WO2021222589A1

WO2021222589A1 - Combination treatment of rhamnolipid and niclosamide

Info

Publication number: WO2021222589A1
Application number: PCT/US2021/029925
Authority: WO
Inventors: Anton Leighton
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-04-29
Filing date: 2021-04-29
Publication date: 2021-11-04
Anticipated expiration: 2022-10-29

Abstract

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.

Description

COMBINATION TREATMENT OF RHAMNOLIPID AND NICLOSAMIDE FIELD OF THE INVENTION 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. Steatosis most often affects the liver – the primary organ of lipid metabolism – where the condition is commonly referred to as fatty liver disease. Nonalcoholic fatty liver disease (NAFLD) 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. As a consequence of the natural history of NALFD, nonalcoholic steatohepatitis (NASH) likewise is increasing in prevalence. Clinical treatment trials for NASH have yielded only modest results. NAFLD has traditionally been considered a consequence of metabolic syndrome. However, the link between NAFLD and metabolic syndrome components, especially type 2 diabetes mellitus (T2DM), hypertension (HTN), and cardiovascular disease (CVD) is more complex than previously thought. A large body of clinical evidence now suggesting that NAFLD may actually precede and/or promote the development of T2DM and cardiovascular disease. The risk of developing these cardiometabolic diseases parallels the underlying severity of NAFLD. Moreover, long-term prospective studies indicate that the presence and severity of NAFLD independently predicts fatal and nonfatal CVD events. (Lonardo A, et al. Hypertension, diabetes, atherosclerosis and NASH: Cause or consequence? J of Hepatology 2018.) www.sciencedirect.com/science/article/abs/pii/S0168B27817323358

Up to 80% of NAFLD patients with T2DM have NASH and the presence of T2DM is an independent risk factor for more-advanced fibrosis, higher rate of fibrosis progression, and increased mortality. Liver-related mortality is increased 10-fold in NASH patients compared with the general population. (Mikolasevic I, et al. Eur. J of Intern. Med. 2020, 82:68-75.)

There is a growing consensus that NASH results from excess free fatty acids generated from lipolysis and de novo lipogenesis in the liver, with subsequent production of lipotoxic species which result in endoplasmic reticulum and oxidant stress, inflammasome activation and fibrogenesis (Friedman et. al. Nature Medicine 2018, 24:908-922). Multiple additional factors are involved in exacerbating the pathophysiology such as changes in the microbiome, liver delivery of gut-derived substances via intestinal permeability, and immune cell mechanisms. This complexity results in heterogeneity of disease phenotypes in humans and strongly implies that targeting multiple drivers of these diseases and disease combinations is warranted.

It is generally recognized that there is a need for cost effective oral therapies that address the multiple complex proinflammatory processes that drive progression of NAFLD to NASH, and the associated decreased glycemic control in T2DM and concomitant increase in cardiovascular complications.

Notably, there are currently no established pharmacotherapies for NASH patients with T2DM. These patients exhibit increased liver inflammation and fibrosis. TGF-β is increased, and in interaction with STAT3 in hepatic stellate cells exacerbates liver injury and fibrosis.

Niclosamide inhibits STATS (IC5Q= 0.25 μM) and stimulates autophagy by reversibly inhibiting mammalian target of rapamycin complex 1 (mTORC1) signaling.

(Balgi, AT)., et al. Screen for chemical modulators of autophagy reveals novel therapeutic inhibitors of mTORC I signaling. PLoS One 2009.) (Ren, X, et al, ACS Med. Chem. Lett. 2010, 1 :454-459)

Niclosamide prevents high-fat diet-induced hepatic steatosis and hepatic insulin resistance and improves glycemic control in db/db mice. (Tao, H, et al. 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. The authors conclude that NEN, due to low oral bioavailability, is limited to activity in the liver, where it improves hepatic steatosis and improves hepatic insulin resistance. (Tao H , et al. 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)

In a PK study performed in prostate cancer patients, plasma levels after a single oral dose of 1,000 mg achieved a maximal plasma concentration of only 182 ng/mL (0.56 μM). The study was prematurely terminated since the maximal daily tolerated dose was only 1500 mg and effective therapeutic plasma levels could not be achieved. (Schweizer MT, et al. (2018) PLoS ONE 13(6): e0198389. https://doi.org/10.1371/jouraal.pone.0198389)

Colorectal cancer patients received 2,000 mg of niclosamide orally once per day. Median plasma Cmax was only 759 ng/ml (2.32 μM). (Burock et ah, BMC Cancer (2018) 18:297).

Niclosamide had limited and transient plasma levels in mice. A gavage of 40 mg/kg of niclosamide led to peak plasma concentrations of only -400-900 ng/ml (-1-2.5 μM). Niclosamide had a half-life of only 1.5 hours. In a model of obesity, T2DM and NASH, mice were continuously dosed by mixing niclosamide into the chow, with an approximate daily dose of 125mg/kg of NEN. (Tao H, et al. Niclosamide ethanoiamine improves blood glycemic control and reduces hepatic steatosis in mice. Nat. Mod. 2014). After oral intake in humans, a median Cmax plasma level of only 0.665 μg/ml was achieved. htps://ascopubs.org/doi/abs/10, 1200/JCO.2018,36. 15 suppl .el 4536

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. There is a need for an efficient and cost-effective formulation of niclosamide that increases ora! bioavailability and tolerability in humans. There is a need for an effective method for treating NAFLD, NASH, and type 2 diabetes mellitus (T2DM), with minimal side effects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the plasma level of TGF-β1 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-1α after 7 days of oral treatment with di-rhamnolipid in the same mice study of Figure 1. Placebo vs. 103 mg/kg: p< 0.01.

Figure 4 shows a decreased in plasma TGF-β1 level in a human subject after oral treatment of di-rhamnolipids.

Figure 5 shows a decreased in plasma IL-1α level in a human subject after oral treatment of di-rhamnolipid s.

DETAILED DESCRIPTION OF THE INVENTION 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. In one embodiment, 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

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. For example, useful niclosamide includes niclosamide ethanolamine, or niclosamide and/or niclosamide salt contained in lipid or other nanoparticles, or in emulsions.

Rhamnolipids

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:

R¹ = H, unsubstituted α-rhamnopyranosyl, α-rhamnopyranosyl substituted at the 2 position with a group of formula

( )

R² =

;

R³ and R⁴ are saturated or mono or polyunsaturated alkyl;

R⁵ and R⁶ 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.

In one embodiment, R¹ = H, unsubstituted α-rhamnopyranosyl;

R² =

R³=- , wherein x = 2-19, preferably 2-10, or 4-8; for example x= 2, 4, 6, 8, or 10;

R⁴¹¹¹ wherein y ^:::: 1-19, preferably 2-10, or 4-8; for example x^:::: 2, 4, 6, 8, or 10;

and

In another embodiment, R¹ = H or unsubstituted α-rhamnopyranosyl,

Useful rhamnolipids of the Formula 1 include α-rhamnopyranosy(i,2)-α- ramnopyranosyl)-3-hydroxydecanoyl-3-hydroxydecanoic acid (di-rhamnolipid, Rha-Rha- C10-C10) and has the following structure (Formula 2):

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.

Some common di-rhamnolipids useful for this invention include: L-rhamnopyranosyl- L-rhamnopyranosyl-β-hydroxydecanoyl-p -hydroxydodecanoate (Rha-Rha-Cl 0-C12); L- rhamnopyranosyl-L-rhamnopyranosyl-b -hydroxydodecanoyl-β-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; and L-rhamnopyranosyl-L-rhamnopyranosyl-β-hydroxytetradecanoyl-β- by droxytetradecanoate (Rha-Rba-C 14-C 14).

Preferred rhamnolipids are L-rhanmosyi-P-hydroxydecanoyl-β-hydroxydeeanoate (mono-rhamnolipid, Rha-C10-C10) and L~rhamnosyl-L~rhamnosyl-β-hydroxydecanoyl-β- hy droxy decan oate (di-rbamnolipid, Rha-Rha-C10-C10), and the mixture thereof.

Increased Bioavailability

The inventor discovered that 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. 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.

Increased Treatment Effects in NAFLD and NASH

The FDA recommends in their Guidance for Industry' for developing drugs for noncirrhotic nonalcoholic steatohepatitis with liver fibrosis that sponsors of clinical development programs consider the following liver histological improvements as endpoints reasonably likely to predict clinical benefit to support accelerated approval under the regulations (www.fda.gov/media/119044):

— Resolution of steatohepatitis on overall histopathological reading and no worsening of liver fibrosis on NASH CRN fibrosis score. Resolution of steatohepatitis 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

^. Improvement in 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

— Both resolution of steatohepatitis and improvement in fibrosis (as defined above).

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.

Rhamnolipids increase expression of HNF4α, which corrects phenotype of cultured end-stage cirrhotic hepatocytes and quickly reverses terminal end-stage liver failure in a mouse model. HNF4α prevents steatosis by modulating lipolysis, ER stress, and lipogenesis. protects against progression of NASH.

Rhamnolipids reduce TGF-β1 in plasma and intrahepatically; TGF-β1 is a driver of fibrosis.

Niclosamide reduces STAT3, which is a key regulator of liver fibrosis.

Xu, et al (Biochim Biophys Acta, 2014, 1842:2237-45) report that STAT3 signaling activation crosslinked with TGF-β1 in hepatic stellate cell to exacerbate liver injury and fibrosis. The present combination treatment of rhamnolipids and niclosamide reduces both TGF-β1 and STAT3 levels, and thus synergistically reduce the liver fibrosis and injury.

Increased Treatment Effects in T2DM

Rhamnolipids significantly reduce IL-1α. IL-1α deficiency reduces adiposity, glucose intolerance and hepatic de-novo lipogenesis in diet-induced obese mice. IL-1α -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-1α level in diet-induced obese mice, and thus reducing adiposity, glucose intolerance and hepatic de-novo lipogenesis in diet-induced obese mice.

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-β and IL-1α, 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. The combination therefore will lead to body weight loss in excess of what is achievable by each drug alone, and niclosamide oral bioavailabiiity is enhanced with rhamnolipid, thus increasing extrahepatic activity of niclosamide, including mTQRCl inhibition, (Guillieri C. et al, mTQRCl Overactivation as a Key Aging Factor in the Progression to Type 2 Diabetes Mellitus. Front. Endocrinol., 16 October 2018)] https :/7d or org/ 10.3389/fendo.2018.00621

Pharmaceutical Compositions

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 (rhamnolipidmiclosamide) is in general 1:2 to 1:100, preferably 1 :2 to 1 :50, or 1 :5 to 1 :20.

For example, 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. For example, 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.

In one embodiment, 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. In one embodiment, the rhamnolipid is at 0.005-20% (w/w), and 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. In one embodiment, the pharmaceutical composition is in a dosage form such as tablets, capsules, granules, fine granules, powders, syrups, injectable solutions, or the like. The above pharmaceutical composition can be prepared by conventional methods. For example, 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. In another embodiment, 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. In another embodiment, 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. In another embodiment, 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.

In another an emulsion of rhamnolipid and niclosamide is administered in a capsule.

In another embodiment, 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.

In one embodiment, 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.

EXAMPLES

The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present invention and are not to be construed as being limiting.

EXAMPLE 1. Oral Treatment of rhamnolipids reduces IL-1α, TGF-β1 and fasting glucose levels in blood of treated 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.

Animal and Materials

Diet-Induced Obese (DIO) C57BL/6J male mice maintained with high fat diet. They were 12 weeks old at arrival. Diet: D 12492 high fat diet

Rhamnolipids, R95Dd, 95% (di-Rhamnoiipid dominant), obtained from AGAE Technologies, Corvallis, OR, USA)

Doses

Treatment Groups (8 mice per group)

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.

1 . Di-rhamnolipid - High Dose (HD):

Doses were escalated over the 7-day treatment phase. Sequential daily dose administrations were: 48 mg/kg, 96 mg/kg, 96 mg/kg, 96 mg/kg, 96 mg/kg, 144 mg/kg, 144 mg/kg. Mean dose was 103mg/kg.

2. Di-rhamnolipid - Low Dose (LD)

Doses were escalated over the 7-day treatment phase. Sequential daily dose administrations were:

24 mg/kg, 48 mg/kg, 48 mg/kg, 48 mg/kg, 48 mg/kg, 72 mg/kg, 72 mg/kg. Mean dose was 51 mg/kg.

3. Matching Placebo (vehicle, no rhamnolipid) TGF-β and IL-1α plasma levels were measured by Eve Technologies Corporation, Calgary, AB. Canada) using TGF-β Multiplex Immunoassay. TGF-β1 and IL-1α were chosen because are elevated in both obesity, type 2 diabetes mellitus, NAFLD and NASH.

Fasting blood glucose determinations were done at IDEXX BioAnaiyties West Sacramento, CA 95605.

Results

Analyses were performed via GraphPad T-tests. A p-value of <0.05 was viewed be viewed to be statistically significant. Figure 1 shows beneficial effects of rhamnolipid on TGF-β1 in C57BL6 mice with diet-induced obesity after 7 days of oral dosing (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).

Figures 1 and 2 show a dose-dependent decrease in plasma TGF-β1 and fasting glucose by oral treatment of DR. A decrease in TGF-β I signaling in obese animals and humans has been associated with improvement in T2DM and NAFLD and NASH.

The results of Figures 1 and 2 show that TGF-β1 reduction is associated with significant increase in glycemic control.

Figure 3 shows a dose dependent significant decrease in plasma IL-1α after 7 days of oral treatment with di-rhamno!ipid.

These results also demonstrate that oral rhamnolipid doses could be escalated up to 144 mg/kg in mice, and are systemieally active in reducing cytokines associated with T2DM, obesity, NAFLD and NASH.

EXAMPLE 2„ Oral administration of rhamnolipids significantly reduces TGF~beta and

IL-lα in plasma of a human volunteer

Objectives/Background

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.

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. (FDA Guidance for Industry. Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. 2005. wwwfda.gov/media/72309/download

Subject

63 year-old adult male with hypertension and a BMI of 28. Co-medications were losartan 100-200 mg daily and hydrochlorothiazide once daily. Medication doses were not changed in the 3 months prior to dosing with rhamnolipids or in the week following dosing. Materials

Di-rbamnolipid C10-C10 (>97% pure) was procured from Prof. Hausmann, Karlsruhe Institute of Technology, University' of Karlsruhe, Germany.

Methods

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.

Results

As demonstrated in Figures 4 and 5, rhamnolipid reduced TGF-β1 and IL-1α plasma levels in a human subject to a similar degree as observed in diet-induced obese mice in Example 1. No adverse effects, especially gastrointestinal effects, were observed.

TGF-beta is increased in T2DM, obesity, NAFLD and NASH. NASH-induced scarring depends on TGF-beta.

The plasma TGF-β profile indicates that the rhamnolipid total dose given in the three days prior to post-dose assessment reduced TGF-β for at least 7 days. The total dose given within that timeframe (the three days of dosing and day 7 plasma value) supports daily dosing of rhamnolipid at 100 mg per day. IL-1α 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-1α profile indicates that the rhamnolipid total dose given in the three days prior to post-dose assessment reduced IL-1α 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-1α deficiency reduces adiposity, glucose intolerance and hepatic de-novo lipogenesis in diet-induced obese mice. ( Almog T, et al. Interleukin-la deficiency reduces adiposity, glucose intolerance and hepatic de-novo lipogenesis in diet-induced obese mice. BMJ Open Diabetes Res Care 2019.) vtavw.ncbi.niin.nih.gov/pmc/aiticles/PMC6827792/

EXAMPLE 3. Assessment of effect of rhamnolipids on solubility and particle size of niclosamide ethanolamine (NEN) in water

The solubility of niclosamide in aqueous solutions of rhamnolipid was assessed.

Materials

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.

Methods

Niclosamide quantification.

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 H₂O 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. As rhamnolipids have some level of absorbance at higher concentrations, samples without niclosamide were prepared and used as controls. Results

As expected, niclosamide was poorly soluble in H₂O 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.

Table 1. Solubility of niclosamide in rhamnolipid solutions.

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.

Conclusions

An oral formulation of rhamnolipid and niclosamide is feasible. In fact, the addition of rhamnolipid enables niclosamide to be sufficiently orally bioavai!able.

EXAMPLE 4. Combination ofrhamnolipids and niclosamide in the treatment of NASH in diabetic mice (prophetic example)

Objectives

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. In addition, 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.

Materials Animals: C57BL/6 mice

Di-rhamnolipids (DB 1-500; Di-rhamno!ipid C10-C10) is obtained from AGAE Technologies. Niclosamide: Niclosamide (ethanolamine salt) is acquired from Cayman Chemical (Cayman Chemical, 1180 East Ellsworth Road, Ann Arbor, Michigan 48108, USA) is used.

Methods

Methodology is in accordance with SMC Laboratories, Inc., Tokyo, Japan.

SMC’s proprietary SEAM™ model is a model that recapitulates the same disease progression as human NASH/HCC. In this model, male C57BL/6 mice aged two days are given a single dose of streptozotocin to reduce insulin secretory capacity. When the mice turn four weeks of age, they start a high-fat diet feeding. 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.

To induce NASH, male C57BL/6J mice (Japan SLC, Inc,, Japan) are receive a single subcutaneous injection of 200 μg 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. At age 6 weeks, 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.

Oral gavage administration of the rhamnolipid and niclosamide: 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.

Study Conduct

All procedures are conducted as described at. https://smccro- lab.com/service/semce _disease_area/pdf/STAM_non- eonfidenti al_January2021.pdf?v210326

Overall study design is summarized in the following Table 2.

Table 2, Dose Groups and Dosing Duration

Mice are terminated at week 92-3 hours post last dosing per gavage in order to assess PK values of niclosamide.

Primary analysis:

Impact of combination treatment on: Body weight, T2DM (HbA1C, OGTT); NASH steatosis and fibrosis (NAS and fibrosis score)

General: Body weight, Liver weight, liver to body weight ratio. Biochemistry: Fasting blood glucose, ALT, liver triglyceride, blood triglycerides and cholesterol. In addition: H1c and OGTT (oral glucose tolerance test)

Histopathological analyses: Paraffin-embedded liver and O.C.T-embedded liver samples are prepared. Hematoxylin and eosin stain (HE staining to assess liver biopsies for NAFLD activity score (NAS score); Sirius Red staining for collagen staining (for assessment of standard fibrosis score; e.g,, 0= no fibrosis and a score of 4 is cirrhosis).

Sample collection: Frozen liver, frozen plasma for pharmacodynamic assessment 2-3 hours post last dose, and determination of plasma levels 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)

Objectives

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).

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.

The planned (to be confirmed by actual results of study Part A) dose groups are:

A: Placebo (standard of care)

B: Niclosamide 1 g BlD

C: (Niclosamide 1 g + 100 mg rhamnolipid) BID D: (Niclosamide 650 mg +50 mg rhamnolipid) TID

The main objectives of study Part B are:

1 . Demonstrate increased glycemic control as based of fasting glucose levels, QGTT and HbAlC levels at end of study vs baseline

2. 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.

3. Assess if there is a significant effect on clinical and laboratory values associated with NAFLD and NASH, including liver transaminases, triglycerides and cholesterol.

4. Document increases plasma niclosamide levels (Cmax) of the combination compared with the niclosamide only DG.

5. Identify significant changes in plasma cytokine values, including TGF-β, IL-1α and IL- 1β, TNFα.

Materials

Rhamnolipids and niclosamide will be used as developed using FDA-approved CMC procedures and prepared in accordance with the IND.

Study Population

1. Adult subjects with T2DM with HgAlc less than 10%

2. Overweight or obese (BMI>=27)

3. On stable medications for T2DM Primary analysis

Impact of combination treatment on: Body weight, T2DM (HbAlC, OGTT): NASH clinical parameters.

Overview of analyses

General: Body weight, Liver weight, liver to body weight ratio.

Biochemistry: Fasting blood glucose, ALT, liver triglyceride, blood triglycerides and cholesterol. In addition: 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.

The invention, and the manner and process of making and using it, are now described in such full, clear, concise and exact terms as to enable any person skilled in the art to which it pertains, to make and use the same. It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the scope of the present invention as set forth in the claims. To particularly point out and distinctly claim the subject matter regarded as invention, the following claims conclude the specification.

Claims

What is Claimed is:

1. A method for treating nonalcoholic steatohepatitis (NASH) in a mammal subject, comprising the step of administering to a subject in need thereof an effective amount of niclosamide or a salt thereof or a derivative thereof and an effective amount of one or more rhamnolipids according to Formula (I),

wherein:

R¹ = H, unsubstituted α-rhamnopyranosyl, or α-rhamnopyranosyl substituted at the 2 position with -G-C (=0)-CH=CH-R⁵;

R^" = H, alkyl, ~CHR⁴~CH₂-COQH or -CHR⁴~CH₂-COQR⁶;

R³ and R⁴ are saturated or mono or polyunsaturated alkyl; and R⁵ and R⁶ are alkyl.

2. A method for treating type 2 diabetes in a patient, comprising the step of administering to a subject in need thereof an effective amount of niclosamide and an effective amount of one or more rhamnolipids according to Formula (I),

wherein:

R^{1 :::} H, unsubstituted a~rhamnopyranosyl, or a~rhamnopyranosyl substituted at the 2 position with

R² = H, alkyl, -CHR⁴-CH₂-CQQH or -CHR⁴-CH₂-COOR⁶; and R³ and R⁴ are saturated or mono or polyunsaturated alkyl,

R⁵ and R⁶ are alkyl.

3. The method of claim 2, wherein said patient also suffers from NAFLD or NASH, or related liver cirrhosis.

4. The method according to any one of Claims 1-3, wherein K^l = H or unsubstituted α- rhamn opy ranosy 1 ,

R² -CHR⁴-CH₂-COOH, R³

or

5. The method according to any one of Claims 1-3, wherein the rhamnolipids are selected from the group consisting of: Rha-Rha-C10-C12; Rha-Rha-C12-C10; Rha-Rha-C8-C10; Rha-Rha-C10-C8; Rha-Rha-C 12: 1 -C1O; Rha-Rha-C10-C12:1; Rha-Rha-C14-C14; Rha-C8- C8; Rha-C8-C10; Rha-C10-C8; Rha-C12: 1 -C10; Rha-C10-C12: 1.

6. The method according to any one of Claims 1-3, wherein the adminitering is by oral administration.