pc expression by Compound #43, and the cooperative action of both Compound #43 and insulin in the inhibition of G6pc expression in AML-12 cells stimulated with diabetic stimuli.
• AML-12 cells were pretreated with Compound #43 in 10% FBS-containing but ITS/Dex-free media for 24 hours followed by retreatment of Compound #43 in the presence or absence of insulin along with 8-CPT/Dex (diabetic stimuli) in serum-free and ITS-free media for 6 hours.
• G6pc mRNA level in each sample was normalized by Actb mRNA level and data are presented as mean ⁇ SEM of indicated number of sample in each group.
• Fig. 12 Chronic treatment of Compound #43 enhanced the phosphorylation of Pdkl/Akt Foxol signaling in the livers of insulin-resistant Lepr db/db mice by Western blot analysis.
• Lepr db/db mice at postnatal day 38 were intraperitoneally injected with saline
• Protein levels in each sample were normalized by Gapdh level and data are presented as mean ⁇ SEM of five mice per group in (B). P value was compared to the saline group.
• FIG. 13 Transient activation of PDK1 and AKT and subsequent inactivation of FOXOl in human liver HepG2 cells by Compound #43. HepG2 cells were serum-starved overnight, incubated without or with Compound #43 (600 ppb) for the indicated time, and then subjected to Western blot analysis.
• A Representative Western blots.
• B-F Quantitative data of protein expressions of (B) pPDKl, (C) pAKT, (D) total AKT, (E) pFOX01T24, and (F) total FOXOl in Western blots are presented as mean ⁇ SEM of 3 samples. * P less than 0.05 when compared to the protein level at 0 min (right before compound treatment) or its control group at each time point.
• FIG. 14 Transient activation of Pdkl and Akt and enhanced Foxol phosphorylation by Compound #43 in mouse liver AML-12 cells cultured under simulated diabetic condition.
• AML-12 cells were cultured in 10% FBS but ITS/Dex-free DMEM/F12 media for 24 hr, and then serum-starved in plain DMEM/F12 media overnight.
• These serum- starved AML12 cells were treated with diabetic stimuli, 8-CPT (0.1 mM) and Dex (0.5 ⁇ ), in combination without (Control) or with 10 nM insulin or Compound #43 (300 ppb) in plain DMEM/F12 media for the indicated time points, and then subjected to Western blot analysis.
• Fig. 15 Effects of Compound #43 and Compound #50 on the expression of the Glut4 gene in the livers of Lepr db/db mice.
• Lepr db/db mice at postnatal day 38 were intraperitoneally injected with saline (containing 0.2% the compound solvent DMSO),
• QRT-PCR analyses were performed on liver RNA samples isolated from these compound-treated Lepr db/db mice. Glut4 mRNA level in each sample was normalized by Actb mRNA level, and data are presented as mean ⁇ SEM of four to five mice per group. P values were calculated for treatment versus the control saline group.
• Fig. 16 Enhanced Glut4 mRNA expression in mouse liver AML-12 cells by
• A QRT-PCR of basal Glut4 expression in AML-12 cells.
• AML-12 cells were amplified, seeded on 24-well plates, and cultured in 10% FBS ITS/Dex-free DMEM/F12 media overnight. These cells were then incubated with vehicle (0.024% DMSO) or Compound #43 (300 ppb) in serum-free DMEM/F12 media for 24 hours (hr).
• B QRT-PCR of Glut4 expression in AML-12 cells cultured under simulated diabetic conditions.
• Amplified AML-12 cells were cultured on 24-well plates in 10% FBS but ITS/Dex-free DMEM/F12 media for 24 hr, and then serum-starved in plain DMEM/F12 media overnight. Serum-starved AML-12 cells were then incubated with vehicle (0.024% DMSO) or with Compound #43 (300 ppb) in the presence of diabetic stimuli, 0.1 mM 8-CPT and 0.5 ⁇ Dex, in serum-free plain DMEM/F12 media for 6 and 24 hr. Glut4 mRNA level in each sample was normalized by Actb mRNA level, and data are presented as mean ⁇ SEM of 3 samples.
• Fig. 17 Enhanced glucose uptake in AML-12 cells after the treatment of insulin and Compound #43 for 1.5 hours. Data are presented as mean ⁇ SEM of the indicated number of samples per group. * P value was less than 0.05, compared to the basal group.
• Fig. 18 Enhanced phosphorylation of insulin downstream signaling molecules-Pdkl, Akt and Foxol- in skeletal muscles of insulin-resistant Lepr db/db mice in response to treatment with Compound #43.
• Lepr mice at postnatal day 38 were intraperitoneally injected with saline (containing 0.2% the compound solvent DMSO) or Compound #43 at the dose of 0.136 mg of Compound #43 per kilogram body weight daily for 52 days.
• Western blots were performed on skeletal muscle protein extracts (100 ⁇ g protein per lane) isolated from saline or Compound #43 -treated Lepr db/db mice.
• A Images of Western blots.
• B Quantitative protein levels (normalized by ⁇ -tubulin protein levels in each sample). Data are presented as mean ⁇ SEM of five mice. P value was derived by comparison to the control (saline) group.
• Fig 19 Effects of insulin and Compound #43 on the glucose uptake in the differentiated mouse C2C12 (skeletal muscle) cells. Equal number of C2C12 cells were seeded on 96-well plates (5000 cells/well), cultured in 10% FBS DMEM media for 5 days, differentiated in 0.5%) horse serum -containing DMEM media for 7 days. The completely differentiated C2C12 cells were pretreated without or with Compound #43 (300 or 600 ppb) in serum/glucose-free DMEM media overnight, and then incubated without (basal) or with insulin, Compound #43, or both in glucose-free DMEM media at 37°C for 1.5 hr.
• Compound #43 300 or 600 ppb
• Fig. 20 Restoration of insulin receptor function (indicated by elevated phosphorylation of InsrP at Tyrosine 1146) in the skeletal muscle of insulin-resistant Lepr db/db mice after chronic treatment with Compound #43.
• Lepr db/db mice at postnatal day 38 were intraperitoneally injected with saline (containing 0.2%> the compound solvent DMSO) or Compound #43 at a dose of 0.136 mg of Compound #43 per kilogram body weight daily for 52 days.
• Western blots were performed on skeletal muscle protein extracts (100 ⁇ g protein per lane) isolated from saline or Compound #43 -treated Lepr db/db mice.
• Equal number of C2C12 cells were seeded on 12-well plates (60,000 cells/well), cultured in 10% FBS DMEM media for 5 days, differentiated in 0.5% horse serum-containing DMEM media for 7 days. Completely differentiated C2C12 cells were serum-starved overnight and then treated without or with Compound #43 (600 ppb) or insulin (200 nM) in serum-free DMEM media at 37°C for (A-B) 5 minutes or (C-D) 30 minutes, and then subjected to Western blot analysis.
• A, C Images of Western blots.
• B, D Quantitative protein levels (normalized by ⁇ -tubulin protein level in each sample). Data are presented as mean ⁇ SEM of three samples per group. *P ⁇ 0.05, ** P ⁇ 0.01, ***P ⁇ 0.001 when compared to the control group (without Compound #43 treatment).
• Fig. 22 Restoration of insulin receptor function (indicated by elevated tyrosine phosphorylation of Insrp) in the livers of insulin-resistant Lepr db/db mice after chronic treatment with Compound #43. Lepr db/db mice at postnatal day 38 were
• Yl 150/1151 Western blot analysis of ⁇ -tubulin.
• A The protein levels of phosphor-Insr ⁇ at Yl 146 (normalized by ⁇ -tubulin protein level in each sample).
• B The protein levels of phospho-Insr ⁇ at Yl 150/1151 (normalized by ⁇ -tubulin protein level in each sample). Data are presented as mean ⁇ SEM of the indicated number of mice. P values were calculated by comparison of treatment values to values in the saline group.
• Fig. 23 Activation of INSR and stimulation of AS160 phosphorylation in human liver HepG2 cells by Compound #43.
• HepG2 cells were seeded on 6-well plates (7 X 10 5 cells/well), cultured in 10% FBS EMEM media for 30 hr, and then serum-starved overnight. These serum-starved HepG2 cells were then treated with Compound #43 (600 ppb) at 37°C for 30 and 60 minutes (min), and then subjected to Western blot analysis.
• A Images of Western blots.
• B Quantitative protein levels (normalized by ACTB protein level in each sample). Data are presented as mean ⁇ SEM of three samples per group. ** P ⁇ 0.01, when compared to the control group (0 min group, before Compound #43 treatment).
• Fig. 24 Chronic treatment of Compound #43 resulted in a decrease of serum insulin and alanine aminotransferase (ALT), but not creatinine, levels in Lepr db/db mice.
• Sera from 3-month-old wild-type (non-diabetic) C57 mice were also collected.
• Fig. 25 Mode of action of Compound #43 against type I and II diabetes.
• Fig. 26 Direct activation of insulin receptor by Compound #43 and insulin in a cell-free system. Equal amounts of native insulin receptor proteins, containing both alpha and beta subunits were incubated with 0.003% DMSO (Compound #43 solvent), Compound #43 or insulin (Ins, 0.5 ⁇ ) in the presence of ATP, and then subjected to Western blot analysis to detect phosphorylated Insrp (activated Insr) at Yl 146, 1150 and 1151. Different alphabetic letter in the bar graph denotes statistically significant changes between those groups.
• Fig. 27 Compound #68 was less effective than Compound #43 in the activation of insulin receptor in the cell-free system. Equal amounts of native insulin receptor were incubated with 0.003% DMSO (Compound #43 solvent), Compound #43 or Compound #68 in the presence of ATP. The activated Insr proteins were detected by Western blot analysis of phosphorylated Insr ⁇ at Yl 146, 1150 and 1151. Different alphabetic letters in the bar graph mean statistically significant changes occurred between those groups.
• Fig. 28 Reduced blood glucose levels in STZ-induced T1D mice after acute treatment of Compound #43.
• STZ-induced T1D mice with unfasted blood glucose levels between 500-550 mg/dL were fasted overnight, and injected intraperitoneally with Compound #43 at a dose of 5.4 mg/kg body weight or with physiological saline containing 2% DMSO (Compound #43 stock solvent, referred to as Control group) for 1, 2 and 3 hours. Mice were then subjected to blood glucose measurement. P values were derived by comparing Compound #43 treatments to the control/saline group at each time point. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS
• Administration typically refers to the administration of a composition to a subject or system.
• routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
• administration may be ocular, oral, parenteral, topical, etc..
• administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e. g.
• administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
• Alzheimer's disease As used herein, the term “Alzheimer's disease”, or “AD”, refers to a progressive disease of the human central nervous system. Brain insulin signaling is important for learning and memory, and insulin resistance in the brain is a major risk factor for AD. The restoration of insulin signaling has emerged as a potential therapy for AD (White MF, Science 2003; 302: 1710-1; De Felice DG et al, Alzheimer's & Dementia 2014; 10: S26-S32). In certain embodiments, it is manifested by dementia typically in the elderly, by disorientation, loss of memory, difficulty with language, calculation, or visual-spatial skills, and by psychiatric manifestations.
• Alzheimer's dementia includes, but is not restricted to, Alzheimer's dementia with or without psychotic symptoms.
• the therapeutic methods provided herein are effective for the treatment of mild, moderate and severe Alzheimer's disease in a subject.
• Phases of Alzheimer's further include “moderately severe cognitive decline,” also referred to as “moderate or mid-stage Alzheimer's disease,” “severe cognitive decline,” also referred to as “moderately severe or mid-stage Alzheimer's disease,” and “very severe cognitive decline,” also referred to as “severe or late-stage Alzheimer's disease.”
• Moderately severe cognitive decline is characterized by major gaps in memory and deficits in cognitive function emerge.
• Late stage Alzheimer's disease or very severe cognitive decline is the final stage of the disease when individuals lose the ability to respond to their environment, the ability to speak and, ultimately, the ability to control movement.
• Biologically activity refers to an observable biological effect or result achieved by an agent or entity of interest.
• a specific binding interaction is a biological activity.
• modulation (e.g., induction, enhancement, or inhibition) of a biological pathway or event is a biological activity.
• presence or extent of a biological activity is assessed through detection of a direct or indirect product produced by a biological pathway or event of interest.
• Combination therapy refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents).
• the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens.
• "administration" of combination therapy may involve administration of one or more agents or modalities to a subject receiving the other agents or modalities in the combination.
• combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).
• Comparable refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
• comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
• Diabetes A central characteristic of diabetes is impaired ⁇ -cell function.
• One abnormality that occurs early in disease progression in both type I and II diabetes is the loss of eating-induced rapid insulin response. Consequently, the liver continues to produce glucose, which adds to glucose that is ingested and absorbed from the basic components of a meal.
• Type II Diabetes One characteristic of type II diabetes is impaired insulin action, termed insulin resistance. Insulin resistance manifests itself as both a reduced maximal glucose elimination rate (GERmax) and an increased insulin concentration required to attain GERmax. Thus, to handle a given glucose load more insulin is required and that increased insulin concentration must be maintained for a longer period of time. Consequently, the diabetic patient is also exposed to elevated glucose concentrations for prolonged periods of time, which further exacerbates insulin resistance. Additionally, prolonged elevated blood glucose levels are themselves toxic to ⁇ -cells. Another characteristic of type II diabetics is a delayed response to increases in blood glucose levels.
• GERmax maximal glucose elimination rate
• Type II diabetics While normal individuals usually begin to release insulin within 2-3 minutes following consumption of food, type II diabetics may not secrete endogenous insulin until blood glucose begins to rise, and then with second-phase kinetics, that is a slow rise to an extended plateau in concentration. As a result, endogenous glucose production is not shut off and continues after consumption and the patient experiences hyperglycemia (elevated blood glucose levels).
• Type II diabetes arises from different and less well understood circumstances. The early loss of early phase insulin release, and consequent continual glucose release, contributes to elevated glucose concentrations. High glucose levels promote insulin resistance, and insulin resistance generates prolonged elevations of serum glucose concentration. This situation can lead to a self-amplifying cycle in which ever greater concentrations of insulin are less effective at controlling blood glucose levels.
• Type I Diabetes occurs as a result of the destruction of insulin- producing cells of the pancreas ( ⁇ -cells) by the body's own immune system. This ultimately results in a complete insulin hormone deficiency.
• Dosage form or unit dosage form refers to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject.
• each such unit contains a predetermined quantity of active agent.
• such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
• a therapeutic dose form or unit dosage form refers to a physically discrete unit of an active agent (e.g., a therapeutic or diagnostic agent) for administration to a subject.
• each such unit contains a predetermined quantity of active agent.
• such quantity is a unit dosage amount (or a whole fraction thereof) appropriate for administration in accordance with a dosing regimen that has been determined to correlate with a desired or beneficial outcome when administered to a relevant population (i.e., with a therapeutic dosing regimen).
• Dosing regimen refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.
• a given therapeutic agent has a
• a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount.
• a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount
• a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
• Excipient refers to a non-therapeutic agent that may be included in a pharmaceutical composition, for example to provide or contribute to a desired consistency or stabilizing effect.
• suitable pharmaceutical excipients may include, for example, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• Honeymoon phase As used herein, the term “honeymoon phase" of type 1 diabetes refers to early stages of the disease characterized by loss of early phase insulin release and the remaining ⁇ -cell function produces some insulin, which is released with second-phase kinetics.
• Hyperglycemia refers to a disease, disorder, or condition characterized by a higher than normal fasting blood glucose concentration. In some embodiments, hyperglycemia is characterized by a blood glucose concentration of 126 mg/dL or higher. In some embodiments, hyperglycemia is characterized by a blood glucose concentration of 280 mg/dL (15.6 mM) or higher.
• hypoglycemia refers to a disease, disorder, or condition characterized by a lower than normal blood glucose concentration.
• hypoglycemia is characterized by a blood glucose concentration of 63 mg/dL (3.5 mM) or lower.
• hypoglycemia causes symptoms such as cognitive impairment, behavioral changes, pallor, diaphoresis hypotonia, flush and weakness that are recognized symptoms of hypoglycemia and that disappear with appropriate caloric intake.
• hypoglycemia is severe such that glucagon injections, glucose infusions, or help by another party are required.
• an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent.
• an appropriate reference measurement may be or comprise a measurement in comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.
• Insulin-related disorder refers to disorders involving production, regulation, metabolism, and action of insulin in a mammal. Insulin-related disorders include, but are not limited to, pre-diabetes, type I diabetes, type II diabetes, hypoglycemia, hyperglycemia, insulin resistance, secretory dysfunction, sarcopenia, loss of pancreatic ⁇ -cell function, and loss of pancreatic ⁇ -cells.
• Non-insulin dependent patients having insulin-related disorders refers to patients with disorders for which therapy with exogenously-provided insulin is not the current standard treatment upon diagnosis. Non-insulin dependent patients having insulin-related disorders which are not treated with exogenously-administered insulin include early type II diabetes, type I diabetes in the honeymoon phase, pre-diabetes and insulin-producing cell transplant recipients.
• Insulin resistance refers to the inability of a patient's cells to respond to insulin appropriately or efficiently. The pancreas responds to this problem at the cellular level by producing more insulin. Eventually, the pancreas cannot keep up with the body's need for insulin and excess glucose builds up in the bloodstream. Patients with insulin resistance often have high levels of blood glucose and high levels of insulin circulating in their blood at the same time.
• Insulin resistance disorder refers to any disease or condition that is caused by or contributed to by insulin resistance. Examples include: diabetes, obesity, metabolic syndrome, insulin-resistance syndromes, syndrome X, insulin resistance, high blood pressure, hypertension, high blood cholesterol, dyslipidemia, hyperlipidemia, dyslipidemia, atherosclerotic disease including stroke, coronary artery disease or myocardial infarction, hyperglycemia, hyperinsulinemia and/or hyperproinsulinemia, impaired glucose tolerance, delayed insulin release, diabetic complications, including coronary heart disease, angina pectoris, congestive heart failure, stroke, cognitive functions in dementia, retinopathy, neuropathy, nephropathy, glomerulonephritis,
• glomerulosclerosis glomerulosclerosis, nephrotic syndrome, hypertensive nephrosclerosis some types of cancer (such as endometrial, breast, prostate, and colon), complications of pregnancy, poor female reproductive health (such as menstrual irregularities, infertility, irregular ovulation, polycystic ovarian syndrome (PCOS)), lipodystrophy, cholesterol related disorders, such as gallstones, cholescystitis and cholelithiasis, gout, obstructive sleep apnea and respiratory problems, osteoarthritis, and prevention and treatment of bone loss, e.g. osteoporosis.
• cancer such as endometrial, breast, prostate, and colon
• PCOS polycystic ovarian syndrome
• lipodystrophy cholesterol related disorders, such as gallstones, cholescystitis and cholelithiasis, gout, obstructive sleep apnea and respiratory problems, osteoarthriti
• Isomer As is known in the art, many chemical entities (in particular many organic molecules and/or many small molecules) can exist in a variety of structural (e.g., geometric, conformational, isotopic) and/or optical isomeric forms. For example, any chiral center can exist in R and S configurations, double bonds can exist in Z and E conformational isomers, certain structural elements can adopt two or more tautomeric forms, certain structures can be substituted with one or more isotopically enriched atoms (e.g., deuterium or tritium for hydrogen, 12 C or 14 C for 13 C, 131 I for 129 I, etc.).
• isotopically enriched atoms e.g., deuterium or tritium for hydrogen, 12 C or 14 C for 13 C, 131 I for 129 I, etc.
• depiction of or reference to a particular compound structure herein may represent all structural and/or optical isomers thereof.
• depiction of or reference to a particular compound structure herein is intended to encompass only the depicted or referenced isomeric form.
• compositions including a chemical entity that can exist in a variety of isomeric forms include a plurality of such forms; in some embodiments such compositions include only a single form.
• compositions including a chemical entity that can exist as a variety of optical isomers include a racemic population of such optical isomers; in some embodiments such compositions include only a single optical isomer and/or include a plurality of optical isomers that together retain optical activity.
• Parenteral As used herein, the terms “parenteral administration” and
• parenteral administration refers to modes of administration other than enteral and topical administration, usually by injection.
• parenteral administration may be or comprise intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular,
• intraarticulare subcapsular, subarachnoid, intraspinal and intrasternal injection and/or infusion.
• Partially unsaturated refers to a ring moiety that includes at least one double or triple bond.
• the term “partially unsaturated” is intended to encompass rings having multiple sites of unsaturation, but is not intended to include aryl or heteroaryl moieties, as herein defined.
• composition refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers.
• the active agent is present in unit dose amount appropriate for administration in a therapeutic regimen that shows a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant population.
• a pharmaceutical composition may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream, or foam; sublingually; ocularly; transdermally; or nasally, pulmonary, and to other mucosal surfaces.
• oral administration for example, drenches (aqueous or non-aqueous solutions
• any composition that is formulated for administration to a human or animal subject may, in some embodiments, be considered to be a pharmaceutical composition, whether or not its administration requires a medical prescription.
• a food or food supplement composition e.g., a liquid or solid consumable composition such as a shake or sports drink or nutritional supplement powder
• a pharmaceutical composition may be a formulation that is specifically regulated and approved for administration to relevant subjects by an appropriate government agency such as, for example, the Food and Drug Administration in the United States.
• a pharmaceutical composition is one that cannot legally be administered without a prescription from a licensed medical practitioner.
• composition as disclosed herein, the term "pharmaceutically acceptable" applied to a carrier, diluent, or excipient used to formulate a composition as disclosed herein means that the carrier, diluent, or excipient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
• compositions or vehicles such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
• a pharmaceutically-acceptable material such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
• Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
• materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ring
• pre-diabetes refers to a disease, disorder, or condition where the patients have impaired fasting glucose, and/or impaired glucose tolerance.
• pre-diabetic patients have a fasting blood glucose level between 100 mg/dL (5.5 mmol/L) and 126 mg/dL (7.0 mmol/L).
• pre- diabetic patients have a 2 hour post-prandial blood glucose level between 140 mg/dL (7.8 mmol/L) and 200 mg/dL (11.1 mmol/L).
• Prodrug refers to a pharmacologically active or more typically an inactive compound that is converted into a pharmacologically active agent by a metabolic transformation.
• Prodrugs of a compound of any of the formulas as described herein are prepared by modifying functional groups present in the compound of any of the formulas in such a way that the modifications may be cleaved in vivo to release the parent compound. In vivo, a prodrug readily undergoes chemical changes under physiological conditions (e.g., are hydrolyzed or acted on by naturally occurring enzyme(s)) resulting in liberation of the pharmacologically active agent.
• Prodrugs include compounds of any of the formulas as described herein wherein a hydroxyl, amino, or carboxyl group is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino or carboxyl group, respectively.
• Examples of prodrugs include, but are not limited to esters (e.g., acetate, formate, and benzoate derivatives) of compounds of any of the formulas as described herein or any other derivative which upon being brought to the physiological pH or through enzyme action is converted to the active parent drug. Conventional procedures for the selection and preparation of suitable prodrug derivatives are described in the art (see, for example, Bundgaard. Design of Prodrugs. Elsevier, 1985).
• Proliferative condition refers to a disease or disorder associated with cell proliferation.
• a proliferative disease or disorder is or comprises cancer.
• a proliferative disease or disorder is an inflammatory disease or disorder.
• a proliferative disease or disorder is an autoimmune disease or disorder.
• a proliferative disease or disorder is a microbial infection (e.g., a bacterial infection).
• reference refers to a standard or control relative to which a comparison is performed.
• an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value.
• a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest.
• a reference or control is a historical reference or control, optionally embodied in a tangible medium.
• a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment.
• risk refers to the likelihood that a particular individual will develop a disease, disorder, and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some embodiments a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.
• Solid form As is known in the art, many chemical entities (in particular many organic molecules and/or many small molecules) can adopt a variety of different solid forms such as, for example, amorphous forms and/or crystalline forms (e.g., polymorphs, hydrates, solvates, etc.). In some embodiments, such entities may be utilized as a single such form (e.g., as a pure preparation of a single polymorph). In some embodiments, such entities may be utilized as a mixture of such forms.
• amorphous forms and/or crystalline forms e.g., polymorphs, hydrates, solvates, etc.
• such entities may be utilized as a single such form (e.g., as a pure preparation of a single polymorph). In some embodiments, such entities may be utilized as a mixture of such forms.
• Subject refers an organism, typically a mammal (e.g., a human, in some embodiments including prenatal human forms).
• a subject is suffering from a relevant disease, disorder or condition.
• a subject is susceptible to a disease, disorder, or condition.
• a subject displays one or more symptoms or characteristics of a disease, disorder or condition.
• a subject does not display any symptom or characteristic of a disease, disorder, or condition.
• a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition.
• a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
• an individual who is "susceptible to" a disease, disorder, or condition ⁇ e.g., influenza
• an individual who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition.
• an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition.
• an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to conditions associated with development of the disease, disorder, or condition.
• a risk of developing a disease, disorder, and/or condition is a population-based risk (e.g., family members of individuals suffering from the disease, disorder, or condition).
• Therapeutically effective amount refers to an amount that produces the desired effect for which it is
• the term refers to an amount that is sufficient, when administered to a population suffering from or susceptible to a disease, disorder, and/or condition in accordance with a therapeutic dosing regimen, to treat the disease, disorder, and/or condition.
• a therapeutically effective amount is one that reduces the incidence and/or severity of, and/or delays onset of, one or more symptoms of the disease, disorder, and/or condition.
• therapeutically effective amount does not in fact require successful treatment be achieved in a particular individual. Rather, a therapeutically effective amount may be that amount that provides a particular desired pharmacological response in a significant number of subjects when
• reference to a therapeutically effective amount may be a reference to an amount as measured in one or more specific tissues (e.g., a tissue affected by the disease, disorder or condition) or fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.).
• tissue e.g., a tissue affected by the disease, disorder or condition
• fluids e.g., blood, saliva, serum, sweat, tears, urine, etc.
• a therapeutically effective amount of a particular agent or therapy may be formulated and/or administered in a single dose.
• a therapeutically effective agent may be formulated and/or administered in a plurality of doses, for example, as part of a dosing regimen.
• treatment refers to administration of a therapy that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of (e.g., relative to an established onset time or period), reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition.
• treatment may be of a subject who does not exhibit signs or symptoms of the relevant disease, disorder and/or condition, and/or of a subject who is not diagnosed suffering the relevant disease, disorder and/or condition, and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition.
• treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition.
• treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition.
• treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.
• such treatment refers to reducing risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition.
• treatment is administration of therapy according to a regimen that has been demonstrated to achieve a relevant result (e.g., to partially or completely alleviate, ameliorate, relieve, inhibit, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms, features, and/or cause of a particular disease, disorder, and/or condition) with statistical significance when applied to a relevant population or system (e.g., model system).
• a relevant result e.g., to partially or completely alleviate, ameliorate, relieve, inhibit, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms, features, and/or cause of a particular disease, disorder, and/or condition
• a relevant population or system e.g., model system
• treatment administered after diagnosis and/or onset of one or more symptoms is considered to be "therapeutic" treatment
• treatment administered prior to diagnosis and/or to onset of symptoms is considered to be "prophylactic" treatment.
• Unit dose refers to an amount administered as a single dose and/or in a physically discrete unit of a pharmaceutical composition.
• a unit dose contains a predetermined quantity of an active agent.
• a unit dose contains an entire single dose of the agent.
• more than one unit dose is administered to achieve a total single dose.
• administration of multiple unit doses is required, or expected to be required, in order to achieve an intended effect.
• a unit dose may be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined quantity of one or more therapeutic agents, a predetermined amount of one or more therapeutic agents in solid form, a sustained release formulation or drug delivery device containing a predetermined amount of one or more therapeutic agents, etc.
• a unit dose may be present in a formulation that includes any of a variety of components in addition to the therapeutic agent(s).
• acceptable carriers e.g., pharmaceutically acceptable carriers
• diluents, stabilizers, buffers, preservatives, etc. may be included as described infra. It will be appreciated by those skilled in the art, in many others.
• a total appropriate daily dosage of a particular therapeutic agent may comprise a portion, or a plurality, of unit doses, and may be decided, for example, by the attending physician within the scope of sound medical judgment.
• the specific effective dose level for any particular subject or organism may depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of specific active compound employed; specific composition employed; age, body weight, general health, sex and diet of the subject; time of administration, and rate of excretion of the specific active compound employed; duration of the treatment; drugs and/or additional therapies used in combination or coincidental with specific compound(s) employed, and like factors well known in the medical arts.
• Alkyl As used herein, the term “alkyl” refers to linear or branched alkyl groups.
• Exemplary groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, isopentyl, neopentyl, and hexyl.
• Heteroatom refers to one or more of oxygen, sulfur, nitrogen, phosphorus, or silicon (including, any oxidized form of nitrogen, sulfur, phosphorus, or silicon; the quaternized form of any basic nitrogen or; a substitutable nitrogen of a heterocyclic ring, for example N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR + (as in N-substituted pyrrolidinyl)).
• Carbocyclic As used herein, the term “carbocyclic” refers to a monocyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point of attachment to the rest of the molecule.
• Heterocyclic refers to a stable monocyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, preferably one to four, heteroatoms, as defined above.
• nitrogen includes a substituted nitrogen.
• the nitrogen may be N (as in 3,4-dihydro-2H- pyrrolyl), NH (as in pyrrolidinyl), or + NR (as in N-substituted pyrrolidinyl).
• a heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted.
• 3-8 membered heterocyclic examples include tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, piperidinyl, pyrrolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl.
• Halogen As used herein, the terms “halogen” and “halo” refer to F, CI, Br, or I.
• Compound C The term "Compound C”, as used herein, refers to 5'-
• Methylselenoadenosine also known as (2R, 4S, 5S)-2-(6-amino-9H-purin-9-yl)-5- ((methylselanyl)methyl)tetrahydrofuran-3,4-diol, CAS Registry Number 5135-40-0, and includes any pharmaceutically acceptable salts thereof.
• Compound D The term "Compound D”, as used herein, refers to 5'-
• Compound E refers to gamnia- glutamyl-methylseleno-cysteine or ⁇ -L-glutanyml-Se-rnethyl-L- cysteine; also known as N5-(l ⁇ carboxy-2-(methylselanyl)ethyl)-L-glutamine, or any pharmaceutically acceptable salt thereof. d E
• Compound CDE refers to a mixture of Compound C, Compound D, and Compound E, or pharmaceutically acceptable salts thereof.
• the present disclosure provides a number of exemplary compounds capable of lowering glucose level, improving glucose tolerance, restoring or activating insulin receptor function and its downstream signaling, enhancing AS 160 phosphorylation for translocation of glucose transporter proteins (GLUTs) from cytosolic vesicles to plasma membrane for glucose uptake, stimualing glucose uptake, and attenuating hyperinsulinemia without impaired kidney function and/or liver damage in diabetic mice, cultured liver and/or skeletal muscle cells after treatment of single compound.
• GLUTs glucose transporter proteins
• Example 2 shows that each of Compounds 43, 50, 53, 69, and 70 lowered glucose production in HepG2 cells. Particularly, Compound 43 exhibited higher potency than Compound CDE.
• Compound 43 When compared to antidiabetic drug metformin in HepG2 and rat H4IIE cells, Compound 43 showed greater potency and lower cell toxicity. It was also shown that Compound #43 is more potent than Compound 50 in the inhibition of the expression of G6pc (a key enzyme gene for liver glucose production) in the liver of diabetic mice, and than Compound CDE, Compound C, Compound D and Compound 50 in mouse liver AML-12 cells (Example 4).
• G6pc a key enzyme gene for liver glucose production
• compound 43 is more potent than compound C, compound 50, compound 69 and compound 70 against hyperglycermia in diabetic mice (Example 3); that Compound 43 significantly improves glucose tolerance (Example 3) and enhances / restores insulin receptor function and its downstream signaling in the livers and skeletal muscles of insulin-resistant diabetic Lepr db/db mice (Examples 5 and 7-8); and that Compound 43 elicits a response to the glucose challenge in diabetic mice which was similar to the response in wild-type mice (Example 3).
• Compound #43 treatment can enhance the phosophorylation of AS 160 (to promote the translocation of GLUTs from cellular vesicles to plasma membrane) for glucose uptake in both liver and skeletal muscle cells (Example 8), stimulate GLUT4 expression in the liver of diabetic mice and cultured mouse liver cells (Example 6), enhance and/or potentiate insulin action to stimulate glucose uptake in the liver and skeletal muscle cells (Example 6-7), and attenuate the hyperinsulinemia without impaired kidney function and/or liver damage in the insulin-resistant diabetic mice (Example 9).
• the present disclosure also provides features of selenium compounds which may contribute to its activity. For example, it was shown that while selenium Compound #43 had a great potency in inhibiting glucose production, its sulfur analog Compound 68 had a low potency (Example 2); and that Compound #43 is more potent than Compound #68 in lowering blood glucose and HbAlc levels and improving glucose tolerance in insulin-resistant diabetic mice (Example 3).
• the present disclosure relates to a compound of formula (1):
• each of R 2 and R 3 is independently H or -C(0)-R, wherein each R is independently Ci. 6 alkyl or 3-8 membered carbocyclic or heterocyclic, wherein R 2 and R 3 cannot be both H;
• R 2 together with R 3 form -(CH2) n -C(0)-(CH2) m -, wherein each of n and m is independently 0-3, and n+m ⁇ 3;
• R 5 is -C 1-6 alkyl or -C 1-6 alkyl-CH( H 2 )COOH;
• R 8 is H or halogen
• X is H or halogen
• each of the carbocyclic, heterocyclic, -(CH 2 ) n -, and -(CH 2 ) m - moieties may optionally be substituted 1-3 times by -OH, halogen, H 2 , CN, or and
• each Ci-6alkyl moiety may optionally be substituted 1-3 times by -OH, halogen, H 2 , or CN.
• R 8 is H.
• R 8 of formula (1) is halogen. In some embodiments of formula (1), R 8 of formula (1) is F.
• X is H. In some embodiments of formula (1), X is H.
• X is halogen. In some embodiments of formula (1), X is F.
• R 5 is -Ci-6alkyl, which may optionally be substituted 1-3 times by -OH, halogen, NH 2 , or CN.
• R 5 is - Ci-6alkyl, which may optionally be substituted 1-3 times by halogen.
• R 5 is unsubstituted -C 1-6 alkyl.
• R 5 is unsubstituted linear -C 1-6 alkyl.
• R 5 is methyl.
• R 5 is ethyl.
• R 5 is propyl.
• R 5 is -C 1-6 alkyl-CH( H 2 )COOH, wherein C 1-6 alkyl may optionally be substituted 1-3 times by -OH, halogen, NH 2 , or CN.
• R 5 is -C 1-6 alkyl-CH( H 2 )COOH, wherein C 1-6 alkyl may optionally be substituted 1-3 times by halogen.
• R 5 is -C 1-6 alkyl- CH( H 2 )COOH, wherein C 1-6 alkyl is unsubstituted.
• R 5 is -CH 2 CH 2 -CH( H 2 )COOH.
• R 5 is -CH 2 -CH( H 2 )COOH.
• R 5 is - CH 2 CH 2 CH 2 -CH( H 2 )COOH.
• R 5 is - CH 2 CH 2 CH 2 -CH( H 2 )COOH.
• R 5 is - CH 2 CH 2 CH 2 -CH( H 2 )COOH.
• R 2 is H
• R 3 is -C(0)-R, wherein R is Ci.
• each of the carbocyclic and heterocyclic moieties may optionally be substituted 1-3 times by -OH, halogen, H 2 , CN, or C 1-6 alkyl; and each C 1-6 alkyl, independently, may optionally be substituted 1-3 times by -OH, halogen, NH 2 , or CN.
• R 3 is H
• R 2 of formula (1) is -C(0)-R, wherein R is or 3-8 membered carbocyclic or heterocyclic, wherein each of the carbocyclic and heterocyclic moieties, independently, may optionally be substituted 1-3 times by -OH, halogen, NH 2 , CN, or C 1-6 alkyl; and each C 1-6 alkyl, independently, may optionally be substituted 1-3 times by -OH, halogen, NH 2 , or CN.
• each of R 2 and R 3 is independently C(0)-R, wherein each R is independently or 3-8 membered carbocyclic or heterocyclic, wherein each of the carbocyclic and heterocyclic moieties, independently, may optionally be substituted 1-3 times by -OH, halogen, NH 2 , CN, or independently, may optionally be substituted 1-3 times by -OH, halogen, NH 2 , or CN.
• each R is independently 3-8 membered carbocyclic or heterocyclic, wherein each of the carbocyclic and heterocyclic moieties, independently, may optionally be substituted 1-3 times by -OH, halogen, NH 2 , CN, or and each independently, may optionally be substituted 1-3 times by -OH, halogen, NH 2 , or CN.
• each R is independently 3-8 membered carbocyclic or heterocyclic, wherein each of the carbocyclic and heterocyclic moieties, independently, may optionally be substituted 1-3 times by halogen.
• each R is independently 3-8 membered carbocyclic or heterocyclic, wherein each of the carbocyclic and heterocyclic moieties, independently, may optionally be substituted 1-3 times by halogen. In some embodiments of formula (1), each R is independently 3-8 membered unsubstituted carbocyclic or unsubstituted heterocyclic. In some embodiments of formula (1), each R is independently 6 membered unsubstituted carbocyclic or unsubstituted heterocyclic. In some embodiments of formula (1), is independently unsubstituted heterocyclic. In some embodiments of formula (1), R is .
• each R is independently C 1-6 alkyl, and each C 1-6 alkyl, independently, may optionally be substituted 1-3 times by -OH, halogen, H 2 , or CN. In some embodiments of formula (1), each R is independently C 1-6 alkyl, and each C 1-6 alkyl, independently, may optionally be substituted 1-3 times by halogen. In some embodiments of formula (1), each R is independently unsubstituted C 1-6 alkyl. In some embodiments of formula (1), each R is independently unsubstituted linear C 1-6 alkyl. In some embodiments of formula (1), each R is independently methyl, ethyl, or propyl. In some embodiments of formula (1), R is
• R 2 together with R 3 form -(CH 2 ) n -C(0)-
• each of the - (CH 2 ) n - and -(CH 2 ) m - moieties may optionally be substituted 1-3 times by -OH, halogen, H 2 , CN, or independently, may optionally be substituted 1-3 times by -OH, halogen, NH 2 , or CN.
• each of the - (CH 2 ) n - and -(CH 2 ) m - moieties independently, may optionally be substituted 1-3 times by halogen.
• the -(CH 2 ) n - and -(CH 2 ) m - moieties are unsubstituted.
• the present disclosure relates to a compound of formula
• R 8 is H or halogen
• X is H or halogen
• each R 5 ' is independently H or halogen
• each R is independently C 1-6 alkyl, each of which, independently, may optionally be substituted 1-3 times by halogen.
• C(R 5 ' )3 is CF 3 , CHF 2 , CH 2 F, or CH 3 .
• the compound is of formula (2'):
• R is H. In some embodiments of formula (2) or (2'), R 8 is halogen. In some embodiments of formula (2) or (2'), R 8 is F.
• X is H. In some embodiments of formula (2) or (2'), X is halogen. In some embodiments of formula (2) or (2'), X is F.
• R is each independently unsubstituted C 1-6 alkyl.
• R is each independently Ci -3 alkyl, each of which, independently, may optionally be substituted 1-3 times by halogen.
• R is each independently unsubstituted Ci -3 alkyl.
• R is each independently -CH 3 , -CH 2 CH 3 , or - CH 2 CH 2 CH 3 .
• the present disclosure relates to a compound of formula (3):
• R 8 is H or halogen
• X is H or halogen
• each R' is independently H or halogen.
• -Se-C(R') 3 is -Se-CH 3 , -Se-CHF 2 ,
• the compound is of formula (3'):
• R 8 is H. In some embodiments of formula (3) or (3'), R 8 is halogen. In some embodiments of formula (3) or (3'), R 8 is F.
• X is H. In some embodiments of formula (3) or (3'), X is halogen. In some embodiments of formula (3) or (3'), X is F. [00132] In some embodiments of formula (3) or (3'), each C(R') 3 is independently CF 3 ,
• each C(R') 3 is
• each C(R') 3 is CH 3 .
• the present disclosure relates to a compound of formula:
• the present disclosure provides compositions that comprise and/or deliver (i.e., upon administration to a system or subject) a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides compositions comprising only a single compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides compositions comprising one or more compounds of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof, and one or more carriers or excipients appropriate for administration to human or animal subjects in accordance with the present disclosure.
• the present disclosure provides compositions that deliver an active moiety of a compound of any one of formulas (l)-(3).
• the composition comprises an active metabolite of a compound of any one of formulas (l)-(3).
• the composition comprises a compound which forms a metabolite of a compound of any one of formulas (l)-(3) upon administration of said composition, which metabolite maintains relevant biological activity.
• compositions that are pharmaceutical compositions in that they contain an active pharmaceutical ingredient (API) and one or more pharmaceutically acceptable carriers or excipients.
• API is or comprises the compound of any one of formulas (l)-(3).
• the API consists of the compound of any one of formulas (l)-(3).
• the API consists of a single compound of any one of formulas (l)-(3).
• the present disclosure provides methods of manufacturing a provided composition, for example by combining one or more appropriate (i.e., one or more appropriate (i.e., one or more appropriate (i.e., one or more appropriate (i.e., one or more appropriate (i.e., one or more appropriate (i.e., one or more appropriate (i.e., one or more appropriate (i.e., one or more appropriate (i.e., one or more appropriate (i.e.,
• the pharmaceutical composition is a solid dosage form.
• the solid dosage form is a tablet, capsule, or lozenge.
• the pharmaceutical composition is a liquid dosage form (e.g., a drink).
• the present disclosure provides pharmaceutical compositions comprising a pharmaceutically acceptable amount of a compound as described herein.
• amount of active ingredient which can be combined with a carrier material to produce a single dosage form may vary depending upon the host being treated, and/or the particular mode of administration.
• the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of active ingredient, from about 5% to about 70%, or from about 10% to about 30%.
• wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
• antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium
• metabi sulfite, sodium sulfite and the like oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
• oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like
• metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
• formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
• the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
• formulations as described herein comprise an excipient selected from the group consisting of cyclodextrins, liposomes, micelle forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides; and a compound as described herein.
• formulations as described herein render orally bioavailable a compound as described herein.
• methods of preparing such formulations may comprise a step of bringing into association a compound as described herein with one or more
• the formulations are prepared by uniformly and intimately bringing into association a compound as described herein with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.
• formulations as described herein suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes, drinks, and the like, each containing a predetermined amount of a compound as described herein as an active ingredient.
• a compound as described herein may alternatively or additionally be administered as a bolus, electuary or paste.
• the active ingredient in solid dosage forms as described herein for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example,
• carboxymethylcellulose alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia;
• humectants such as glycerol
• disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate
• solution retarding agents such as paraffin
• absorption accelerators such as quaternary ammonium compounds
• wetting agents such as, for example, cetyl alcohol, glycerol monostearate, and non-ionic surfactants
• absorbents such as kaolin and bentonite clay
• lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof
• coloring agents such as glycerol
• disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate
• solution retarding agents such as paraffin
• absorption accelerators such as quaternary ammonium
• compositions may also comprise buffering agents.
• solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such carriers as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
• a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
• compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
• molded tablets may be made in a suitable machine in which a mixture of the powdered compound is moistened with an inert liquid diluent.
• compositions as described herein may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art.
• they may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres.
• they may be formulated for rapid release, e.g., freeze-dried.
• they may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
• sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
• compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or preferentially, in a certain portion of the
• the active ingredient can be in micro-encapsulated form, if appropriate, with one or more of the above- described excipients.
• liquid dosage forms for oral administration of the compounds as described herein include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
• the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
• inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and
• the oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
• adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
• suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
• suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
• formulations as described herein for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more compounds as described herein with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
• suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound.
• formulations as described herein which are suitable for vaginal administration include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such carriers as are known in the art to be appropriate.
• dosage forms for the topical or transdermal administration of a compound as described herein include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants.
• the active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier, and with any preservatives, buffers, or propellants which may be required.
• the ointments, pastes, creams and gels may contain, in addition to a compound as described herein, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
• excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
• powders and sprays can contain, in addition to a compound as described herein, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
• sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
• transdermal patches have the added advantage of providing controlled delivery of a compound as described herein to the body.
• dissolving or dispersing the compound in the proper medium can make such dosage forms.
• absorption enhancers can be used to increase the flux of the compound across the skin.
• either providing a rate controlling membrane or dispersing the compound in a polymer matrix or gel can control the rate of such flux.
• the present disclosure provides ophthalmic formulations, eye ointments, powders, solutions and the like.
• compositions as described herein suitable for parenteral administration comprise one or more compounds as described herein in combination with one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
• aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
• polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
• vegetable oils such as olive oil
• injectable organic esters such as ethyl oleate.
• proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
• the compositions as described herein may contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
• adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
• prevention of the action of microorganisms upon the subject compounds may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like.
• isotonic agents such as sugars, sodium chloride, and the like into the compositions.
• prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
• the rate of absorption of the drug then depends upon its rate of dissolution, which in turn, may depend upon crystal size and crystalline form.
• delayed absorption of a parenterally-administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
• injectable depot forms are made by forming
• microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide.
• biodegradable polymers such as polylactide-polyglycolide.
• the rate of drug release can be controlled.
• depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissue.
• drug-eluting forms include coated or medicated stents and implantable devices.
• drug-eluting stents and other devices may be coated with a compound or pharmaceutical preparation and may further comprise a polymer designed for time-release.
• a compound or pharmaceutical preparation is administered orally. In some embodiments, the compound or pharmaceutical preparation is administered intravenously. In some embodiments, a compound is attached via a cleavable linker to a solid support that is administered with a catheter. In some embodiments, routes of administration include sublingual, intramuscular, and transdermal administrations.
• the compounds as described herein are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, 0.1% to 99.5%, or 0.5% to 90%, of active ingredient in combination with a pharmaceutically acceptable carrier.
• the compounds as described herein may be given orally, parenterally, topically, or rectally. In some embodiments, they are of course given in forms suitable for each administration route. In some embodiments, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc. administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories.
• the compounds as described herein may be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, an aerosol, a spray, rectally, intravaginally, parenterally,
• the compounds as described herein, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions as described herein, are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art.
• actual dosage levels of the active ingredients in the pharmaceutical compositions as described herein may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
• a selected dosage level will depend upon a variety of factors including the activity of the particular compound as described herein, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
• the physician or veterinarian could start doses of the compounds as described herein in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and then gradually increasing the dosage until the desired effect is achieved.
• chronic treatments include any form of repeated administration for an extended period of time, such as repeated
• a chronic treatment involves administering a compound or pharmaceutical composition as described herein repeatedly over the life of the subject.
• chronic treatments involve regular administrations, for example one or more times a day, one or more times a week, or one or more times a month.
• a suitable dose such as a daily dose of a compound as described herein will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described herein.
• doses of the compounds as described herein for a patient when used for the indicated effects, will range from about 0.0001 to about 100 mg per kg of body weight per day. In some embodiments, the daily dosage will range from 0.001 to 50 mg of compound per kg of body weight. In some embodiments, the daily dosage will range from 0.01 to 10 mg of compound per kg of body weight. However, lower or higher doses can be used. In some embodiments, the dose administered to a subject may be modified as the physiology of the subject changes due to age, disease progression, weight, or other factors.
• the effective daily dose of the active compound may be administered as two, three, four, five, six, or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
• a compound as described herein is administered alone. In some embodiments, a compound as described herein is administered as a pharmaceutical formulation (composition) as described herein.
• the compounds as described herein may be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceuticals.
• a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof may be prepared in whole or in part by chemical synthesis; in some embodiments, a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof, prepared in part by chemical synthesis is prepared using semi-synthetic methodologies. In some embodiments, a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof, may be prepared by isolation.
• the present disclosure provides methods for preparing a
• compositions comprising assaying one or more samples, for example to detect bioactivity therein.
• one or more of the samples comprise a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• methods provided herein comprise a step of detecting and/or confirming presence of detectable bioactivity in one or more samples.
• methods provided herein comprise a step of confirming absence of detectable bioactivity in one or more samples.
• the bioactivity is inhibition of glucose production.
• the bioactivity is tested in HepG2 cells.
• the bioactivity is tested in H4IIE cells.
• the bioactivity is reduction of serum HbAlc level. In some embodiments, the bioactivity is tested in insulin-resistant and diabetic mice.
• the bioactivity is enhanced glucose tolerance. In some embodiments, the bioactivity is tested in insulin-resistant and diabetic mice.
• the bioactivity is inhibition of G6pc expression.
• the bioactivity is tested in AML-12 cells.
• the bioactivity is tested in AML-12 cells stimulated with diabetic stimuli.
• the bioactivity is tested in human HepG2 cells.
• the bioactivity is tested in the liver of insulin-resistant and diabetic mice.
• the bioactivity is enhanced phosphorylation of Pdkl, Akt,
• the bioactivity is tested in the liver. In some embodiments, the bioactivity is tested in the skeletal muscle.
• the bioactivity is enhanced Glut4 expression.
• the bioactivity is tested in mouse liver AML-12 cells.
• the bioactivity is tested in the liver of insulin-resistant and diabetic mice.
• the bioactivity is activation and/or restoration of insulin signaling in a subject in insulin-resistant state.
• the subject is
• the insulin- resistant state is characterized by reduced level and/or activity of phosphorylated insulin receptor in the subject.
• the subject has diabetes, and/or diabetes associated disease, disorders, or conditions.
• the bioactivity is enhanced glucose uptake.
• the cells are liver cells and skeletal muscle cells.
• the bioactivity is reduction of serum insulin level.
• the bioactivity is tested in insulin-resistant and diabetic mice.
• the present disclosure provides methods for identifying and/or characterizing a compound and/or a composition as described herein.
• a method comprises steps of testing a plurality of samples, each of which comprises a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof, for bioactivity therein; and determining presence and/or level of said bioactivity in one or more such samples.
• a provided method comprises detecting said bioactivity associated with presence and/or level of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• a provided method comprises a step of identifying and/or characterizing a particular compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof, by detecting said bioactivity of the compound.
• the bioactivity is inhibition of glucose production. In some embodiments, the bioactivity is tested in HepG2 cells. In some embodiments, the bioactivity is tested in H4IIE cells.
• the bioactivity is reduction of serum HbAlc level. In some embodiments, the bioactivity is tested in insulin-resistant and diabetic mice.
• the bioactivity is enhanced glucose tolerance. In some embodiments, the bioactivity is tested in insulin-resistant and diabetic mice.
• the bioactivity is inhibition of G6pc expression.
• the bioactivity is tested in AML-12 cells.
• the bioactivity is tested in AML-12 cells stimulated with diabetic stimuli.
• the bioactivity is tested in human HepG2 cells.
• the bioactivity is tested in the liver of insulin-resistant and diabetic mice.
• the bioactivity is enhanced phosphorylation of Pdkl, Akt,
• the bioactivity is tested in the liver. In some embodiments, the bioactivity is tested in the skeletal muscle.
• the bioactivity is enhanced Glut4 expression.
• the bioactivity is tested in mouse liver AML-12 cells.
• the bioactivity is tested in the liver of insulin-resistant and diabetic mice.
• the bioactivity is activation and/or restoration of insulin signaling in a subject in insulin-resistant state.
• the subject is characterized by significant levels of circulating insulin.
• the insulin- resistant state is characterized by reduced level and/or activity of phosphorylated insulin receptor in the subject.
• the subject has diabetes, and/or diabetes associated disease, disorders, or conditions.
• the bioactivity is enhanced glucose uptake into cells in a subject. In some embodiments, the bioactivity is enhanced glucose uptake into liver cells and skeletal muscle cells. In some embodiments, the bioactivity is reduction of serum insulin level. In some embodiments, the bioactivity is tested in insulin-resistant and diabetic mice.
• the compounds as described herein, for example compound 43 can mimic insulin to inhibit glucose production (see for example, Example 2); lower blood glucose and HbAlc levels, attenuate the development of hyperglycemia and improve glucose tolerance in insulin-resistant diabetic subjects (see for example, Example 3); inhibit G6pc expression in the liver of insulin-resistant diabetic subjects, and mimic but bypass insulin to inhibit G6pc expression in cultured mouse and human liver cells and potentiate insulin action (see for example, Example 4); mimic but bypass insulin to activate Pdkl and Akt and enhance Foxol phosphorylation in the liver (see for example, Example 5); enhance Glut4 expression in the liver of insulin-resistant diabetic subjects, and mimic but bypass insulin to enhance Glut4 expression in mouse liver cells (see for example, Example 6), the phosphorylation of AS 160 (a key event for GLUT4 transportation from cytosolic vesicles to plasma membrane to facilitate glucose uptake) in human liver cells (see for example, Example 8), and glucose
• the present disclosure provides methods for modulating glucose metabolism and/or treating glucose metabolism disorders, comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a
• glucose metabolism disorders involve a blood glucose level which is not within the normal range.
• glucose metabolism disorders relate to defective glucose uptake and/or transport.
• glucose metabolism disorders are Diabetes Mellitus, glyceraldehyde-3 -phosphate dehydrogenase deficiency, glycosuria, hyperglycemia,
• hypoglycemia hyperinsulinism, or hypoglycemia.
• the present disclosure provides methods for treating disorders of glucose transport, comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• disorders of glucose transport are glucose-galactose malabsorption, Fanconi-Bickel syndrome, or De Vivo disease (GLUTl deficiency syndrome (GLUT IDS)).
• the present disclosure provides methods for enhancing
• AS 160 phosphorylation for translocation of glucose transporter proteins (GLUTs) from cytosolic vesicles to plasma membrane for glucose uptake comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for enhancing glucose uptake in both liver and skeletal muscles, comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for treating an insulin-related disorder, comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the insulin-related disorders are selected from the group consisting of pre-diabetes, type I diabetes, type II diabetes, hypoglycemia, hyperglycemia, insulin resistance, secretory dysfunction, loss of pancreatic ⁇ -cell function, and loss of pancreatic ⁇ -cells.
• the patients of insulin-related disorders are non-insulin dependent patients having insulin-related disorders.
• the present disclosure provides methods for treating insulin resistance disorder comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof. Certain examples of insulin resistance disorders are described above. In some embodiments, provided methods are for treating Type II diabetes, hyperinsulinemia,
• hyperproinsulinemia retinopathy, neuropathy, or nephropathy.
• provided methods attenuate hyperinsulinemia without impairing kidney function and/or resulting in liver damage.
• the present disclosure provides methods for treating obesity comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for treating diabetes, comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the diabetes is type I diabetes. In some embodiments, the diabetes is type II diabetes.
• the present disclosure provides methods for treating hyperglycemia comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for inhibiting glucose production, comprising administering a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for reducing serum HbAlc level, comprising administering a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof. [00206] In some embodiments, the present disclosure provides methods for increasing glucose tolerance, comprising administering a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for inhibiting
• G6pc expression comprising administering a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for enhancing phosphorylation of Pdkl, Akt, and Foxol in the liver and/or in the skeletal muscle, comprising administering a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for increasing
• Glut4 expression comprising administering a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for activating and/or restoring insulin signaling in a subject in insulin-resistant state, comprising administering a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the subject is characterized by significant levels of circulating insulin.
• the insulin-resistant state is characterized by reduced level and/or activity of phosphorylated insulin receptor in the subject.
• the subject has diabetes, and/or diabetes associated disease, disorders or conditions.
• the subject has type II diabetes.
• the present disclosure provides methods for enhancing glucose uptake into cells in a subject, comprising administering a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the cells are skeletal muscle cells.
• PCOS polycystic ovary syndrome
• AD Alzheimer's disease
• sarcopenia a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• PCOS is a hormone imbalance that can cause irregular periods, unwanted hair growth, and acne. Young women with PCOS often have elevated insulin levels which can cause the ovaries to make more androgen hormones, resulting in increased body hair, acne, and irregular or few periods. Having PCOS can cause insulin resistance and the development of type 2 diabetes. Metformin is a medication often prescribed for women with PCOS to improve insulin sensitivity and prevent the development of type 2 diabetes. The results demonstrate that compound #43 is more potent than metformin in the inhibition of glucose production in cultured liver cells, and can restore insulin receptor function in insulin-resistant diabetic mice. Thus, Compound #43 can be potentially useful for the treatment of PCOS.
• the present disclosure provides methods for treating
• AD Alzheimer's disease
• a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof Brain insulin signaling is important for learning and memory, and insulin resistance in the brain is a major risk factor for AD.
• the restoration of insulin signaling has emerged as a potential therapy for AD (White MF, Science 2003; 302: 1710-1; De Felice DG et al, Alzheimer's & Dementia 2014; 10: S26-S32).
• Compound #43 exhibited insulin-like activity and was able to restore insulin receptor function in insulin-resistant subjects, it can be potentially useful for the treatment of AD.
• the present disclosure provides methods for treating sarcopenia comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• Sarcopenia is characterized by the progressive loss of skeletal muscle mass with increasing age, leading to decreased muscle strength, decreased mobility and function, increased fatigue, an elevated risk of fall-related injury, and, often, frailty (Candow and chilibeck, 2005; Sakuma and Yamaguchi, 2012).
• Insulin signaling Insr/PI3K/Akt
• Akt/Foxol -mediated inhibition of the expression of two atrophy genes Fbxo32 and Trim63 insulin signaling
• Optimal insulin signaling attenuates muscle wasting processes, including sarcopenia (Glass and Roubenoff, 2010; Ryall et al., 2008; Sakuma and Yamaguchi, 2012).
• the present disclosure also teaches that provided compounds likely enhance mitochrodrial function, and are therefore useful for treating mitochrodrial diseases and/or dysfunction.
• the present disclosure provides methods for treating mitochondria-associated diseases (e.g., caused by dysfunctional mitochondria), comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• mitochondria-associated diseases can be degenerative diseases (e.g., cancer, cardiovascular disease and cardiac failure, type 2 diabetes, Alzheimer's and Parkinson's diseases, fatty liver disease, cataracts, osteoporosis, muscle wasting, sleep disorders and inflammatory diseases such as psoriasis, arthritis and colitis).
• the present disclosure provides methods for enhancing mitochondrial function, comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• the present disclosure provides methods for enhancing gluconeogenesis in the brain, comprising administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• provided methods increase glucose uptake in the brain.
• provided methods are for maintaining or restoring brain functions including memory and learning.
• the present disclosure provides use of the compounds and/or compositions as described herein for combination therapy of a disease, disorder, or condition as described herein.
• the present disclosure provides methods for treating patients with a disease, disorder, or condition, who have received, are receiving, or will receive one or more different therapies for said disease, disorder, or condition, wherein the methods comprise administering a therapeutically effective amount of a compound of any one of formulas (l)-(3), or a pharmaceutically acceptable salt, prodrug, or isomer thereof.
• one or more therapies for patients with insulin-related disorders are selected from the group consisting of insulin therapy, for example, for type I diabetes; diet and exercise, for example, for incipient type II diabetes; oral antidiabetic agents, for example, for early stage type II diabetes; metformin; insulin secretagogues, for example, sulfonylureas; glitazones; long-acting basal insulin; intermediate acting insulin; and short (rapid) acting insulin.
• insulin therapy involves administration subcutaneously (SC), intravenously, and/or by inhalation.
• one or more therapies for patients with insulin-related disorders may be therapies currently under development, for example, insulin-fumaryl diketopiperazine (FDKP).
• FDKP insulin-fumaryl diketopiperazine
• the reaction mixture was stirred for 6 hours at 5-10 °C. Quenched the excess acetic anhydride by adding ice-cold water (100 ml), and then pH adjusted to 7 by adding 10 wt% NaHCCh aqueous solution. The aqueous mixture was extracted with ethyl acetate (2 x 100 nil). The combined ethyl acetate extracts are dried over anhydrous Na 2 SO 4 0Igr), filtered into a 250 ml round-bottomed flask.
• the reaction mixture was stirred for 6 hours at 5-10 °C. Quenched the excess acetic anhydride by adding ice-cold water (100 ml), and then pH adjusted to 7 by adding 10 wt% NaHC0 3 aqueous solution. The aqueous mixture was extracted with ethyl acetate (2 x 100 ml). The combined ethyl acetate extracts are dried over anhydrous Na 2 S0 4 (Igr), filtered into a 250 ml round-bottomed flask.
• the human hepatoma HepG2 and rat hepatoma H4IIE cells were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). HepG2 cells and H4IIE cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS.
• EMEM Eagle's Minimum Essential Medium
• Equal numbers of Human HepG2 or rat H4IIE cells (1-1.5 X10 5 cells/well) were seeded on 96 well plates in 10% FBS-containing media for 24 hr. Cells were then washed twice with PBS, and treated with various concentrations of compounds, metformin or insulin in 100 ⁇ of glucose production media (glucose-free, phenol red-free DMEM media supplemented with 20 mM sodium lactate, 2 mM sodium pyruvate and 5 mM HEPES) at 37°C for 24 hr (H4IIE cells only) or 48 hr (HepG2 cell only). Cells were also incubated with 0.24% DMSO (the maximal volume of DMSO solvent used in the experiments).
• Glucose production in the culture cells were obtained by normalizing the glucose concentration in culture media by cell viability in each well. At least 3 samples per each treatment were examined for the above analysis. Data are presented as Mean ⁇ SEM of those samples.
• IC50 values The half maximal inhibitory concentration (IC50 values) of Compound #43 or metformin for the inhibition of glucose production were determined using the ED50 Plus vl .O online software.
• Compound #43 at a dose of 3.8 ⁇ was as potent as the CDE combination product (which contained 3.8 ⁇ of each individual compound) in inhibiting glucose production in HepG2 cells.
• Compound #43 at a dose of 7.6 ⁇ or higher was more potent than CDE in inhibiting glucose production in HepG2 cells.
• C, D, and E inhibited glucose production in human HepG2 cells at the indicated doses.
• the compound concentration indicated on the X-axis of Fig. 1 refers to the Se- concentration of each Se-containing compound in the mixture
• the total Se-concentration is actually three-times higher than that is indicated on the X-axis. This was done to facilitate direct comparison of the mixture components with each of the single molecule candidates tested in those experiments.
• Fig. 1 shows that each of compounds C, D, 43, 50, 53, 69 and 70 inhibited glucose production, among which compound 43 was the most potent. Further, compound E was found to stimulate glucose production in HepG2 cells.
• glucose production in some cases, selenium-containing, structurally similar compounds exhibited lower or opposite effect
• Comparison of Compounds C and D indicates that 5' methyl seleno group and 5' seleno homocysteine may provide similar inhibition of glucose production.
• Comparison of Compounds 43, 50, 53, 69, and 70 indicates that diacetyl ester at 2' and 3' position (#43) provides higher inhibition of glucose production than cyclic carbonate (Compound #50), morpholino carboxylate (#53), dipropanoyl ester (#69), and dibutanoyl ester (#70).
• Adenine, adenosine and several chemical variants of adenosine did not inhibit glucose production in HepG2 cells.
• rat H4IIE liver cells can respond to metformin leading to lowered glucose production.
• this rat liver cell line was used to further confirm the inhibitory activity of Compound #43 on glucose production, and to compare the potency of Compound #43 with metformin.
• treatment with Compound #43 and metformin, respectively resulted in a dose-dependent decrease in glucose production in H4IIE cells under serum-free conditions. No toxic effect of these compounds was observed at the tested doses on cell viability (data not shown).
• the IC50 of Compound #43 was 17.8 ⁇ , which is nearly identical to its IC50 in HepG2 cells.
• metformin at a dose of 36.25 ⁇ showed little or no inhibitory activity, and the IC50 of metformin in this experiment was 275 ⁇ .
• Compounds #43, 50, 53, 69 and 70 all displayed activity in the inhibition of glucose production in cultured liver cells.
• Compound #43 was by far the most potent single compound tested with an inhibitor activity against glucose production which exceeded that of a high insulin dose (100 nM) in HepG2 cells.
• Lepr db/db mice (C57BL/6J strain) were purchased from The Jackson Laboratory (Bar Harbor, Maine), and housed in a pathogen-free vivarium with free access to chow and water.
• mice at 38 days of age were intraperitoneally (ip) injected daily with physiological saline (0.09% NaCl) containing 0.2% DMSO, Compound #C, Compound #50, Compound #68, and/or Compound #43 (25 ⁇ g selenium or sulfur equivalents of each compound per kilogram body weight, diluted in sterile physiological saline) for periods ranging from 43-90 days.
• physiological saline 0.09% NaCl
• Compound #50 Compound #68
• Compound #43 25 ⁇ g selenium or sulfur equivalents of each compound per kilogram body weight, diluted in sterile physiological saline
• Body weights of the above treated mice were recorded daily using a balance and any visible abnormal animal gross morphology and walking behavior were monitored daily. After the treatments, animals were fasted overnight and then subjected to blood glucose or HbAlc assays, glucose tolerance tests or tissue collections.
• mice After overnight fasting, 8-10-week-old Lepr db/db mice were intraperitoneally injected with physiological saline (0.09% NaCl) containing 0.2% DMSO, or Compound #43 (0.0054, 0.054, 0.54, or 5.4 mg Compound #43 (the compound stock was diluted in sterile physiological saline) per kilogram of body weight. Following the injection of saline or compound, mice were returned to their cages with free access to water but not chow for 1, 2, 3, 5 and 8 hr. At each time point after injection, a small drop of blood was collected from the tail of each mouse for glucose assay.
• Glucose tolerance tests were performed as described previously (Li et al, Int J Biol Sci 2008; 4:29-36). Briefly, overnight-fasted Lepr db/db mice after saline or compound treatments were injected intraperitoneally with 2 grams/kg body weight of 20% D-glucose. Blood glucose levels at time 0 (immediately before the injection of glucose), 0.25, 0.5, 1 and 2 hours after injection of glucose were determined using a glucometer with a maximum glucose measurement capacity of 600 mg/dL. Because of this, blood glucose levels over 600 mg/dL were counted as 600 mg/dL inthe data analysis.
• a small drop of blood from the mouse tail was collected in an EDTA-coated eppendorf tube (to prevent blood coagulation) (Fisher Scientific), and then subjected to a HbAlc assay using the Crystal Chem's or Kamiya's mouse glycated hemoglobin Ale ELISA kit, according to the manufacturer's protocol. Also after the final treatments, mouse serum was collected and subjected to HbAlc assay using the Kamiya Biomeidcal Company's mouse HbAlc kit, according to the manufacturer's protocol.
• Lepr db/db mice lack all known isoforms of the leptin receptor gene (Lepr).
• This homozygous mouse model is an aggressive Type II diabetic mouse model with impaired glucose tolerance, reduced insulin sensitivity, hyperglycemia and hyperinsulinemia. These mice display gross obesity at around 3 to 4 weeks of age, elevation of plasma insulin beginning at 10 to 14 days and hyperglycemia (i.e., high blood sugar levels) developed at about 4-8 weeks of age (Coleman DL. 1978 Diabetologia 14: 141-8).
• Compound #43 can mimic but bypass insulin to inhibit glucose production with a much greater potency than closely related compounds such as Compound #C or #50. Furthermore, the sulfur-containing analog of Compound #43 (Compound #68) had little or no inhibitory effect on glucose production in HepG2 cells (FIGS. 1-2).
• the insulin-resistant Lepr db/db mouse is an ideal in-vivo model system to investigate the use of experimental compounds in potentially lowering glucose in the bloodstream and improving insulin sensitivity and glucose tolerance against a severe diabetic background.
• mice were administered treatments daily by intraperitoneal injection of Compounds around the onset of hyperglycemia (developed at about 4-8 week after birth).
• the three compounds i.e., Compound #43, Compound #C and Compound #50, delivering identical concentrations of selenium was injected daily for 43-52 days to investigate if these seleno- organic compounds have measureable effects against hyperglycemia in the insulin-resistant mice.
• HbAlc levels in the blood samples from Lepr db/db mice after Compound #43 treatment for 3 months were also significantly decreased (by about 30%), compared to the control (saline-treated) group.
• Compound #68 treatments did not affect blood HbAlc levels in Lepr db/db mice.
• the extent of decreased HbAlc levels in Lepr db/db mice after this 90-day-treatment with Compound #43 appears to be more pronounced than in Lepr db/db mice following 42 days of Compound #43 treatment (Fig. 3).
• Lepr db/db mice display elevated plasma insulin beginning at 10 to 14 days-of-age and hyperglycemia (i.e., high blood sugar levels) at approximately 4-8 weeks of age (Coleman DL. 1978 Diabetologia 14: 141-8).
• hyperglycemia i.e., high blood sugar levels
• a single dose of Compound #43 was administered via an acute injection in younger mice.
• 6-week-old Lepr db/db male mice under normal feeding conditions were intraperitoneally injected once with saline containing 0.2% DMSO or Compound #43 at a dose of 5.4 mg/kg body weight. 24 hr after the treatment, blood glucose levels were determined on these mice while they had ad-libitum access to food and water.
• the glucose tolerance test identifies abnormalities in the way the body handles glucose after a high and rapid rise of blood sugar (e.g., usually after a meal). Insulin plays a critical role not only in the inhibition of glucose production in the liver, but also in glucose uptake, storage and metabolism in muscle, liver, and fat cells, causing lower glucose levels in the bloodstream.
• Diabetic patients have a very low glucose tolerance either due to their inability to produce insulin or to respond to insulin efficiently to maintain glucose homeostasis.
• the in vitro studies described herein indicate that Compound #43 not only can mimic insulin but also can bypass insulin to inhibit glucose production (Fig. 1-2).
• Lepr db/db mice are the ideal mouse Type II diabetic model to investigate the role of Compound #43 in maintaining glucose homeostasis, considering the fact that impaired glucose tolerance and insulin-resistance are displayed in these mutant mice. Therefore, the effect of Compound #43 and other structurally similar related selenium and sulfur compounds on improved glucose tolerance in Lepr db/db mice after intraperitoneal injection of the respective compounds into mice was investigated.
• mice were injected intraperitoneally with physiological saline (containing 0.2% DMSO), Compound #C, #43, or #50 (25 ⁇ g selenium of each compound per kilogram body weight) for 43 days.
• physiological saline containing 0.2% DMSO
• Compound #C, #43, or #50 25 ⁇ g selenium of each compound per kilogram body weight
• mice were fasted overnight, injected with glucose (2 g/kg body weight) and blood glucose levels were measured at 0.25 hours (15 minutes), 0.5 hours (30 minutes), 1 hour (60 minutes) and 2 hours (120 minutes) post-glucose injection.
• the blood glucose levels immediately before the glucose injection (referred to as the zero time point) were also recorded.
• Compound #43 at the tested doses dramatically improves glucose tolerance, and the replacement of the selenium atom in Compound #43 with sulfur almost completely destroys its ability to facilitate glucose clearance in these insulin-resistant diabetic Lepr db/db mice.
• Compound #43-treated Lepr db/db mice were also much lower than Saline-, Compound #69- or Compound #70-treated mice.
• the decrease in glucose levels in Compound #43-treated Lepr db/db mice at 2 hours after glucose injection was significantly different when compared to saline- treated mice (P ⁇ 0.05).
• the extent of the decrease of glucose levels in Compound #43-treated mice relative to the other treatments at each time point after glucose injection was likely much more dramatic than that shown in FIG. 8C. Regardless,the results further demonstrate that Compound #43 at the tested dose can dramatically improve glucose tolerance in these insulin-resistant diabetic mice.
• Compound #43 in this process is likely mediated through the improvement of insulin sensitivity in the clearance of glucose in the skeletal muscle, liver and the adipose tissues. Furthermore, while selenium is essential for the action of Compound #43 its presence is not sufficient on its own to confer glucose clearance ability on a diabetic subject. The selenium atom must be presented in a very specific chemical form. This is evidenced by the lower activity of Compound #50 and the lack of activity of Compound C; both of which are structurally very similar to Compound #43. In addition, the acetyl groups at both 2' and 3' positions of the ribose group of Compound #43 are also required for maintaining its activity in glucose clearance.
• Example 4 Inhibition of the expression of the gluconeogenic enzyme gene G6pc in the liver of diabetic leptin receptor (Lepr) spontaneous null mutant mice and in cultured liver cells after Compound #43 treatment together with the potentiation of insulin action in the inhibition of G6pc expression in cultured liver cells
• Liver is the main organ for producing glucose to maintain normal glucose levels in the blood stream.
• Glucose-6-Phosphatase Catalytic subunit (G6pc) is an essential enzyme for gluconeogenesis in the liver.
• the effect of Compound #43 on the regulation of G6pc expression was studied both in vivo and in vitro.
• mice Male Lepr db/db mice (C57BL/6J strain, purchased from The Jackson Laboratory) at 38-days of age were intraperitoneally injected daily with physiological saline (0.09% NaCl) containing 0.2% DMSO, Compound #50, or Compound #43 (25 ⁇ g selenium equivalent of each compound per kilogram body weight, diluted in the sterile physiological saline) for 52 days. After the treatment, livers were collected and subjected RNA analysis.
• HepG2 and mouse liver AML-12 cells were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). HepG2 cells were amplified in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS. AML-12 cells were amplified in Dulbecco's modified Eagle's medium and Ham's F12 (DMEM/F12) media supplemented with 10% fetal bovine serum (FBS), 40 ng/ml dexamethasone (Dex, Sigma) and IX ITS (containing 0.01 mg/ml bovine insulin, 0.0055 mg/ml human transferrin, 5 ng/ml sodium selenite) solution (Sigma).
• EMEM Eagle's Minimum Essential Medium
• DMEM/F12 Dulbecco's modified Eagle's medium and Ham's F12
• FBS fetal bovine serum
• Dex 40 ng/ml dexamethasone
• IX ITS containing 0.01 mg/ml bovine insulin
• RNA analysis of basal G6pc expression (without the presence of diabetic stimuli: 8-CPT/Dex), amplified AML-12 and HepG2 cells were cultured on 24-well plates (0.5- 2 X 10 5 cells/well) overnight in 10% FBS ITS- and Dex-free DMEM/F12 media and 10% FBS EMEM media, respectively. These cells were rinsed twice with PBS to remove residual sera. Then, the PBS-washed HepG2 cells were treated without or with insulin or Compound #43 in serum-free EMEM media for 40 hr. In some experiments, the PBS-washed AML-12 cells were incubated without or with Compound #43 or other selenium compounds in serum-free
• amplified AML-12 cells were pretreated without or with Compound #43 (150 or 300 ppb) in 10% FBS but ITS/Dex-free DMEM/F12 media for 24 hr. After 24 hr treatment, AML-12 cells were washed twice with PBS (to remove any residual serum in the culture) and then treated with insulin, Compound #43 or both, in the serum-free DMEM/Dex media for 6 hr.
• RNA analysis of the diabetic stimuli-induced G6pc expression the AML-12 cells were pretreated without or with Compound #43 (150 or 300 ppb) in 10% FBS but ITS/Dex- free DMEM/F12 media for 24 hr. Then these cells were washed twice with PBS remove any residual sera, and incubated with Compound #43 (150 or 300 ppb) in the presence or absence of insulin (10 or 100 nM), or 0.1 mM 8-CPT (Sigma) and 0.5 ⁇ Dex in serum-free plain
• RNA from saline- or selenium compound-treated Lepr db/db mice was isolated using a Qiagen RNAeasy RNA isolation kit according to the Manufacturer's protocol.
• Total RNA from cultured cells was isolated using Trizol (Invitrogen) according to the manufacturer's protocol, and then incubated with DNase I to remove any potential contaminating genomic DNA.
• RNA samples were subjected to real-time PCR (QRT-PCR) analysis using Applied-Bioscience's RT kit and predesigned Taqman probes (Invitrogen), as described previously (Lan et al EMBO J 2003). Data were normalized by Actin B (Actb) mRNA levels in each sample, and are presented as mean ⁇ SEM of 3-5 samples.
• G6pc mRNA levels in the liver was slightly, but not significantly, decreased in Lepr db/db mice after the treatment of Compound #50.
• treatment of Compound #43 resulted in a dramatic decrease (about 56% reduction) of G6pc mRNA levels in the livers of Lepr db/db mice, when compared to saline-treated controls (Fig. 9).
• mice liver AML-12 cells were treated without (Control), with compound
• AML-12 cells were pretreated with Compound #43 in serum-containing but ITS/Dex-free media for 24 hr followed by retreatment of this compound in FBS/ITS/Dex- free media in the presence or absence of insulin for 6 hr to further investigate whether
• Compound #43 can inhibit G6pc expression and whether there is additive or synergistic effect between insulin and Compound #43 in the downregulation of G6pc expression.
• treatment of 10 nM of insulin resulted in a significant decrease (about 65%) of G6pc mRNA levels when compared to Control group (1 st bar in Fig. IOC).
• treatment of Compound #43 (at both 150 and 300 ppb) also resulted in a significant decrease of G6pc expression with the decrease levels comparable to 10 nM insulin.
• Cyclic AMP (8-CPT) and Dex are well known stimuli of G6pc expression and glucose production in the liver, which mimics diabetic conditions in vivo.
• G6pc mRNA expression in AML-12 cells co- treated with cell-permeable 8-(4-chlorophenylthio) cAMP (8-CPT) and Dexamethasone (Dex) were examined.
• AML-12 liver cells were pretreated without or with 150 ppb or 300 ppb of Compound #43 in 10% FBS but ITS/Dex-free DMEM/F12 media for 24 hours.
• AML-12 liver cells treated with 8-CPT/Dex resulted in a
• Example 5 Compound #43 mimics but bypasses insulin to activate phosphoinositide-dependent protein kinase 1 (PDKl) and protein kinase B (AKT) signaling to enhance the phosphorylation of Forkhead box protein Ol (FOXOl) in the liver in vivo and in vitro
• PDKl phosphoinositide-dependent protein kinase 1
• AKT protein kinase B
• the Forkhead transcription factor FOXOl plays a critical role in metabolism, gluconeogenesis and insulin sensitivity in the liver. Intracellular activity of FOXOl is tightly regulated by post-translational modification. In particular, phosphorylation of FOXOl excludes FOXOl from the nucleus, thereby blocking its access to its target genes such as G6pc in the liver for glucose production. In insulin-resistant or diabetic individuals, there is no signal to exclude FOXOl from the nucleus, so it remains present in the nucleus and stimulates the transcription of G6pc. Increased expression of G6pc drives gluconeogenesis, leading to hyperglycemia.
• mice Male Lepr db/db mice (C57BL/6J strain, purchased from The Jackson Laboratory) at postnatal day 38 were intraperitoneally injected daily with physiological saline (0.09% NaCl) containing 0.2% DMSO, Compound #43 (25 ⁇ g selenium or sulfur equivalent of each compound per kilogram body weight, diluted in sterile physiological saline) for 52 days. After the treatment, livers were collected and stored at -80°C.
• physiological saline 0.09% NaCl
• Compound #43 25 ⁇ g selenium or sulfur equivalent of each compound per kilogram body weight, diluted in sterile physiological saline
• Frozen liver tissues were minced in sterile ice-old PBS containing complete proteinase and phosphatase inhibitors (Thermo-Fisher Scientific, Waltham, MA) and subjected to homogenization using a tissue homogenizer (Thermo-Fisher Scientific, Waltham, MA). These tissue homogenates were diluted in Themo-Fisher's premade RIPA buffer (1 part homogenate/2 part of RIPA buffer) containing complete proteinase/phosphatase inhibitors to extract the proteins. Proteins in the homogenates were extracted in RIPA buffer at 4°C overnight.
• HepG2 and mouse liver AML-12 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). HepG2 cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS. AML- 12 cells were amplified in Dulbecco's modified Eagle's medium and Ham's F12 (DMEM/F12) media supplemented with 10% fetal bovine serum (FBS), 40 ng/ml dexamethasone (Dex, Sigma) and IX ITS (containing 0.01 mg/ml bovine insulin, 0.0055 mg/ml human transferrin, 5 ng/ml sodium selenium) solution (Sigma).
• EMEM Eagle's Minimum Essential Medium
• FBS fetal bovine serum
• Dex 40 ng/ml dexamethasone
• IX ITS containing 0.01 mg/ml bovine insulin, 0.0055 mg/ml human transferrin, 5 ng/ml sodium selenium
• HepG2 cells were seeded on 6-well plates (7 X 10 5 cells/well) and cultured in 10% FBS EMEM media for 30 hr. Then these cells were washed twice with PBS to remove residual sera, and serum-starved in plain EMEM media overnight. These serum-starved HepG2 cells were treated without or with Compound #43 (600 ppb) for 0 minute (right before the treatment), 30 minutes, 60 minutes, 90 minutes , 24 hr, 30 hr and 40 hr.
• Compound #43 600 ppb
• AML-12 cells were used to investigate whether Compound #43 can regulate Pdkl/Akt/Foxol signaling molecules in the liver cells following induction with diabetic stimuli.
• Amplified AML-12 cells were seeded on 6-well (1 X 10 6 cells/well) plates and cultured in 10% FBS but ITS/Dex-free DMEM/F12 media for 24 hr. Then these cells were washed twice with PBS to remove residual sera, and serum-starved in plain DMEM/F12 media overnight.
• serum-starved AML12 cells were treated with the diabetic stimuli 8-CPT (0.1 mM) and Dex (0.5 ⁇ ) in combination, without (Control group) or with 10 nM insulin or Compound #43 (300 ppb) in serum-free plain DMEM/F12 media for 60 minutes, 90 minutes, and 6 hr, respectively.
• cultured HepG2 and AML-12 cells were rinsed twice with ice-cold PBS and lysed in ice-cold RIP A buffer containing complete proteinase and phosphatase inhibitors (Thermo-Fisher Scientific, Waltham, MA) on ice for 30 min.
• Cell ly sates were collected using a cell scraper and transfer pipette, and then centrifuged at 12000 x g for 30 min at 4°C to remove the DNA pellet and obtain the protein extract. Protein levels in the supernatant of these cell lysates were determined using the Pierce Micro-BCA protein assay kit (Thermo Scientific-Piece Biotechnology, Rockford, IL) according to the manufacturer's protocol.
• Compound #43 can reduce blood glucose and HbAlc levels and inhibit liver G6pc expression in Lepr db/db mice (Fig. 3-7, 9).
• the reduced fasting glucose levels and blood HbAlc levels are at least in part attributed to the attenuated expression of the gluconeogenic G6pc gene in the livers of Lepr mice.
• G6pc expression in the liver is controlled by the insulin signaling Pdkl/Akt/Foxol cascade.
• the application investigates whether the chronic treatment of Compound #43 can restore, at least to some extent, the insulin signaling (i.e., enhancing the phosphorylation of Pdkl/Akt/Foxo) in the livers of these insulin-resistant Lepr db/db mice.
• Compound #43 mimics but bypasses insulin to transiently activate PDK1/AKT and subsequently inactivate FOXOl in human HepG2 cells cultured in serum-free media
• Compound #43 in the liver to regulate the phosphorylation of PDK1, AKT and FOXOl, serum- starved human HepG2 cells were treated with control, and 600 ppb of Compound #43 in serum- free media for various times, ranging from 30 min to 48 hours. Treated cells were subjected to Western blot analysis.
• Compound #43 mimics but bypasses insulin to transiently activate Pdkl/Akt and subsequently inactivate Foxol in AML-12 cells cultured under simulated diabetic conditions (stimulated by both 8-CPT and Dex)
• Compound #43 can mimic but bypass insulin to inhibit 8-CPT/Dex-induced G6pc expression in AML-12 cells (Fig. 11). This effect could be due to the potential insulin-like activity of Compound #43 to inactivate Foxol in these mouse liver cells.
• the application examined the protein expression of insulin signaling molecules in AML-12 cells, cultured under simulated diabetic conditions (stimulated with 8-CPT and Dex).
• Akt and Foxol at 60 and 90 minutes, indicating that AML-12 cells cultured under simulated diabetic conditions were responsive to insulin.
• Compound #43 also significantly induced the phosphorylation of Pdkl, Akt and Foxol in these 8-CPT/Dex-treated AML-12 cells after 60 minutes of compound treatment (Fig. 14A-B).
• Fig. 14A-B At 90 minutes after compound treatment, a significant increase in pFoxol protein levels was observed in these AML-12 cells, while the protein levels of all other tested molecules including pPdkl, pAkt, Akt, and Foxol were not significantly alteredafter Compound #43 treatment (Fig. 14A,C).
• #43 can mimic but bypass insulin to transiently activate Pdk/Akt and then inactive Foxol in the liver.
• Example 6 Enhanced Glut4 (SLC2A4) expression in the livers of Lepr db/db mice and cultured liver cells, and enhanced glucose uptake in culture liver cellsafter Compound #43 treatment
• mice Male Lepr db/db mice (C57BL/6J strain, purchased from The Jackson Laboratory) at postnatal day 38 were intraperitoneally injected daily with physiological saline (0.09% NaCl) containing 0.2% DMSO, Compound #43, or Compound #50 (25 ⁇ g selenium of each compound per kilogram body weight, diluted in sterile physiological saline) for 52 days. After treatment, livers were collected and subjected to RNA analysis.
• physiological saline 0.09% NaCl
• Compound #43, or Compound #50 25 ⁇ g selenium of each compound per kilogram body weight, diluted in sterile physiological saline
• Mouse liver AML-12 cells were purchased from the American Type Culture
• DMEM/F12 Dulbecco's modified Eagle's medium and Ham's F12
• FBS fetal bovine serum
• Dex 40 ng/ml dexamethasone
• IX ITS containing 0.01 mg/ml bovine insulin, 0.0055 mg/ml human transferrin, 5 ng/ml sodium selenium
• RNA analysis of basal Glut4 (Slc2a4) expression in the absence of diabetic stimuli 8-CPT/Dex
• amplified AML-12 cells were cultured on 24-well (IX 10 5 cells/well) plates overnight in 10% FBS ITS- and Dex-free DMEM/F12 media. These cells were washed twice with PBS to remove residual sera and then were incubated with vehicle (0.024% DMSO) or with Compound #43 (300 ppb) in serum-free DMEM/F12 media for 24 hours.
• RNA analysis of Glut4 expression in AML-12 cells cultured under simulated diabetic conditions amplified AML-12 cells were cultured on 24-well (2 X 10 5 cells/well) plates in 10% FBS ITS- and Dex-free DMEM/F12 media overnight. Then these cells were washed twice with PBS to remove any potential residual sera and then were serum-starved in plain DMEM/F12 media overnight.
• RNA samples were subjected to real-time PCR analysis using Applied- Bioscience's RT kit and predesigned Taqman probes (Invitrogen), as described previously (Lan et al EMBO J 2003). Data were normalized by Actin B (Actb) mRNA levels in each sample and are presented as mean ⁇ SEM of 3-5 samples.
• Equal numbers of amplified AML-12 cells were seeded on 96 well plates (1.5 X 10 5 /well) and cultured in 10% FBS but ITS/Dex-free DMEM/F12 media overnight. Then these cells were washed twice with PBS (to remove any potential residual sera), and serum-starved in plain DMEM/F12 media overnight. These serum-starved AML-12 cells were treated without (basal), with insulin (10 and 100 nM) or Compound #43 (150, 300 and 600 ppb) in
• glucose/phenol red-free DMEM media at 37°C for 1.5 hr. After treatment, media was removed and cells were washed once with PBS and incubated with 1 mM 2-deoxyglucose (2DG) at room temperature for 30 min. The 2DG-treated cells were then subjected to glucose uptake measurement using Promega's Glucose Uptake-Glo Assay kit, according to the manufacturer's protocol. Luminescent signals were recorded using a Bio-Tek luminometer.
• mice at postnatal day 38 were intraperitoneally injected daily with physiological saline (0.09% NaCl) containing 0.2% DMSO, Compound #43, or Compound #50 (25 ⁇ g selenium equivalent of each compound per kilogram body weight, diluted in sterile physiological saline) for 52 days. After these compound treatments, livers were collected and subjected to RNA analysis of Glut4 and Actb mRNA.
• Compound #50 treatments numerically increased Glut4 mRNA levels in the livers of Lepr db/db mice (when compared to the saline-treated group).
• Glut4 expression in the liver of insulin-resistant Lepr db/db mice was due to the potential systemic effect of Compound #43 or the potential direct effect of Compound #43 on the liver tissue.
• cultured liver cells were used to examine whether Compound #43 can directly regulate Glut4 expression in the liver.
• Compound #43 mimics but bypasses insulin to enhance glucose uptake in mouse liver AML-12 cells
• Enhanced Glut4 expression suggests that Compound #43 likely can mimic, yet bypass insulin to enhance glucose uptake in the liver.
• glucose uptake experiments were conducted on mouse liver AML-12 cells. In brief, equal numbers of AML-12 cells were seeded on 96 well plates, cultured in 10% FBS but ITS/Dex-free DMEM/F12 media for 24 hr, and then serum-starved in plain DMEM/F12 media overnight. These serum-starved AML-12 cells were treated with insulin (10 and 100 nM) or Compound #43 (150, 300 and 600 ppb) in glucose/phenol red/serum-free DMEM media at 37°C for 1.5 hr.
• cells were incubated with 1 mM 2-deoxy glucose (2DG) at room temperature for 30 minutes, and then subjected to luminescence analysis using Promega's Glucose Uptake-Glo Assay kit.
• the detected luminescent signals represent the glucose uptake into the cultured cells.
• Example 7 Enhanced expression of the key downstream molecules of insulin signaling, phosphorylated Pdkl, Akt and Foxol, in the skeletal muscle of insulin-resistant Lepr db/db mice, and the cooperative action of both insulin and Compound #43 in the stimulation of glucose uptake in the differentiated C2C12 (skeletal muscle) cells
• mice Male Lepr db/db mice (C57BL/6J strain, purchased from The Jackson Laboratory) at 38 days of age were intraperitoneally injected daily with physiological saline (0.09% NaCl) containing 0.2% DMSO or 0.136 mg of Compound #43 per kilogram body weight, diluted in the sterile physiological saline) for 52 days. After the treatment, gastrocnemius skeletal muscle samples were collected and stored at -80°C. Skeletal muscle protein preparation and Western blot analysis
• Frozen skeletal muscles were minced in sterile ice-old PBS containing complete proteinase and phosphatase inhibitors (Thermo-Fisher Scientific, Waltham, MA) and subjected to homogenization using a tissue homogenizer (Thermo-Fisher Scientific, Waltham, MA). These tissue homogenates were diluted in Themo-Fisher's RTPA buffer (1 part homogenate/2 part of RIPA buffer) containing complete proteinase/phosphatase inhibitors to extract the proteins. Proteins in the homogenates were extracted in RIPA buffer at 4°C overnight.
• mouse myoblast C2C12 cells were purchased from the American Type
• C2C12 cells were amplified in DMEM media supplemented with 10% FBS. Equal number of C2C12 cells were then seeded on 96-well plates (5000 cells/well) and cultured in 10% FBS DMEM media at 37°C for 5 days. Cells were replenished daily with fresh 10% FBS DMEM media. At day 5 of culture, C2C12 cells were differentiated using 0.5% horse serum (Sigma)-containing DMEM media (differentiation media) continuing for 7 days with daily replacement of fresh differentiation media, as previously described (Misu et al, Cell Metabolism 12, 483-495, 2010).
• differentiated C2C12 cells were rinsed with PBS twice and pretreated without or with 0.006% DMSO (Compound #43 solvent) or Compound #43 (300 or 600 ppb) in serum-free glucose-free DMEM media overnight. Then these cells were washed once with PBS, and then treated without (basal) or with insulin (200 nM), Compound #43 (300 and 600 ppb), or both insulin and
• Luminescent signals were recorded using a Bio-Tek luminometer.
• skeletal muscle is the other major organ critical for glucose homeostasis in response to systemic insulin.
• Glucose uptake in skeletal muscle plays a key role in maintaining normal glucose levels in the bloodstream.
• the animal studies revealed that Compound #43 can reduce blood glucose and HbAlc levels, as well as significantly improving glucose tolerance in the insulin-resistant Lepr db/db mice (Fig. 3-8). These effects could be due to restoration of insulin signaling (i.e., Pdkl/Akt) in the skeletal muscle to stimulate glucose uptake. Therefore, it was investigated whether chronic treatment of Compound #43 could partially repair damaged insulin signaling in the skeletal muscles of these insulin-resistant
• mice at postnatal day 38 were intraperitoneally injected with saline (containing 0.2% DMSO) or Compound #43 at the dose of 0.136 mg of Compound #43 per kilogram body weight daily for 52 days. After the above treatments, skeletal muscles were collected and subjected to Western blot analysis using specific antibodies against those insulin signaling molecules.
• Insulin/Pdkl downstream signaling molecules, pAkt and pFoxol were significantly elevated in the skeletal muscles of Lepr db/db mice after treatment with Compound #43 (Fig. 18B). In contrast, there was no significant change of total Akt and Foxol protein levels in the skeletal muscles of Compound #43-treated mice. The increased phosphorylation of Pdkl and the significant increase of phosphorylated Akt and phosphorylated Foxol suggest that the insulin downstream signaling cascade is active in the skeletal muscles of Lepr db/db mice after the chronic treatment of
• Compound #43 even though Lepr db/db mice are known to be unable to respond to insulin.
• the results demonstrate that Compound #43 can restore insulin action, bypass insulin or both, to activate PI3K to induce the phosphorylation of Pdkl/Akt in skeletal muscle, thus allowing them to perform their key functions in the regulation of glucose homeostasis in these severely insulin-resistant type II diabetic mice.
• skeletal muscle In humans and other mammals, skeletal muscle normally accounts for 75% of whole body insulin-stimulated glucose uptake. Impaired ability of skeletal muscle to respond to insulin is severely disruptive to systemic glucose homeostasis. It is well documented that the process of glucose uptake in skeletal muscle is mainly mediated through the PI3K/Pdkl/Akt signaling cascade in response to insulin. The activation of PI3K/Pdkl/Akt signaling molecules in the skeletal muscle of the insulin-resistant Lepr db/db mice (Fig. 18) suggests that Compound #43 likely can directly regulate glucose uptake in the skeletal muscle and potentiate insulin action in the process. To test these possibilities, glucose uptake experiments were conducted on the differentiated mouse C2C12 (skeletal muscle) cells.
• C2C12 cells were seeded on 96-well plates (5000 cells/well), and cultured in 10% FBS DMEM media at 37°C for 5 days. These cells were differentiated using 0.5% horse serum (Sigma)-containing DMEM media for 7 days to become skeletal muscle cells, as previously described (Misu et al, Cell Metabolism 12, 483-495, 2010). The completely differentiated C2C12 cells were pretreated without or with 0.006% DMSO (Compound #43 solvent) or Compound #43 (300 or 600 ppb) in serum-free glucose-free DMEM media overnight.
• DMSO Compound #43 solvent
• Compound #43 300 or 600 ppb
• Example 8 Activation of insulin receptor (Insr) signaling in the skeletal muscle and liver of insulin-resistant Lepr db/db mice and in cultured mouse skeletal muscle and human liver cells after Compound #43 treatment and insulin-like effects of Compound #43 on the
• Insr/PDk/Pdkl/Akt signaling in response to insulin phosphorylates AS160, promoting the translocation of GLUT4 proteins from cytosolic vesicles to the plasma membrane to facilitate glucose uptake in skeletal muscle cells as well as into liver cells.
• Compound #43 to cause tyrosine phosphorylation of Insr ⁇ was investigated in skeletal muscle and liver of insulin-resistant Lepr db/db mice and in differentiated mouse C2C12 (skeletal muscle) cells as well as human liver HepG2 cells. In addition, it was investigated whether Compound #43 could mimic but bypass insulin to activate Akt, the downstream signaling molecule of insulin receptor in differentiated mouse C2C12 cells. Further, the effects of Compound #43 on the phosphorylation of the Akt target substrate, AS 160, were investigated in both differentiated mouse C2C12 and human liver HepG2 cells.
• mice Male Lepr db/db mice (C57BL/6J strain, purchased from The Jackson Laboratory) at postnatal day 38 were intraperitoneally injected daily with physiological saline (0.09% NaCl) containing 0.2% DMSO,or 0.136 mg of Compound #43 per kilogram body weight, diluted in the sterile physiological saline) for 52 days. After the treatment, gastrocnemius skeletal muscle and liver samples were collected and stored at -80°C.
• physiological saline 0.09% NaCl
• DMSO 0.136 mg of Compound #43 per kilogram body weight
• Frozen skeletal muscle and liver tissues were minced in sterile ice-old PBS containing complete proteinase and phosphatase inhibitors (Thermo-Fisher Scientific, Waltham, MA) and subjected to homogenization using a tissue homogenizer (Thermo-Fisher Scientific, Waltham, MA). These tissue homogenates were diluted in Themo-Fisher's premade RIPA buffer (1 part homogenate/2 part of RIPA buffer) containing complete proteinase/phosphatase inhibitors to extract the proteins. Proteins in the homogenates were extracted in RIPA buffer at 4°C overnight.
• HepG2 and C2C12 cells Cell culture of HepG2 and C2C12 cells, differentiation of C2C12 cells, cell treatments and preparation of cultured cell protein extracts [00364]
• the human hepatoma HepG2 cells and mouse myoblast C2C12 cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). HepG2 cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% FBS, while C2C12 cells were amplified in DMEM media supplemented with 10% FBS.
• EMEM Eagle's Minimum Essential Medium
• HepG2 cells were seeded on 6-well plates (7 X 10 5 cells/well) and cultured in 10% FBS EMEM media for 30 hr. Then these cells were washed twice with PBS to remove residual sera, and serum-starved in plain EMEM media overnight. These serum-starved HepG2 cells were treated without or with Compound #43 (600 ppb) in serum-free media for 0 minute (right before the treatment), 30 and 60 minutes.
• C2C12 cells To differentiate the C2C12 cells, equal number of these myoblast cells were firstly seeded on 12-well plates (60,000 cells/well) and cultured in 10% FBS DMEM media at 37°C for 5 days. These cells were replenished daily with fresh 10% FBS DMEM media. At day 5 after the culture, C2C12 cells were differentiated using 0.5% horse serum (Sigma)-containing DMEM media (differentiation media) for 7 days with daily replacement of fresh differentiation media, similar to previous described (Misu et al, Cell Metabolism 12, 483-495, 2010). At day 7 after the differentiation, differentiated C2C12 cells were rinsed with PBS twice and incubated in serum-free DMEM media overnight. Then these serum-starved cells were treated without (basal) or with insulin (200 nM), Compound #43 (600 ppb), or both insulin and Compound #43 in serum-free DMEM media at 37°C for 5 and 60 minutes.
• cultured HepG2 cells or differentiated C2C12 cells were rinsed twice with ice-cold PBS and lysed in ice-cold RIP A buffer containing complete proteinase and phosphatase inhibitors (Thermo-Fisher Scientific, Waltham, MA) on ice for 30 min.
• Cell lysates were collected using a cell scraper and transfer pipette, and then centrifuged at 12000 x g for 30 min at 4°C to remove the DNA pellet and obtain the protein extract. Protein levels in the supernatant of these cell lysates were determined using the Pierce Micro-BCA protein assay kit (Thermo Scientific-Piece Biotechnology, Rockford, IL) according to the manufacturer's protocol.
• Enzyme-linked immunosorbent assay (ELISA) of phospho-InsrP at Y1146 or Yl 150/51 [00369] Liver protein samples from Saline- or Compound #43 -treated Lepr db/db mice were subjected to ELISA assay using the PathScan Phospho-Insulin Receptor ⁇ (Tyrl 146 or
• the level of the internal protein control, ⁇ -tubulin, in each sample was determined by Western blot analysis using 100 ⁇ g of protein extract and a specific ⁇ -tubulin monoclonal antibody, followed by quantitative analysis of ⁇ -tubulin protein band density in the Western blot using the NIH Image J software. The OD450 of each tested sample was then normalized by its ⁇ -tubulin protein level to obtain the level of phospho-Insr at Yl 146 or Yl 150/1151.
• Compound #43 can reduce blood glucose and HbAlc levels, and improve the glucose tolerance in the insulin- resistant Lepr db/db mice (Fig. 3-8). These effects could be due to the potential restoration of insulin receptor functioning in skeletal muscle, allowing glucose uptake to occur in these insulin- resistant Lepr db/db mice.
• the enhanced phosphorylation of Pdkl and Akt in the skeletal muscle of Compound #43-treated insulin-resistant Lepr db/db mice (Fig. 18) suggest that Compound #43 may bypass but mimic insulin or restore the insulin action to activate the insulin signaling cascade molecules upstream of the Pdkl/Akt cascade in the skeletal muscle.
• Tyrosine phosphorylation of Insr ⁇ at Yl 146 reflects the first step of activated insulin receptor signaling following the binding of insulin to Insra, and this is the key event upstream of PI3K/Pdkl/Akt signaling in skeletal muscle. Therefore, it was investigated whether chronic treatment with Compound #43 could regulate the phosphorylation of Insr ⁇ in the skeletal muscle of these insulin-resistant Lepr db/db mice.
• Tyrosine 1146 but not total Insr , were much higher in the skeletal muscle of Compound #43- treated Lepr db/db mice than saline-treated mice. Quantitative analysis of these Western blots showed that phosphorylated Insr ⁇ protein levels were robustly increased (about a 2.5 fold- increase) in the skeletal muscle of Lepr db/db mice after treatment with Compound #43, when compared to saline-treated mice (Fig. 20B). In contrast, there was no significant change of total Insr ⁇ protein levels in the skeletal muscles of Compound #43-treated mice (Fig. 20C).
• Compound #43 mimics but bypasses insulin to stimulate phosphorylation of InsrP at Y1146, Pdkl, Akt, and AS160 in differentiated mouse C2C12 (skeletal muscle) cells
• Compound #43 treatment is indeed a transient event and that there exists a negative feedback to regulate Insr ⁇ tyrosine phosphorylation in skeletal muscle cells. Regardless, the enhanced tyrosine phosphorylation of Insr ⁇ at Yl 146 observed in these cultured skeletal muscle cells after the short-time (5 minutes) treatment of Compound #43 is consistent with the activation of Insr in the insulin-resistant of Lepr db/db mice described above (Fig. 20). Since these differentiated C2C12 cells were serum-starved, cultured and treated with Compound #43 under totally serum- free conditions,the results suggest that Compound #43 can mimic but bypass insulin to directly and quickly activate Insr ⁇ in these differentiated skeletal muscle cells.
• Compound #43 could mimic insulin to regulate Akt phosphorylation in these differentiated C2C12 skeletal muscle cells. As shown in Fig. 21 A, treatment of insulin for 5 minutes resulted in a significant increase of phosphorylated Akt, but not total Akt, protein levels. However, the increased phosphorylation of Akt was not observed in these differentiated C2C12 cells after 60 minutes of insulin treatment (Fig. 21C-D). In contrast, treatment of Compound #43 for 5 minutes did not cause a significant increase of phosphorylated Akt protein levels in these C2C12 cells, while a robust increased of phosphorylated Akt proteins levels was observed in these cells after the treatment of Compound #43 for 60 minutes (Fig. 21C-D).
• the results indicate that there was no synergistic action between insulin and Compound #43 in the stimulation of Akt phosphorylation in these differentiated skeletal muscle cells.
• AS 160 is an Akt target substrate and is required for keeping GLUT4 proteins in the vesicles inside the skeletal muscle cells. Phosphorylation of AS 160 at S588 in response to insulin/Insr /PI3K/Akt signaling will cause the translocation of GLUT4 proteins from cytosolic vesicles to the plasma membrane to facilitate glucose uptake.
• Compound #43 can lower blood glucose levels and improve glucose tolerance in the insulin-resistant Lepr db/db mice diabetic mice (Fig. 3-8).
• increased glucose uptake was observed following Compound #43 treatment, especially after co-treatment with both insulin and Compound #43 (Fig. 19).
• Compound #43 can regulate GLUT4 translocation in skeletal muscle cells for glucose uptake through the phosphorylation of AS 160. Therefore, the protein levels of phosphorylated AS160 at S588 were measured in the differentiated C2C12 (skeletal muscle) cells after the treatments of insulin, Compound #43, or both in serum-free media for 5 and 60 minutes.
• mice at postnatal day 38 were intraperitoneally injected with saline (containing 0.2% compound solvent DMSO) or Compound #43 at the dose of 0.136 mg of Compound #43 per kilogram body weight daily for 52 days.
• liver samples were collected and subjected to ELISA assays of phosphor-Insr ⁇ at Yl 146 and phosphor-Insr ⁇ at Yl 150/1151 (to obtain OD450) and Western blot analysis of internal control ⁇ -tubulin.
• the level of phosphor-Insr ⁇ at Yl 146 or at Yl 150/1151 in each sample was obtained after the OD450 in each sample was normalized by its ⁇ -tubulin protein level.
• Compound #43 mimics but bypasses insulin to stimulate phosphorylation of InsrP at Y1146 and AS160 at S588 in human liver HepG2 cells
• AS 160 is another AKT target substrate that plays a critical role for glucose translocation from cellular vesicles to the plasma membrane in insulin-target tissues such as adipose cells and skeletal muscle.
• the in vivo and in vitro studies showed that Compound #43 can lower blood glucose levels and improve glucose tolerance in diabetic mice (Fig. 3-8) and can enhance glucose uptake in cultured AML-12 liver cells (Fig. 17), indicating that enhanced glucose uptake in the liver could be one of the mechanisms of Compound #43 in lowering blood glucose levels and improving glucose tolerance against type I and II diabetes.
• the enhanced glucose uptake elicited after Compound #43 treatment may well result from the enhanced GLUT4 expression (indicated by Figures 15-16), the potential enhanced GLUT4 translocation, or both, to stimulate glucose uptake into liver cells. To address the latter scenario,
• phosphorylated AS 160 an AKT targeted substrate protein levels were measured in Compound #43-treated HepG2 cells.
• Enhanced phosphorylation of AS 160 promotes GLUT4 translocation from cytosolic vesicles to plasma membrane to enhance glucose uptake in both the liver and skeletal muscle cells resulting in lower blood glucose levels and improved glucose tolerance in diabetic situations.
• Compound #43 through its ability to restore the insulin signaling cascade in these tissues, could be of immense therapeutic value in the treatment of type II diabetes.
• Compound #43 can function as an insulin-mimetic in insulin-responsive cells to which no insulin has been added, there exists a strong possibility that it could be an effective treatment for type I diabetes also.
• Example 9 Chronic treatments of Compound #43 resulted in a decrease of serum insulin and alanine aminotransferase (ALT) levels but not serum creatinine levels in insulin- resistant diabetic db/db mice
• Lepr db/db mice (C57BL/6J strain) were purchased from The Jackson Laboratory (Bar Harbor, Maine), and housed in a pathogen-free vivarium with free access to chow and water. 3 -month- old wild-type (non-diabetic) C57 mice were also purchased from the Jackson Laboratory.
• mice at 38 days of age were intraperitoneally (ip) injected daily with physiological saline (0.09% NaCl) containing 0.2% DMSO, and Compound #43 (0.136 mg per kilogram body weight, diluted in sterile physiological saline) for 52 days.
• Sera from 3- month-old wild-type (non-diabetic) C57 mice were also collected. After the treatments, these serum samples were collected and subjected to insulin, ALT and creatinine assays.
• Serum insulin, ALT and creatinine assays [00395] The serum levels of insulin, ALT and creatinine were determined using Themo-
• ALT alanine aminotransferase
• PI3K/PDK1/AKT signaling cascade in both liver and skeletal muscle In other words, normal insulin signaling can be restored without a need for insulin or active cell surface insulin receptor to be present.
• activation of AKT causes a robust increase in FOXOl
• phosphorylation of the AKT target substrate AS 160 (TBC1D4), which is key for GLUT4 translocation from cytosolic vesicles to the plasma membrane, are enhanced in liver cells after Compound #43 treatment, resulting in more GLUT4 transport proteins in the liver cell membrane and improved glucose uptake from the bloodstream. All of this points to improved glucose tolerance in type I and II diabetic subjects. In skeletal muscle cells, Compound #43 can also mimic, yet bypass, insulin to activate INSRJ3/PDK1/AKT signaling, leading to phosphorylation of AS 160 (TBC1 D4).
• Compound #43 can restore insulin receptor function in the skeletal muscle of insulin-resistant diabetic mice, and can potentiate insulin action to stimulate glucose uptake in cultured skeletal muscle cells. Together, these results indicate great potential for the use of Compound #43 against type I and II diabetes in humans.
• Example 10 Direct activation of insulin receptor proteins by Compound #43 in a cell-free system
• Compound #43 in the activation of Insr which may explain the lower efficacy of Compound #68 in the inhibition of liver glucose production (Fig. 1) and against hyperglycemia in T2D mice (Fig. 4).
• Example 11 Deceased blood glucose levels in streptozotocin (STZ)-induced Type 1 diabetic (TIP) mice after acute treatment of Compound #43
• Streptozotocin (STZ) was purchased from Sigma.
• Compound #43 was synthesized in the Chemistry Laboratory of Alltech, Inc. The purity of Compound #43 was verified to be > 99%, as determined by UPLC.
• Type 1 diabetic (TID) mouse model and effects of Compound #43 on blood glucose levels in these TID mice TID mice
• mice Five-week-old C57/BL6 male mice were intraperitoneally injected with streptozotocin (STZ, 55 mg/kg mouse body weigh) daily for 5 days, and then housed in the vivarium for another 14 days for recovery. Blood glucose levels of these mice were measured using a glucometer. Those animals with a blood glucose level higher than 500 mg/dL were considered to be type I diabetic (TID). These TID mice with unfasted blood glucose levels between 500-550 mg/dL were fasted overnight and injected intraperitoneally with Compound #43 at a dose of 5.4 mg/kg body weight or physiological saline containing 2% DMSO

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