WO2021222589A1 - Traitement combiné à base de rhamnolipide et de niclosamide - Google Patents

Traitement combiné à base de rhamnolipide et de niclosamide Download PDF

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WO2021222589A1
WO2021222589A1 PCT/US2021/029925 US2021029925W WO2021222589A1 WO 2021222589 A1 WO2021222589 A1 WO 2021222589A1 US 2021029925 W US2021029925 W US 2021029925W WO 2021222589 A1 WO2021222589 A1 WO 2021222589A1
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niclosamide
rha
rhamnolipids
rhamnolipid
nash
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Anton Leighton
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Anton Leighton
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages

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  • the present invention relates to treatment of non-alcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), including liver cirrhosis, and type 2 diabetes mellitus (T2DM).
  • the method comprises the step of administering to a subject in need thereof an effective amount of rhamnolipids and an effective amount of niclosamide or niclosamide derivative.
  • BACKGROUND OF THE INVENTION Steatosis, also called fatty change, is abnormal retention of fat (lipids) within a cell or organ.
  • Nonalcoholic fatty liver disease is defined as the presence of fat in the liver (hepatic steatosis) either on imaging or on liver histology after the exclusion of secondary causes of fat accumulation in the liver (e.g., significant alcohol consumption, certain medications, and other medical conditions).
  • NAFLD is further categorized histologically into non-alcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH).
  • NAFL is defined as hepatic steatosis with no evidence of hepatocellular injury in the form of hepatocyte ballooning.
  • NASH is defined as the presence of hepatic steatosis and inflammation with hepatocyte injury (ballooning) with or without fibrosis.
  • NAFLD can progress to liver cirrhosis and liver failure.
  • Non-alcoholic fatty liver disease (NAFLD) has become one of the most common forms of chronic liver disease worldwide and its prevalence is expected to continue rising.
  • NALFD nonalcoholic steatohepatitis
  • Clinical treatment trials for NASH have yielded only modest results.
  • NAFLD has traditionally been considered a consequence of metabolic syndrome.
  • NAFLD type 2 diabetes mellitus
  • HTN hypertension
  • CVD cardiovascular disease
  • Niclosamide prevents high-fat diet-induced hepatic steatosis and hepatic insulin resistance and improves glycemic control in db/db mice.
  • Niclosamide ethanolamine-induced mild mitochondrial uncoupling improves diabetic symptoms in mice. Nat. Med. 2014
  • Translation of animal data to humans is challenging due to low oral bioavailability and increased potential for gastrointestinal adverse events associated with chronic oral niclosamide doses of greater than 1500 mg/day in humans. While liver fat content was significantly reduced by niclosamide treatment, liver fibrosis, which leads to cirrhosis, was not significantly affected in mice. Further, the mice dosed with niclosamide did not show a reduction in inflammatory cytokines.
  • NEN due to low oral bioavailability, is limited to activity in the liver, where it improves hepatic steatosis and improves hepatic insulin resistance.
  • Niclosamide ethanoiamine improves blood glycemic control and reduces hepatic steatosis in mice. Nat. Med. 2014
  • Niclosamide and niclosamide salts are crystalline solids that are sparingly soluble in aqueous solutions. In the lab setting, niclosamide is often first be dissolved in DMSQ and then diluted with an aqueous buffer, and then has a solubility of only 0.5 mg/ml. (Product information, Cayman Chemical 2021)
  • U.8. Patent No. 9,968,626 discloses methods for reducing excessive body weight, treating unwanted localized fat deposits, and treating an obesity -related condition in a subject by administering to the subject an effective amount of rhamnolipid, said obesity -related condition includes metabolic syndrome, hypertension, T2DM, kidney disease, or non- alcoholic fatty liver disease.
  • Figure 1 shows the plasma level of TGF- ⁇ 1 in C57BL6 mice with diet-induced obesity after 7 days of oral dosing of di-rhamnolipids (mean daily dose for the low dose group was 51 mg/kg and mean daily dose for the high dose group was 103 mg/kg). Placebo vs. 103 mg/kg: p ⁇ 0.001.
  • Figure 2 shows the plasma level of glucose after fasteing of the same mice treated with di-rhamnolipids as Figure 1. Placebo vs. 103 mg/kg: p ⁇ 0.05.
  • Figure 3 shows a dose-dependent significant decrease in plasma IL-1 ⁇ after 7 days of oral treatment with di-rhamnolipid in the same mice study of Figure 1.
  • Figure 4 shows a decreased in plasma TGF- ⁇ 1 level in a human subject after oral treatment of di-rhamnolipids.
  • Figure 5 shows a decreased in plasma IL-1 ⁇ level in a human subject after oral treatment of di-rhamnolipid s.
  • the reported NASH clinical trials with positive effects of drug over placebo have often shown only modest results, with a large percentage of patients who did not respond to therapy.
  • No drugs have been approved for NASH and especially for NASH with T2DM.
  • the present invention is directed to a combination therapy of rhamnolipids and niclosamide, which targets multiple pathways and improves the outcomes in higher percentages of patients in this heterogenous disorder.
  • the combination of rhamnolipids with niclosamide increases the water solubility of niclosamide and therefore increases the oral bioavailability of niclosamide.
  • the present invention is directed to a method for treating nonalcoholic fatty liver disease (NAFLD) in a subject.
  • the method comprises the steps of first identifying a subject suffering from NAFLD and administering to the subject an effective amount of rhamnolipids and an effective amount of niclosamide.
  • the present invention is directed to a method for treating NASH in a subject.
  • the method comprises the steps of first identifying a subject suffering from NASH and administering to the subject an effective amount of rhamno!ipids and an effective amount of niclosamide.
  • the present invention is directed to a method for treating T2DM in a subject.
  • the method comprises the steps of first identifying a subject suffering from T2DM and administering to the subject an effective amount of rhamnolipids and an effective amount of niclosamide.
  • the subject further suffers from NASH or NAFLD.
  • An effective amount of rhamnolipids and niclosamide, is an amount effective to treat a disease by ameliorating the condition or reducing the symptoms of the disease.
  • Niclosamide sold under the brand name Niclocide among others, is a medication used to treat tapeworm infestations and has an excellent safety profile. It is taken by mouth.
  • Niclosamide has a molar mass: 327.119 g/mol; Formula: C13H8C12N204.
  • Niclosamide useful for the present invention including niclosamide or a salt thereof, or a derivative thereof.
  • useful niclosamide includes niclosamide ethanolamine, or niclosamide and/or niclosamide salt contained in lipid or other nanoparticles, or in emulsions.
  • Rhamnolipids are biosurfactants containing glycosyl sugar molecules and b- hydroxyalkanoic acids.
  • Rhamnolipids suitable to be used in the present invention include natural rhamnolipids, for example, obtained from Pseudomonas aeruginosa; rhamnolipids produced by any Pseudomonad, including P, chlororaphis, Burkholdera pseudomallei, Burkholdera (Pseudomonas) plantarii, and any recombinant Pseudomonad.
  • Suitable rhamnolipids also include those produced by other bacteria or by plants either naturally or through (genetic) manipulation.
  • Suitable rhamnolipids further include rhamnolipids and their analogs prepared by chemical synthesis or expression by mammalian cells.
  • Suitable rhamnolipids include those disclosed in U.S. Patent Nos. 7,262,171 and 5,514,661, in which the structures of rhamnolipids are incorporated herein by reference.
  • Suitable rhamnolipid formulations contain one or more rhamnolipids of formula (I) wherein:
  • R 1 H, unsubstituted ⁇ -rhamnopyranosyl, ⁇ -rhamnopyranosyl substituted at the 2 position with a group of formula ( )
  • R 3 and R 4 are saturated or mono or polyunsaturated alkyl
  • R 5 and R 6 are alkyl
  • Alkyl refers to groups of from 1 to 12 carbon atoms, either straight chained or branched, preferably from 1 to 8 carbon atoms, or 1 to 6 carbon atoms.
  • R 1 H, unsubstituted ⁇ -rhamnopyranosyl
  • R 1 H or unsubstituted ⁇ -rhamnopyranosyl
  • Useful rhamnolipids of the Formula 1 include ⁇ -rhamnopyranosy(i,2)- ⁇ - ramnopyranosyl)-3-hydroxydecanoyl-3-hydroxydecanoic acid (di-rhamnolipid, Rha-Rha- C10-C10) and has the following structure (Formula 2):
  • Some common mono-rbamnolipids useful for this invention include: Rha-C8-C8; Rha-C8-C10; Rha-C10-C8; Rha-C12:l-C10; Rha-C10-C12:l.
  • L-rhamnopyranosyl- L-rhamnopyranosyl- ⁇ -hydroxydecanoyl-p -hydroxydodecanoate Rha-Rha-Cl 0-C12
  • L- rhamnopyranosyl-L-rhamnopyranosyl-b -hydroxydodecanoyl- ⁇ -hydroxydecanoate Rha-Rha- C 12-C 10
  • Rha-Rha-C8-C 10 Rha-Rha-C 10-C8 ; Rha-Rha-C 12:1 -C 10; Rha-Rha-C 10-C 12:1
  • L-rhamnopyranosyl-L-rhamnopyranosyl- ⁇ -hydroxytetradecanoyl- ⁇ - by droxytetradecanoate Rha-Rba-C 14-C 14
  • Preferred rhamnolipids are L-rhanmosyi-P-hydroxydecanoyl- ⁇ -hydroxydeeanoate (mono-rhamnolipid, Rha-C10-C10) and L ⁇ rhamnosyl-L ⁇ rhamnosyl- ⁇ -hydroxydecanoyl- ⁇ - hy droxy decan oate (di-rbamnolipid, Rha-Rha-C10-C10), and the mixture thereof.
  • rhamnolipids increases the solubility of niclosamide in an aqueous solution. Increased solubility is an important factor in improving oral and systemic bioavailability of niclosamide.
  • the inventor also discovered that rhamnolipids in solution with niclosamide decrease the particle size of suspended niclosamide. Reduction in particle size of a sparingly soluble material results in an increased rate of solution. (Bucton G, et al. International J of Pharmaceutics, 1992: 82: Volume 82, Issue 3, 25 May 1992, Pages R7-R10.) Therefore, by decreasing particle size of niclosamide in a solution, rhamnolipids further increases the oral and systemic bioavailability of niclosamide.
  • rhamnolipids Due to the increased bioavailability of niclosamide when dosed with rhamnolipids, the combination administration ofrhamnolipid and niclosamide in humans results in both tolerable (maximally 2000 mg/day) and efficacious niclosamide treatment.
  • the inventor has established that oral doses of rhamnolipid daily for 7 or more days, at levels that are systemically active and increase permeability across tight junctions, are well tolerated. Therefore, rhamnolipids can be administered daily at effective doses of tablets and capsules that increase niclosamide bioavai lability without adversely affecting gut wall integrity.
  • Resolution of steatohepatitis on overall histopathological reading and no worsening of liver fibrosis on NASH CRN fibrosis score is defined as absent fatty liver disease or isolated or simple steatosis without steatohepatitis and a NAS score of 0-1 for inflammation, 0 for ballooning, and any value for steatosis; or
  • liver fibrosis greater than or equal to one stage (NASH CRN fibrosis score) and no worsening of steatohepatitis (defined as no increase in NAS for ballooning, inflammation, or steatosis); or
  • the present combination therapy provides a significant improvement on both fibrosis and steatohepatitis due to the following reasons.
  • Niclosamide demonstrates a significant effect on liver fat content without effect on fibrosis, whereas rhamnolipids significantly improve liver fibrosis, and to a lesser extent steatohepatitis.
  • the combination of niclosamide and rhamnolipid improves both steatohepatitis and fibrosis in nonalcoholic fatty liver disease.
  • the combination therapy increases number of functional liver cells, reduces the progression to NASH, cirrhosis and reduces the progression to hepatocellular cancer (HCC).
  • Rhamnolipids and niclosamide modulate both inter-dependent and independent pathways to block inflammatory pathways associated with disease progression in both NAFLD/NASH and T2DM.
  • HNF4 ⁇ Rhamnolipids increase expression of HNF4 ⁇ , which corrects phenotype of cultured end-stage cirrhotic hepatocytes and quickly reverses terminal end-stage liver failure in a mouse model.
  • HNF4 ⁇ prevents steatosis by modulating lipolysis, ER stress, and lipogenesis. protects against progression of NASH.
  • TGF- ⁇ 1 is a driver of fibrosis.
  • Niclosamide reduces STAT3, which is a key regulator of liver fibrosis.
  • Rhamnolipids significantly reduce IL-1 ⁇ .
  • IL-1 ⁇ deficiency reduces adiposity, glucose intolerance and hepatic de-novo lipogenesis in diet-induced obese mice.
  • IL-1 ⁇ -mediated deterioration of insulin signaling is largely due to the IL-6 production and SOCS3 induction in 3T3-L1 adipocytes.
  • the inventor has shown that rhamnolipids significantly reduce plasma IL-1 ⁇ level in diet-induced obese mice, and thus reducing adiposity, glucose intolerance and hepatic de-novo lipogenesis in diet-induced obese mice.
  • mTORC1 Mammalian target of rapamycin complex 1 (mTORC1) plays a critical role in coupling nutrient sensing to anabolic and catabolic processes. Type 2 diabetes gives rise to greater mTORC 1 activity. If mTORC1 remains chronically overactivated, pancreatic beta cell death occurs and insulin secretion compromised, leading to decreased glycemic control.
  • Niclosamide inhibits mTORC1 and improves hepatic insulin resistance. However, niclosamide is not effective as a systemic mTORC1 inhibitor without increased oral bioavailability.
  • Niclosamide improves hepatic insulin resistance and hepatic steatosis.
  • Rhamnolipids reduce plasma cytokines, including TGF- ⁇ and IL-1 ⁇ , associated with liver fibrosis and increased insulin resistance.
  • the combination therapy of niclosamide with rhamnolipids increases the oral bioavailability of niclosamide and improves the efficacy of treatment T2DM, NA FLD and NA SH.
  • Body weight loss of >5% in overweight and obese patients is associated with significant improvement of T2DM, NAFLD and NASH.
  • Niclosamide increases calorie burning and rhamnolipids decrease food intake.
  • Rbamnolipids and niclosamide can be prepared in two separate pharmaceutical formulations or they can be mixed together as one pharmaceutical formulation.
  • the pharmaceutical composition comprises a pharmaceutically acceptable carrier and is in a form of a liquid, a solid, a semi-solid, or a suspension in rhamnolipid.
  • Effective and safe doses of rbamnolipids are between 10 and 1000 mg a day, or 30 and 500 mg a day, or 50 and 300 mg per day.
  • Effective and safe doses of niclosamide in combination with rbamnolipids are between 500 and 2000 mg, preferably between 1000 and 2000 mg per day.
  • the weight ratio of rhamnolipid to niclosamide is in general 1:2 to 1:100, preferably 1 :2 to 1 :50, or 1 :5 to 1 :20.
  • the daily doses of rhamnolipid and niclosamide or niclosamide salt are given maximally 1000:2000 mg (rhamnolipidmiclosamide), which can be given once or twice daily.
  • the daily doses are 200:1000, 100:750, 100:500 mg, 50:500 mg, or 20- 40:400 mg (rhamnolipidmiclosamide), which may be given once, or 2 - 4 times per day.
  • Pharmaceutically acceptable carriers can be selected by those skilled in the art using conventional criteria.
  • Pharmaceutically acceptable carriers include, but are not limited to, sterile water or saline solution, aqueous electrolyte solutions, isotonicity modifiers, water polyethers such as polyethylene glycol, polyvinyls such as polyvinyl alcohol and povidone, cellulose derivatives such as methylcellulose and hydroxypropyl methyl cellulose, polymers of acrylic acid such as carboxypolymethylene gel, nanoparticles, polysaccharides such as dextrans, and glycosarninoglycans such as sodium hyaluronate and salts such as sodium chloride and potassium chloride.
  • the pharmaceutical composition of the present invention provides an aqueous solution comprising water, rbamnolipids and niclosamide; the composition optionally comprises suitable ionic or non-ionic tonicity modifiers and suitable buffering agents.
  • the rhamnolipid is at 0.005-20% (w/w)
  • the aqueous solution has a tonicity of 200-400 mOsm/kG and a pH of 4-9.
  • the pharmaceutical composition is preferably formulated to have a pH between 4.5-8, more preferably 5-7.4.
  • the pharmaceutical composition may optionally contain a buffer to facilitate a stable pH of 5-7.4.
  • the pharmaceutical composition optionally contains non-ionic tonicity agents such as mannitol, sucrose, dextrose, glycerol, polyethylene glycol, propylene glycol, or ionic tonicity agent such as sodium chloride.
  • the pharmaceutical composition can further contain ionic or non-ionic surfactants, bile salts, phospholipids, cyclodextrins, micelles, liposomes, emulsions, polymeric microspheres, nanoparticles, other biodegradable microsphere technology, deoxycholic acid or their combination.
  • the pharmaceutical composition is in a dosage form such as tablets, capsules, granules, fine granules, powders, syrups, injectable solutions, or the like.
  • a tablet formulation or a capsule formulation may contain other excipients that have no bioactivity and no reaction with rhamnolipids or niclosamide.
  • Excipients of a tablet may include fillers, binders, lubricants and glidants, disintegrators, wetting agents, and release rate modifiers.
  • Binders promote the adhesion of particles of the formulation and are important for a tablet formulation. Examples of binders include, but not limited to, carboxymethylcellulose, cellulose, ethylcellulose, hydroxypropylmethylcellulose, methylcellulose, karaya gum, starch, starch, and tragacanth gum, poly(acrylic acid), and polyvinylpyrrolidone.
  • a tablet or capsule formulation may contain as active pharmaceutical ingredients 10-90% of rhamnolipids and the rest may contain niclosamide.
  • the pharmaceutical composition comprises one or more rhamnolipids and niclosamide imbedded in a solid or semi-solid matrix, and is in a liquid, solid, or semi-solid form.
  • the pharmaceutical composition can be injected subcutaneously to a subject and then the active ingredients slowly released in the subject.
  • the pharmaceutical composition comprises one or more rhamnolipids in one chamber of a dual chamber syringe and a niclosamide solution is contained in a second chamber. The rhamnolipids and/or niclosamide are imbedded in a semi-solid matrix, in a liquid or semi-solid form.
  • the pharmaceutical composition can be injected subcutaneously to a subject and then the active ingredients slowly released in the subject.
  • a hygroscopic inactive agent is added to a tablet or capsule to provide an oral solid dosage form suitable for oral administration.
  • This formulation has either a semi-permeable membrane and/or at least one passageway in the semi-permeable membrane so that water enters the capsule or tablet and the rhanmoiipid and subsequently the niclosamide or niclosamide salt is subsequently released.
  • an emulsion of rhamnolipid and niclosamide is administered in a capsule.
  • a solid of semisolid matrix containing rhanmoiipid, niclosamide and one or more hygroscopic agents is administered in a capsule, tablet or other form.
  • the pharmaceutical compositions of the present invention can be prepared by aseptic technique. The purity levels of all materials used in the preparation preferably exceed 90%.
  • the pharmaceutical composition of the present invention is applied by systemic administration to a subject.
  • Systemic administration includes oral, intranasal, subcutaneous, percutaneous, or intravenous administration.
  • Oral and subcutaneous administration are the preferred routes of administration for the present invention.
  • the pharmaceutical composition is applied once, twice, three or four times daily.
  • the present invention is useful in treating a mammalian subject, such as humans, dogs and eats.
  • the present invention is particularly useful in treating humans.
  • EXAMPLE 1 Oral Treatment of rhamnolipids reduces IL-1 ⁇ , TGF- ⁇ 1 and fasting glucose levels in blood of treated mice.
  • mice Diet-induced obese 14 weeks old, male C57BL/6 mice received daily oral administration of rhamnolipids. Body weight and overall health were monitored daily. Three groups of 8-9 mice received treatments for a total of 7 consecutive days.
  • Di-rhamnolipid (DR) was added to water to produce a solution of 8 mg/ml and mixed in a 300ml plastic bottle and stored in 4°C refrigerator. The suspension was sonicated to prepare a homogenous suspension before each treatment.
  • TGF- ⁇ and IL-1 ⁇ plasma levels were measured by Eve Technologies Corporation, Calgary, AB. Canada) using TGF- ⁇ Multiplex Immunoassay. TGF- ⁇ 1 and IL-1 ⁇ were chosen because are elevated in both obesity, type 2 diabetes mellitus, NAFLD and NASH.
  • Figures 1 and 2 show a dose-dependent decrease in plasma TGF- ⁇ 1 and fasting glucose by oral treatment of DR.
  • a decrease in TGF- ⁇ I signaling in obese animals and humans has been associated with improvement in T2DM and NAFLD and NASH.
  • Figure 3 shows a dose dependent significant decrease in plasma IL-1 ⁇ after 7 days of oral treatment with di-rhamno!ipid.
  • the objective of this example was to explore if rhamnolipids would generate a similar cytokine profile as in rodents. These cytokines are key to efficacy in T2DM, obesity, NAFLD and NASH.
  • mice The effects of oral rhamnolipids on cytokines were explored in order to estimate the human dose of rhamnolipid required to significantly reduce plasma cytokines associated with progression ofT2DM, obesity, NAFLD and NASH.
  • the standard conversion rate of mice to human dose is 12.3, i.e., the mouse dose is divided by 12.3 to estimate the probable human dose equivalent.
  • Di-rbamnolipid C10-C10 (>97% pure) was procured from Prof. Hausmann, Düsseldorf Institute of Technology, University' of Düsseldorf, Germany.
  • Oral doses were prepared by filling standard gelatin capsules, each containing 100 mg of di-rhamno!ipids. Three capsules (300 mg) were ingested on day 1, 3 capsules (total of 300mg) were swallowed on day 2, and 4 capsules (containing a total of 400mg) were ingested on day 3, for a total ingested dose of 1000 mg within the 3 -day dosing period.
  • the daily doses were ingested with a glass of water (about 240 ml). Blood draws were taken 4 days prior to first dose, on day of last dose, and 7 days post-last dose. Plasma samples were prepared and sent on dry ice to and analyzed by Eve Technologies (Eve Technologies Corporation, Calgaiy, AB. Canada) using their standard human Multiplex Immunoassay. Each sample analysis was done in triplicate.
  • TGF-beta is increased in T2DM, obesity, NAFLD and NASH. NASH-induced scarring depends on TGF-beta.
  • the plasma TGF- ⁇ profile indicates that the rhamnolipid total dose given in the three days prior to post-dose assessment reduced TGF- ⁇ for at least 7 days.
  • the total dose given within that timeframe supports daily dosing of rhamnolipid at 100 mg per day.
  • IL-1 ⁇ deficiency reduces adiposity', glucose intolerance and hepatic de-novo hpogenesis in diet-induced obese mice. (Almog T, et al . BMJ Open Diabetes Res Care 2019.) www.ncbi .nlffi .nih.gov/pmc/artides/PMC6827792/
  • the plasma IL-1 ⁇ profile indicates that the rhamnolipid total dose given in the three days prior to post-dose assessment reduced IL-1 ⁇ for at least 7 days.
  • the total dose given within that timeframe (the three days of dosing and 7-day plasma value) supports daily dosing of rhamnolipid at 100 mg per day.
  • the error bars depict standard deviations computed by using the results of the triplicate sample analyses.
  • IL-1 ⁇ deficiency reduces adiposity, glucose intolerance and hepatic de-novo lipogenesis in diet-induced obese mice. ( Almog T, et al.
  • EXAMPLE 3 Assessment of effect of rhamnolipids on solubility and particle size of niclosamide ethanolamine (NEN) in water
  • Rhamnolipids Highly purified di-rhamnolipid C10-C10 (AGAE Technologies, USA) was used.
  • Niclosamide niclosamide (ethanolamine salt). Acquired from Cayman Chemical (Cayman Chemical, 1180 East Ellsworth Road, Ann Arbor, Michigan 48108, USA) is used.
  • Niclosamide ethanolamine salt (niclosamide) was dissolved and titrated (2-fold) in methanol to generate standard curves.
  • the linear range of niclosamide quantification using UV spectrometry ( 33nm) was established to be 0.006-0.1 m.M.
  • Niclosamide solubility m the presence of rhamnolipid.
  • a suspension of niclosamide was prepared in H2O at a concentration of 32.7mg/ml (100mM) by vigorous vortexing.
  • a stock solution of rhamnolipids were prepared in H 2 O at a concentration of 400mg/mi and diluted appropriately prior to the addition of niclosamide.
  • Niclosamide was added to a final concentration of 10mM.
  • the solutions were vortexed vigorously, warmed at 37°C for 5 minutes, vortexed vigorously then centrifuged at 17,000 RCF for 5 minutes to remove any large particulates.
  • the supernatant of each sample was assayed for soluble niclosamide by diluting 1 : 10 to 1 : 100 in methanol and comparing to a niclosamide stand curve.
  • samples without niclosamide were prepared and used as controls.
  • niclosamide was poorly soluble in H 2 O and was readily pelleted upon centrifugation.
  • Niclosamide was present in solution (1.06 m M) after centrifugation as determined by UV spectroscopy (see Table 1 below).
  • Rhamnolipids increased the solubility of niclosamide in a concentration dependent manner. The highest concentration of rhamnolipid, 200mg/ml, increased niclosamide solubility by 253% (3.74 mM) compared to water alone.
  • Oral bioavailability is dependent on solubility and particle size.
  • Table 1 shows that the solubility of niclosamide was increased and particle size was decreased by including rhamnolipid in the solutions.
  • rhamnolipid and niclosamide An oral formulation of rhamnolipid and niclosamide is feasible.
  • the addition of rhamnolipid enables niclosamide to be sufficiently orally bioavai!able.
  • the objectives of this study are to determine the extent of improvement in both fat content and fibrosis in an established mouse model of NASH and NASH cirrhosis.
  • the study is designed to confirm that the impact of the combination treatment provides improvement in glycemic control and reduction in body weight far in excess to rhamnolipid alone since co-administration of rhamnolipid facilitates effective hepatic and systemic concentrations of niclosamide at tolerable doses of up to 2g.
  • Niclosamide has been shown to improve steatohepatitis, but does not affect degree of fibrosis, and effects were limited to the liver due to the low oral bioavailability of niclosamide. (Tao H, et al. Nat Med. : 1263-1269.)
  • Rhamnolipids have been shown to significantly reduce fibrosis in rodent N ASH models.
  • the objective of this study is to demonstrate a significant effect of the combination of niclosamide and rhamnolipid to improve both steatohepatitis and fibrosis in nonalcoholic fatty liver disease and progression to hepatocellular cancer (HCC).
  • a further objective of the study is to demonstrate that the combination provides glycemic control in this murine model diabetic model of liver disease.
  • Di-rhamnolipids (DB 1-500; Di-rhamno!ipid C10-C10) is obtained from AGAE Technologies.
  • Niclosamide ethanolamine salt
  • Cayman Chemical Cayman Chemical, 1180 East Ellsworth Road, Ann Arbor, Michigan 48108, USA
  • SMC SMC’s proprietary SEAMTM model is a model that recapitulates the same disease progression as human NASH/HCC.
  • male C57BL/6 mice aged two days are given a single dose of streptozotocin to reduce insulin secretory capacity.
  • streptozotocin to reduce insulin secretory capacity.
  • This model has a background of late type 2 diabetes which progresses into fatty liver, NASH, fibrosis and consequently liver cancer (HCC).
  • mice are all fed a high fat diet.
  • mice Female C57BL/6J mice (Japan SLC, Inc,, Japan) are receive a single subcutaneous injection of 200 ⁇ g streptozotocin (Sigma- Aldrich Co. LLC., USA) solution 2 days after birth, followed by feeding with a high fat diet (HFD, 57 kcal% fat, cat#: HFD32, CLEA Japan, Inc., Japan) starting at 4 weeks of age.
  • HFD high fat diet
  • mice are treated for 4 weeks with vehicle (0.5% methylcellulose) OCA (10 mg/kg) or EDP-305 (3 or 10 mg/kg), administered in the high fat diet.
  • Liver samples are collected for histological analysis and NAS evaluation.
  • Di-rhamnolipid is added to water to produce a solution of 8mg/ml and mixed in a 300ml plastic bottle and stored in 4°C refrigerator. The suspension is sonicated to prepare a homogenous suspension before each treatment.
  • Niclosamide ethanolamine at 40mg/kg is added to the rhamnolipid solution, heated to 40 degrees C, and vortexed for 60 minutes.
  • mice are manually restrained and administered a volume of prepared dosing solution and sediment corresponding with the following doses of di-rhamnolipid C10-C10 and niclosamide (Table 2). Doses are administered by gavage twice (BID) or 3 times (TID) daily.
  • mice are terminated at week 92-3 hours post last dosing per gavage in order to assess PK values of niclosamide.
  • a p-value less than 0.05 was considered statistically significant. Histological assessments of liver tissue, clinical and clinical labs and hematology will be compared across dose groups and assessment time points.
  • EXAMPLE 5 Combination of rhamnolipids and niclosamide in the treatment of NASH in diabetic humans (prophetic example)
  • the primary objective is to identify a dose regimen that increases oral bioavailability of niclosamide in combination with rhamnolipid that is well-tolerated, improves glycemie control, and clinical and laboratory parameters of NASH.
  • This study is to assess single ascending doses of rhamnolipid and niclosamide and multiple dosing of rhamnolipid and niclosamide combination capsules in regard to their pharmacokinetic, tolerability and pharmacodynamics.
  • Part A Single Ascending dose study
  • Dose groups will be dosed sequentially. Placebo subjects will be added to each Dose Group (DG).
  • DG Dose Group
  • DG1 10 subjects receive niclosamide 1g BID: and 2 receive matching placebo
  • DG 2 10 subjects receive niclosamide 1g BE) + 100 mg rhamnolipid BE); and 2 subjects receive matching placebo
  • DG 3 10 subjects receive niclosamide 650 mg + 50 mg rhamnolipid ; 2 subjects receive matching placebo
  • DG 4 10 subjects receive niclosamide 650 mg + 100 mg rhamnolipid ; 2 subjects receive matching placebo Part B: Multiple ascending dose study
  • the objective of study Part B is to identify two dose regimens evaluated in Part A that demonstrate increases in oral bioavailability of a combination of niclosamide and rhamnolipid, are well -tolerated, and do not show any adverse effects or dose limiting toxicity .
  • Dosing is for 12 weeks. Each subject will continue to receive their standard medications.
  • Liver Multi Scan imaging software is used for characterizing liver tissue at baseline and at study end. It is designed to be used with MRI to help clinicians to diagnose and stage liver disease. MRI is sensitive to subtle differences in tissue composition. It can scan the entire liver to provide measurements to help in the diagnosis and management of liver disease. No contrast agent is needed.
  • Rhamnolipids and niclosamide will be used as developed using FDA-approved CMC procedures and prepared in accordance with the IND.
  • Biochemistry Fasting blood glucose, ALT, liver triglyceride, blood triglycerides and cholesterol.
  • H1c and OGTT ora! glucose tolerance test
  • a p-value less than 0.05 was considered statistically significant. Histological assessments of liver tissue, clinical and clinical labs and hematology will be compared across dose groups and assessment time points.

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  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
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Abstract

La présente invention concerne le traitement de la maladie du foie gras non alcoolique (NAFLD), de la stéatohépatite non alcoolique (NASH), notamment de la cirrhose du foie, et du diabète sucré de type 2 (T2DM). Le procédé comprend l'étape d'administration à un sujet qui en a besoin d'une quantité efficace de rhamnolipides et d'une quantité efficace de niclosamide ou d'un dérivé de niclosamide.
PCT/US2021/029925 2020-04-29 2021-04-29 Traitement combiné à base de rhamnolipide et de niclosamide WO2021222589A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130310330A1 (en) * 2011-06-21 2013-11-21 Rhamnopharma Inc. Method for treating obesity
US20190083638A1 (en) * 2017-07-25 2019-03-21 Duke University Niclosamide-conjugated polypeptide nanoparticles
WO2020247808A1 (fr) * 2019-06-07 2020-12-10 Dale Biotech, Llc Procédé de traitement de la maladie de dupuytren

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130310330A1 (en) * 2011-06-21 2013-11-21 Rhamnopharma Inc. Method for treating obesity
US20190083638A1 (en) * 2017-07-25 2019-03-21 Duke University Niclosamide-conjugated polypeptide nanoparticles
WO2020247808A1 (fr) * 2019-06-07 2020-12-10 Dale Biotech, Llc Procédé de traitement de la maladie de dupuytren

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE PUBCHEM SUBSTANCE 1 November 2019 (2019-11-01), ANONYMOUS : "SUBSTANCE RECORD FOR SID 386518225", XP055870481, Database accession no. SID 386518225 *

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