WO2023164752A1 - Pharmaceutical product for the treament of obesity and associated metabolic syndrome diseases, uses and methods of treatment - Google Patents

Pharmaceutical product for the treament of obesity and associated metabolic syndrome diseases, uses and methods of treatment Download PDF

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WO2023164752A1
WO2023164752A1 PCT/BR2022/050072 BR2022050072W WO2023164752A1 WO 2023164752 A1 WO2023164752 A1 WO 2023164752A1 BR 2022050072 W BR2022050072 W BR 2022050072W WO 2023164752 A1 WO2023164752 A1 WO 2023164752A1
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extract
ilex paraguariensis
standardized
leaves
animals
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PCT/BR2022/050072
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French (fr)
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João BATISTA CALIXTO
Rodrigo MARCON
Cristina SETIM FREITAS
Edinéia LEMOS DE ANDRADE
Adara ÁUREA DOS SANTOS
Melina HELLER
Sérgio José MACEDO JÚNIOR
Jarbas MOTA SIQUEIRA JUNIOR
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Centro De Inovação E Ensaios Pré-Clínicos
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Priority to PCT/BR2022/050072 priority Critical patent/WO2023164752A1/en
Publication of WO2023164752A1 publication Critical patent/WO2023164752A1/en

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    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps

Definitions

  • the present invention generally relates to pharmaceutical product for human or veterinary use for the preventive or therapeutic treatment of obesity and related metabolic syndrome diseases, comprising a standardized aqueous or ethanolic extracts containing a mixture of natural compounds such as rutin, caffeine, theobromine, theophylline and chlorogenic acids, particularly obtained from the leaves of the plants from genus Magnoliopsida, family Aquifoliaceae, especially from Ilex paraguariensis , as well its preventive and therapeutic uses, methods of preparation, in vitro and in vivo pharmacological effects, pharmacokinetic, safety and its molecular mechanism of action.
  • Obesity currently represents a major health burden as it substantially increases the risk of many chronic diseases such as type 2 diabetes mellitus, fatty liver disease (steatosis), hypertension, myocardial infarction, stroke, dementia, osteoarthritis, obstructive sleep apnea and several cancers, among others, thereby contributing to a decline in both quality of life and life expectancy.
  • diseases such as type 2 diabetes mellitus, fatty liver disease (steatosis), hypertension, myocardial infarction, stroke, dementia, osteoarthritis, obstructive sleep apnea and several cancers, among others, thereby contributing to a decline in both quality of life and life expectancy.
  • Obesity and metabolic diseases can be interrelated and the prevention and treatment strategies for both have not been successful in a long term.
  • Lifestyle and behavioral interventions aimed at reducing calorie intake and increasing energy expenditure have limited effectiveness because the complex and persistent hormonal, metabolic and neurochemical adaptations defend against weight loss and promote weight regain. Reducing the obesity burden requires approaches that combine appropriated drug intervention combined with changes in the environment and society.
  • the present invention provides the development method for preparation of aqueous and ethanolic standardized extracts and presents the use of such combination of natural compounds, particularly the standardized aqueous extract obtained from the leaves of the plants of genus Magnoliopsida, preferably Ilex paraguariensis , in the manufacture of a pharmaceutical product.
  • Standardized aqueous and ethanolic extract obtained from the leaves of the plant of the genus Magnoliopsida, family Aquifoliaceae, more especially from Ilex paraguariensis also called in this invention as ST-076, ST-076.1, ST-076.2, ST-076.3, ST-076.4, ST-076.7 or ST-076.8, contain a mixture of compounds including rutin, caffeine, theobromine, theophylline, and caffeoylquinic acid and its isomers quantified as chlorogenic acid, among others.
  • the present invention provides the development of a pharmaceutical product for human or veterinary use, composed of standardized and very stable aqueous extracts from the leaves of Ilex paraguariensis .
  • the extract given orally was found to be very effective given either in a preventive or in a therapeutic manner for the treatment of obesity and related metabolic syndrome diseases, such as type 2 diabetes, hypertension, dyslipidemia, steatosis, inflammation, among others.
  • the standardized aqueous extract obtained from the leaves of Ilex paraguariensis can be considered safe as it was not genotoxic nor toxic according to acute and repeated dose 90-day oral toxicity assessments.
  • Fig.2 Pharmacokinetic for oral (p.o.) administration of ST-076 and evaluation of plasma concentration profile of caffeine, theobromine and theophylline after acute administration according to the present invention (1,000 mg/Kg, p.o.).
  • the blood was collected at 0.083, 0.5, 1, 2, 4, 8, 12, 16 and 24 hours after ST-076 oral administration for the determination of pharmacokinetic profiles (AUC last , T max , C max, T 1/2 and Ke).
  • Data are represented as mean ⁇ standard error of mean (S.E.M.) of 5 animals for each time point.
  • FIG.3 Plasma concentration profile of caffeine, theobromine and theophylline following a 7-day treatment with ST-076 according to the present invention (1,000 mg/Kg, p.o.).
  • the blood was collected at 0.083, 0.5, 1, 2, 4, 8, 12, 16 and 24 hours after ST-076 administration for the determination of pharmacokinetic profiles (AUC last , T max , C max, T 1/2 and Ke).
  • Data are represented as mean ⁇ S.E.M. of 5 animals for each time point.
  • mice body weight gain induced by high-fat diet in mice.
  • Body weight gain was evaluated from week 1 to week 16. Animals were fed with normal chow diet (NCD) or with high-fat diet (HFD) during 16 weeks. From week 8 until week 16, mice that were fed with HFD began to be daily treated with vehicle (10 mL/Kg, p.o.), ST-076 (500 or 1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c. used as a positive control drug). Body weight was measured once a week. Data are presented as mean ⁇ S.E.M. Two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test were performed.
  • ANOVA analysis of variance
  • NCD Normal Chow Diet
  • HFD High-fat Diet. 9-10 animals per group.
  • mice Representative images of mice phenotype in the end of the study of high-fat diet induced obesity. Animals were fed with NCD or HFD during 16 weeks. From week 8 until week 16, mice that were fed with HFD began to be daily treated with vehicle (10 mL/Kg, p.o.), ST-076 (500 or 1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.). NCD: Normal Chow Diet; HFD: High-fat Diet. [Fig.9]. Effect of ST-076 on high-fat diet induced fat accumulation in mice. Animals were fed with normal chow diet (NCD) or high-fat diet (HFD) during 16 weeks.
  • NCD Normal Chow Diet
  • HFD High-fat Diet.
  • mice that were been fed with HFD began to be daily treated with vehicle (10 mL/Kg, p.o.), ST-076 (1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.).
  • vehicle 10 mL/Kg, p.o.
  • ST-076 1,000 mg/Kg, p.o.
  • Liraglutide 0.2 mg/Kg, s.c.
  • FIG.10 Histological micrograph showing the effect of ST-076 on high-fat diet induced liver fat accumulation in mice.
  • Animals were fed with NCD or HFD during 16 weeks. From week 8 to week 16, mice that have been fed with HFD began to be daily treated with vehicle (10 mL/Kg, p.o.), ST-076 (1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.). On week 16 animals were euthanized and liver were removed. H&E staining of liver tissue was performed in all groups. A ⁇ 100; B ⁇ 400. NCD: Normal Chow Diet; HFD: High-fat Diet.
  • NCD Normal Chow Diet
  • HFD High-fat Diet. 9-10 animals per group.
  • FIG. 12A and 12B Effect of ST-076 on liver and inguinal (ingWAT) white tissue weights.
  • NCD Normal Chow Diet
  • HFD High-fat Diet. 9-10 animals per group.
  • FIG.15 Effect of ST-076 on body weight gain induced by high-fat diet in rats. Body weight gain from week 1 to week 11. Animals were fed with NCD or HFD during 11 weeks and were treated daily with vehicle (10 mL/Kg, p.o.), ST-076 (1,000 mg/Kg, p.o.) or with liraglutide (0.2 mg/Kg, s.c.). Body weight was measured once a week. Data are presented as mean ⁇ S.E.M. Two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test were performed. # indicates significant statistical difference (p ⁇ 0.05) when compared to NCD group. * indicates significant statistical difference (p ⁇ 0.05) when compared to vehicle HFD. NCD: Normal Chow Diet; HFD: High-fat Diet. 7-9 animals per group.
  • FIG. 17A and 17B Effect of ST-076 on blood glucose in Type 2 Diabetes Model (T2DM).
  • T2DM Type 2 Diabetes Model
  • One experimental group received saline (non-diabetics, ND), while the other groups received nicotinamide (180 mg/kg, i.p.) and then streptozotocin (65 mg/kg, i.v.) (DB).
  • ND animals were treated with vehicle; DB animals were treated with vehicle, Liraglutide (0.2 mg/kg; s.c.), ST-076 (500 mg/kg; p.o.) or ST-076 (1,000 mg/kg; p.o.).
  • FIG. 18A, 18B and 18C Effect of ST-076 on cardiovascular parameters of animals with Type 2 Diabetes Model (DMT2).
  • DMT2 Type 2 Diabetes Model
  • One experimental group received saline (non-diabetics, ND), while the other groups received nicotinamide (180 mg/kg, i.p.) and then streptozotocin (65 mg/kg, i.v.) (DB).
  • ND animals were treated with vehicle
  • DB animals were treated with vehicle, Liraglutide (0.2 mg/kg; s.c.), ST-076 (500 mg/kg; p.o.) or ST-076 (1,000 mg/kg; p.o.).
  • PCR-array validation using RT-qPCR in white adipose tissue RT q-PCR analysis of gene expression in white adipose tissue was performed for the following genes: (A) Monoacylgliceride Lipase - Mgll , (B) NLR Family Pyrin Domain Containing 3 – Nlrp3 , (C) Neuropeptide Y – Npy. The mRNA expression levels were normalized to the expression level of 18S housekeeping gene using the 2 ⁇ Ct formula. The results were expressed as the means ⁇ S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed.
  • ANOVA Analysis of variance
  • NCD Normal Chow Diet
  • HFD High-fat Diet
  • n 3-4 animals per group.
  • FIG.31 Representative scheme of the ST-076 mechanism of action in adipose tissue obtained from high-fat diet-induced obesity in mice.
  • ST-076 administration showed a decrease in AMPK activity following the increase of ERK1/2 signaling and increase of the lipolysis.
  • ST-076 induces activation of PI3K and downstream stimulation of JNK and PPAR pathways, resulting in tissue lipolysis.
  • the effect of ST-076 seems to be related with release of GLP-1 in serum, which reduces the food intake, changes feeding behavior and tissue lipolysis.
  • NCD Normal Chow Diet
  • HFD High-fat Diet.
  • n 7-9 animals per group.
  • the present invention provides a method of preparation of a very stable pharmaceutical product useful for human and veterinary health for the preventive or therapeutic treatment of obesity and associated metabolic syndrome diseases comprising a combination of selected phytochemical compounds obtained from the standardized aqueous extract obtained from the leaves of Ilex paraguariensis plant including: rutin, caffeine, theobromine, theophylline, and chlorogenic acids.
  • the method of preparing an Ilex paraguariensis extract comprises: (a) contacting Ilex paraguariensis material with water or a solvent at a temperature below boiling to produce a mixture, and milling the mixture to obtain a crude extract; (b) filtering the crude extract to obtain a purified extract; and (c) drying and standardizing the alcoholic or aqueous extract to produce the Ilex paraguariensis extract.
  • an Ilex paraguariensis extract prepared according to methods provided herein the standardized extract preferentially comprising: At least rutin: about 100 mg to 1,000 mg per 100 grams of standardized extract of Ilex paraguariensis ; At least caffeine: about 100 mg to 3,000 mg per 100 grams of standardized extract of Ilex paraguariensis ; At least theobromine: about 100 mg to 500 mg per 100 grams of standardized extract of Ilex paraguariensis ; At least theophylline: about 2 to 10 mg per 100 grams of standardized extract of Ilex paraguariensis ; At least chlorogenic acids: about 100 mg to 10,000 mg per 100 grams of standardized extract of Ilex paraguariensis ;
  • the present invention is a combination of compounds such as Rutin (861.01 mg per 100 grams of ST-076), Caffeine (2,219.43 mg per 100 grams of ST-076), Theobromine (370.92 mg per 100 grams of ST-076), Theophylline (6.89 mg per 100 grams of ST-076) and Chlorogenic acids (6,450.22 mg per 100 grams of ST-076), preferably obtained from a standardized aqueous extract of Ilex paraguariensis .
  • Rutin 861.01 mg per 100 grams of ST-076)
  • Caffeine 2,219.43 mg per 100 grams of ST-076)
  • Theobromine 370.92 mg per 100 grams of ST-076)
  • Theophylline (6.89 mg per 100 grams of ST-076)
  • Chlorogenic acids 6,450.22 mg per 100 grams of ST-076), preferably obtained from a standardized aqueous extract of Ilex paraguariensis .
  • the phytochemical constituents of the standardized aqueous extract obtained from the leaves of Ilex paraguariensis according to the present invention were quite stable for up to 20 months at a temperature of 20 to 30 °C since Rutin, Chlorogenic Acid, Theophylline, Theobromine and Caffeine were measured by HPLC (High Performance Liquid Chromatography).
  • the standardized aqueous extract obtained from the leaves of Ilex paraguariensis according to the present invention is useful as it can be given either preventively or therapeutically to a patient in need via oral route, or to be used in the preparation of a pharmaceutical product in the form of pills, liquid, soft capsule, among others, for the treatment of obesity and associated metabolic syndrome diseases.
  • the pharmaceutical product according to the present invention additionally comprises pharmaceutically acceptable excipients (for human or veterinary use).
  • Suitable excipients for the invention are, for example, and without any limitation, those cited in the book Remington's Pharmaceutical Sciences, by the American publisher Mack Publishing, European Pharmacopoeia or Brazilian Pharmacopoeia.
  • the excipients are selected according to the pharmaceutical form to be composed.
  • Suitable pharmaceutical forms according to the present invention include, without limitations, pill, tablet, capsule, syrup, etc. Such forms are adapted according to the standard route of administration, for instance as approved by The Food and Drug Administration (FDA) at the following website: https://www.fda.gov/drugs/data-standards-manual-monographs/route-administration.
  • FDA Food and Drug Administration
  • the present invention includes the use of the combination of compounds or the standardized aqueous of the plant Ilex paraguariensis in the manufacturing of a pharmaceutical stable product for the treatment of obesity and associated metabolic syndrome diseases.
  • the standardized aqueous extract obtained from the leaves of Ilex paraguariensis according to the present invention can be administered directly to the patient in need via oral route.
  • the present invention includes methods for the treatment of obesity and/or metabolic diseases comprising the administration of an effective dosage of the pharmaceutical product of the standardized aqueous extract obtained from the leaves of Ilex paraguariensis to a patient in need.
  • the dosage of the standardized aqueous/ethanolic extracts range from about 1 to about 5,000 mg/kg/day, one or at different times a day.
  • the present invention deals with a method for preventing manifestations involved in the obesity and diseases associated with the metabolic syndrome, as well as in the prophylaxis or treatment of diseases resulting from the syndrome, such as heart disease, lipid problems (steatosis), hypertension, type 2 diabetes, dementia, cancer, polycystic ovarian syndrome, non-alcoholic fatty liver disease, among others.
  • diseases resulting from the syndrome such as heart disease, lipid problems (steatosis), hypertension, type 2 diabetes, dementia, cancer, polycystic ovarian syndrome, non-alcoholic fatty liver disease, among others.
  • the standardized aqueous/ethanolic extract is manufactured according to a specific method of preparing comprising the steps of 1) material cleaning; 2) solvent used, 3) time of maceration, 4) filtration, 5) evaporation, 6) drying and 7) stability and storage.
  • the step “1” covers the use of different parts of the plant, preferably leaves and branches, separated into “fresh”, “aged” and “mix of fresh and aged leaves”.
  • the process according to the present invention uses a “mix of fresh and aged leaves” and “pulverized plant in natura ”.
  • the plant material composed by leaves and branches was washed in running water to eliminate impurities and kept for 1-3 days drying at room temperature. Then, leaves were weighed and placed in an incubator at 50-70 °C for approximately 2-4 days.
  • the steps “2” and “3” cover preferably the use of 1 part of plant material to 10 parts of Extractor Liquid, which was a mixture of ethanol and water in 50% ratio (v/v) or pure water heated at 90 °C.
  • the extract is left macerating at room temperature (approximately 20 °C) for 3 to 10 days, preferably in the dark, with occasional agitation.
  • the step “4” comprises the filtration of the extracts by gravity with the aid of “voil” fabric filter to remove the thicker part of the plant material (larger particles), followed by vacuum filtration using filter paper of 80.0 g/m2, porosity of 3 microns, to remove smaller particulate material.
  • the step “5” includes the evaporation of ethanol only from hydroalcoholic extracts, particularly by rotary evaporation.
  • the steps “6” and “7” comprise the drying/storage of aqueous or hydroalcoholic extracts.
  • the aqueous and hydroalcoholic extracts that would be dried in the incubator were distributed into containers previously weighed and placed in the incubator at 50-70 °C; then the extracts were weighed and stored in a freezer at -20 °C until further analysis.
  • the extracts that would be lyophilized were light protected and stored in a freezer at -20 °C.
  • the lyophilization process took between 7 - 10 days, depending on the volume of extract in each container. After completion of lyophilization, the extracts were homogenized and aliquoted in flasks wrapped with light protection and stored in a freezer at -20 °C until further analysis.
  • the first aim was to produce standardized aqueous or hydroalcoholic extracts from plant leaves (ST-076: plant material of Ilex paraguariensis , same as ST-076.1, ST-076.2, ST-076.3, ST-076.4; ST-076.7: an extract of Ilex paraguariensis , same as ST-076.8) on a small scale, but viable for large scale production, and free of potentially toxic substances, such as organic solvents (e.g. methanol, hexane, among others). All extracts yielded an average of 17% in mass.
  • organic solvents e.g. methanol, hexane, among others. All extracts yielded an average of 17% in mass.
  • the chemical characterization of the extracts resulting from this invention was performed by identifying the major phytochemical components using selective and sensitive analytical techniques, such as liquid chromatography coupled to mass spectrometry.
  • an Ultra Performance Liquid Chromatography (UPLC) equipment coupled to a mass spectrometer with a triple-quadrupole analyzer (XevoTM TQ-S, Waters) was used.
  • Theobromine, theophylline, caffeine, rutin and caffeoylquinic acid and its isomers are among the compounds identified.
  • the procedures involved in the extraction process were: 1) material cleaning; 2) maceration, 3) filtration, 4) evaporation, 5) drying and 6) storage.
  • the leaves and branches were selected and damaged leaves and/or stems were discarded.
  • the remaining leaves were separated into “fresh” (ST-076.1), “aged” (ST-076.2) and “mix of fresh and aged leaves” (ST-076.3).
  • the plant material was washed in running water to eliminate impurities and was kept for two days drying at room temperature.
  • the leaves were weighed and placed to dry further in an incubator at 60 °C, for approximately three days. Subsequently, the dried plant material was packed in clean plastic bags and stored at room temperature and protected from light, until further analysis.
  • the extraction tests with leaves were accomplished using portions of approximately 500 grams of manually crushed material.
  • the extraction proportion was of 1 part of plant material to 10 parts of Extractor Liquid, which was a mixture of absolute ethanol and water in 50% ratio (v/v).
  • the extract was left to macerate at room temperature (approximately 20 °C) for 5-10 days in the dark, with occasional agitation.
  • the extraction tests with Pulverized plant in natura were performed in three optimization tests: i. Selection of the extractor liquid: two types of extracts were produced: a heated aqueous extract, and a hydroalcoholic extract in which the extractor liquid was a mixture of deionized water and absolute Ethanol in 1:1 ratio (v/v); ii. Maceration time: periods of three and six days were tested; iii. Drying procedure: incubator at 60 °C or Lyophilization.
  • the extraction tests with pulverized plant material were carried out using 25 grams portions (approximately), in the proportion of 1 part of plant material to 10 parts of extractor liquid. This proportion was maintained for both extractor liquid tests (heated aqueous and hydroalcoholic).
  • the procedure was the following: approximately 25 grams of the plant material was weighed in a glass beaker wrapped in light protection, and 250 mL of deionized water, heated in a microwave at approximately 90 °C, was added to it. The beaker with aqueous extract was allowed to cool until room temperature was reached with occasional agitation. Finally, after cooling, the beaker containing the aqueous extract was sealed and was left macerating by the stipulated time in a refrigerator at 4 °C.
  • the hydroalcoholic extract was produced as described above, except that instead of 250 mL of hot water, 125 mL of deionized water at room temperature with 125 mL of absolute ethanol were used. For both extracts, two replicates were made, one for each drying process (incubation or lyophilization), and thus periods of 3-6 days of maceration were tested.
  • Rotary Evaporation Stage Following the filtration process, both hydroalcoholic extracts (ST-076.3 and ST-076.4) were submitted to the rotary evaporation process in order to eliminate the organic solvent.
  • the volume of solution placed in the evaporation flask was always less than half of the total volume of the flask.
  • a rotary evaporator (model R-100, Buchi) with heating bath, chiller for cooling and vacuum pump was used for evaporating ethanol. Heating bath and chiller temperatures were set at 60 °C and 10 °C, respectively.
  • the resulting solution was fractionated into borosilicate glass containers protected of light.
  • the amount of extract placed in each container corresponded to at most half its volume (for example, approximately 125 ml of extract was placed in a 250 mL beaker).
  • extracts were frozen at -20 oC for at least 24 hours before the Lyophilization process.
  • Drying/Lyophilization/Storage Stage The aqueous and hydroalcoholic extracts that would be dried in the incubator were weighed and placed in the incubator for drying at 60 °C. After completely dried, the extracts were left at room temperature to cool. Next, the extracts were weighed and stored in a freezer at -20 °C until further analysis.
  • the extracts that were lyophilized were distributed into beakers, protected from light and placed in a freezer at -20 °C until Lyophilization. However, the aqueous extracts were frozen right after the filtration step, while the hydroalcoholic extracts were placed in the freezer following the rotary evaporation step.
  • lyophilization is a process were water changes from a solid state (ice) to a gaseous state. This change in physical state only happens with water at very low temperatures, approximately -55 °C, and under vacuum conditions.
  • a lyophilizer model LS3000, Terroni
  • the procedure consisted of freezing the extract for at least 24 hours and placing the container in the machine, with the temperature stabilized beforehand.
  • the Lyophilization process took between 7 - 10 days until the extract was completely dry, depending on the volume of extract in each container.
  • the yields of aqueous and hydroalcoholic extracts of ST-076.4 (Pulverized plant in natura ) dried in the incubator were the following: ST-076.4-hidroalcholic started with 12.71 grammas of pulverized plant, resulted in 2.32 grammas of dry extract, yielding 18.25%; ST-076.4-aqueous used 26.60 grammas of pulverized plant produced 4.88 grams of dried extract, which yielded 18.34%.
  • Table 1 presents the amount of dried extract obtained in each extraction of ST-076. Table 1. Yield as percentage of dried extract mass of ST-076.
  • Phytochemical Extract Characterization An investigative method was applied for identifying the phytochemical constituents, such as alkaloids and other constituents present in the dried extract. The method was built from the compilation of literature data referring to precursor and product ions, both in positive and negative ionization modes, of compounds commonly found in plant material. Thus, initially, samples of the extract were submitted to a screening analysis.
  • Lyophilized extracts of ST-076.4 (pulverized plant in natura ), both aqueous and hydroalcoholic, were also not considered for phytochemical characterization. In order for the extract to be produced on a larger scale, the lyophilization step would not be optimal for industrial use.
  • Table 2 Chromatographic area of compounds identified in aqueous and hydroalcoholic extracts prepared with pulverized plant in natura , dried in an incubator (ST-076.4) using Liquid Chromatography Coupled to Mass Spectrometry.
  • the samples were analyzed by using UPLC-MSMS type equipment (Ultra Performance Liquid Chromatography; MSMS, Mass Spectrometry in tandem) whose system is composed of a Xevo TQS mass spectrometer with a Waters (United Kingdom) triquadrupole mass analyzer.
  • the mass spectrometer is coupled to a high performance liquid chromatograph (Acquity H-Class) equipped with a degasser, quaternary pump system, chromatographic column oven, temperature-controlled sampler and automatic injector, also branded Waters.
  • the data acquisition and treatment were performed with the MassLynx software, version 4.1.
  • the mass spectrometer was calibrated in both positive and negative ion modes with a standard phosphoric acid calibration solution. Nitrogen was used as nebulizer gas (flow rate of 150 L/hour) and drying gas (flow rate of 1,000 L/hour). Other parameters: ionization source, electrospray; capillary voltage, 4,000 V; source temperature, 500 °C; cone voltage, 40 V. The parameters of the collision cell were: collision gas flow (argon), 0.15 mL/min; collision energy, 28 eV.
  • Analytical method for the determination of the phytochemical constituents of the extract For the quantification of the major compounds identified in the extract (Rutin, Chlorogenic Acid, Theophylline, Theobromine and Caffeine), a C18 reverse phase chromatographic column (Kinetex 50 mm x 2.1 mm, particle size 2.6 ⁇ m, brand Phenomenex, USA) was used.
  • the constituent solvents of the mobile phase were 0.1% formic acid in deionized water (solvent A) and acetonitrile (solvent C), respectively.
  • the linear gradient elution mode provided the best separation of components from the matrix.
  • the mobile phase flow rate was set at 400 ⁇ L min -1 . In all runs, the injected volume was 1 ⁇ L.
  • Linearity/Calibration curve For the linearity evaluation, a methanol calibration curve was constructed in the application range of 50 a 1,000 ng mL -1 .
  • the calibrators were prepared as follows: first, individual stock solutions 1 mg mL - 1 were made with each reference substance; second, a working solution 50 ⁇ g mL -1 with five compounds together was made by diluting of the stock solutions with methanol; lastly, the calibrators were prepared in methanol in the desired range of application.
  • the procedure adopted for analyzing the samples was the following: at each selected time point, an aliquot of approximately 20 mg of the extract was weighed. After weighing, this amount of material was solubilized in enough volume of deionized water to result in a concentration of approximately 20 mg/mL. Then, the sample was centrifuged for 5 min at 14,000 rpm and 200 ⁇ L of the supernatant were removed, placed in insert vials and injected into the LC-MSMS equipment on the same preparation day. Stability was assessed by monitoring variation of the chromatographic peak areas of compounds (rutin, chlorogenic acid, caffeine, theobromine and theophylline) from May 2018 to February 2020. May 2018 was considered as zero time (Table 8). Table 8.
  • the evaluation of long-term stability was performed by analyzing the percentage variation between the analytical signals (peak chromatographic area) of the compounds at zero time compared to the analytical signal in the months of evaluation. According to RDC No 26 of the National Health Surveillance Agency (ANVISA), a Brazilian FDA-like agency, and the sample is considered stable when the variability between analytical markers is not greater than 20%.
  • ANVISA National Health Surveillance Agency
  • the pharmacokinetic profile was evaluated after acute and repeated oral administration with ST-076 for 7 days in Sprague Dawley rats. Animals were treated orally (p.o.) by gavage with the extract according to the present invention (1,000 mg/Kg). Plasma was collected by caudal vein puncture at the following time points: 0.083, 0.5, 1, 2, 4, 8, 12, 16 and 24 hours after administration of the extract according to the present invention.
  • the ST-076 was prepared and quantified using an analytical method previously developed and validated by means of UPLC-MSMS. Caffeine, theobromine and theophylline were assessed. All compounds were very well orally absorbed and reach micromolar plasma concentration (Table 10).
  • This study aimed to evaluate the possible CYP3A4-mediated interaction with ST-076 in vivo .
  • the study was performed using rats treated with vehicle or with ST-076 (1,000 mg/kg, p.o) for 7 consecutive days. On the last day, animals were treated with midazolam (10 mg/kg, p.o. a compound that is metabolized mainly by CYP3A4) and the animal’s blood was collected by caudal vein puncture at the following time points: 0.083, 0.25, 0.5, 1, 2 and 4 hours.
  • Midazolam plasma concentration was quantified using a previously validated UPLC-MS/MS analytical method.
  • Table 11 The pharmacokinetics parameters obtained for the animals treated with ST-076 are presented in and table 11. Table 11.
  • the efficacy of the ST-076 according to the present invention on body weight gain in a high-fat diet-induced obesity mouse model was investigated, and its efficacy was compared with Liraglutide, a reference drug used in type 2 diabetes and obesity treatments.
  • Animals received normal chow diet (NCD) or high-fat diet (HFD) for 8 consecutive weeks. From week 8 to week 16, animals fed with HFD were treated with ST-076 according to the present invention (500 or 1,000 mg/Kg, p.o.), Liraglutide (0.2 mg/Kg, s.c. - subcutaneously), a reference item, or with vehicle (10 ml/Kg, p.o.), once a day, until week 16. The evaluation of body weight change was assessed weekly from week 8 to week 16.
  • OGTT oral glucose tolerance test
  • blood was collected for chemistry analysis of total cholesterol, triglyceride, glucose, insulin, GLP-1 and leptin.
  • liver, inguinal (IngWAT) and epididymal white adipose tissue (EpiWAT) were removed and weighed.
  • the ST-076 according to the present invention 500 or 1,000 mg/Kg, p.o. or Liraglutide (0.2 mg/Kg, s.c.) significantly prevented body weight gain induced by HFD when compared to the HFD vehicle group (p ⁇ 0.05) ([Fig. 5]).
  • the extract according to the present invention (1,000 mg/Kg, p.o.) or Liraglutide (0.2 mg/Kg, s.c.) were able to significantly reduce food intake from week 8 to week 16 ([Fig. 6]).
  • daily treatment with the extract according to the present invention 500 or 1,000 mg/Kg, p.o. or with Liraglutide (0.2 mg/Kg, s.c.), improved glucose oral tolerance ([Fig. 7]).
  • the effect of ST-076 was evident through images of animals in the end of study ([Fig. 8]) and histology of white adipose tissue ([Fig. 9]) and liver ([Fig. 10]).
  • the effect of ST-076 on body weight gain in a high-fat diet (HFD) model in rats also was studied, and the activity compared with liraglutide, a reference drug used for clinical obesity treatment.
  • Animals received normal chow diet (NCD) or HFD, during 11 consecutive weeks.
  • Animals fed with HDF were treated with ST-076 according to the present invention (1,000 mg/Kg, p.o.), liraglutide (0.2 mg/Kg, s.c.), a reference item, or with vehicle (10 ml/Kg, v.o.), once a day, for 11 consecutive weeks. Body weight changes were evaluated weekly until the 11 th week.
  • liver and epididymal white adipose tissue were removed and weighed.
  • the extract according to the present invention 1,000 mg/Kg, p.o.
  • liraglutide 0.2 mg/Kg, s.c.
  • This effect was evident in the 5 th week and lasted until the 11 th week.
  • the effect of ST-076 was evaluated in a rat model of Type 2 Diabetes Mellitus (T2DM).
  • T2DM Type 2 Diabetes Mellitus
  • the hypoglycemic activity, changes in body weight, water intake, diuresis and possible interactions with blood pressure were investigated.
  • fasted Sprague Dawley rats were treated orally with nicotinamide and, 15 minutes later, streptozotocin was administered through caudal vein for induction of experimental T2DM.
  • glucose levels were evaluated to confirm the induction of T2DM, and animals considered diabetic (blood glucose above 150 mg/dL) were randomly assigned to different experimental groups and treated for 4 weeks. Weekly assessments of body weight and fasting blood glucose as well as urine volume and water consumption were performed.
  • T2DM leads to cardiovascular changes
  • parameters such as blood pressure were evaluated in animals with T2DM and the effect of the extract according to the present invention on these parameters was observed. Over the 4 weeks of the study, the maintenance of fasting hyperglycemia was observed. These data indicate that animals with T2DM remained diabetic for the entire experimental period.
  • T2DM Type 2 Diabetes Model
  • mice were treated daily with the Vehicle and presented glycemic levels of 217.1 ⁇ 27.4, 198.1 ⁇ 25.0, 185.6 ⁇ 24.2 and 230.2 ⁇ 26.6 mg/dL after the 1 st , 2 n d, 3 rd and 4 th weeks of treatment, respectively.
  • Blood glycemia in T2DM animals treated with the vehicle was higher than the group of non-diabetic animals (p ⁇ 0.05), in all evaluations after induction of T2DM, indicating that animals remained diabetic until the end of the experimental protocol.
  • the second group of diabetic animals showed basal glycemia (before T2DM induction) of 94.6 ⁇ 3.6 mg/dL, and after diabetes induction, glycemic levels reached 230.9 ⁇ 21.6 mg/dL.
  • animals were treated daily with the Test Article according to the present invention (500 mg/kg) and presented glycemic levels of 145.6 ⁇ 27.7, 164.0 ⁇ 29.5, 131.8 ⁇ 22.7 and 221.8 ⁇ 39.3 mg/dL, following the 1 st , 2 nd , 3 rd and 4 th week of treatment, respectively.
  • the third group of diabetic animals showed basal glycemia (before T2DM induction) of 93.0 ⁇ 4.1 mg/dL, and after diabetes induction the glycemia was 234.7 ⁇ 26.2 mg/dL.
  • animals were treated daily with the ST-076 extract according to the present invention (1,000 mg/kg) and presented glycemic levels of 114.6 ⁇ 24.2, 125.0 ⁇ 22.9, 101.7 ⁇ 9.1 and 252.0 ⁇ 59.6 mg/dL, following the 1 st , 2 nd , 3 rd and 4 th week of treatment, respectively (Figure 17A).
  • the fourth group of diabetic animals showed basal glycemic values (before T2DM induction) of 101.0 ⁇ 4.2 mg/dL, and after diabetes induction the glycemia was 263.3 ⁇ 26.5 mg/dL.
  • animals were treated daily with Liraglutide (0.2 mg/kg, s.c.) and presented glycemic levels of 113.4 ⁇ 27.6, 113.6 ⁇ 11.6, 116.4 ⁇ 15.4 and 166.8 ⁇ 2011 mg/dL, after the 1 st , 2 nd , 3 rd and 4 th week of treatment, respectively (Figure 17A).
  • the reduction of glycemia in general was more evident in the area under the curve ( Figure 17B).
  • RT-qPCR was performed in liver, hypothalamus and adipose tissue, which are important tissues in regulation of energy homeostasis and feeding behavior.
  • PCR-array allowed the evaluation of the expression of most genes related to lipid metabolism and its possible modulation with the daily use of ST-076.
  • Results obtained with PCR-array in liver and adipose tissue were validated using RT-qPCR. For these analyzes, total RNA from tissues was isolated and transcribed in cDNA using random primers and probes.
  • the PCR-assay data in the adipocyte tissue showed an increase in the expression of Growth Hormone Secretagogue Receptor ( Ghsr ) gene promoted by ST-076.
  • Ghsr Growth Hormone Secretagogue Receptor
  • This gene is related to energy homeostasis and body weight regulation.
  • treatment with ST-076 induced an increase in transcription of the following genes: Ramp3 (Receptor activity-modifying protein 3), Insulin-2 (Ins2), Peptide YY (Pyy), Somatostatin Receptor 2 (Sstr2) and Urocortin (Ucn).
  • These genes are related to amylin modulation, glycemia control and satiety induction.
  • Zinc Finger Protein Atypical E3 Ubiquitin Ligase Zfp91
  • Zfp91 Zinc Finger Protein Atypical E3 Ubiquitin Ligase
  • NFkB non-canonical Nuclear Factor kappa B
  • RT-qPCR revealed an increase in the expression of Cocaine-And Amphetamine-Regulated Transcript Protein ( Cartpt ) ( Figure 21A), Fatty Acid Amide Hydrolase ( Faah ) ( Figure 21B), Glucagon Receptor (Gcgr) (Figure 21C), Neuropeptide Y (Npy) ( Figure 21C), Monoacylgliceride Lipase (Mgll) ( Figure 22A), Peroxisome Proliferator Activated Receptor alpha (Ppara) ( Figure 23A), Peroxisome Proliferator Activated Receptor gamma (Pparg) ( Figure 23B) and Receptor Activity-Modifying Protein 3 (Ramp3) ( Figure 23C), genes expression showed an increase by ST-076.
  • the NLR Family Pyrin Domain Containing 3 (Nlrp3) ( Figure 22B) gene expression showed a significant reduction in Nlrp3 gene expression promoted by ST-076.
  • RT-qPCR revealed that the invention ST-076 promoted increased Fatty Acid Amide Hydrolase ( Faah ) ( Figure 24A), Monoglyceride Lipase ( Mgll ) ( Figure 24B) and Peroxisome Proliferator-Activated Receptor alpha (Ppara) (figure 24C) gene expression.
  • Faah Fatty Acid Amide Hydrolase
  • Mgll Monoglyceride Lipase
  • Ppara Peroxisome Proliferator-Activated Receptor alpha
  • the ST-076 treatment decreased inflammation and NF ⁇ B plays a critical role in regulating inflammation.
  • Many NF ⁇ B target genes [Fig. 30]), including TNF- ⁇ and MCP-1, are also implicated in the development of obesity-induced insulin resistance (Lee and Lee, 2013).
  • treatment with ST-076 reduces plasma levels of TNF- ⁇ and MCP-1.
  • the PCR-array ( Figures 19 and 20) and PCR results ( Figures 21 – 24) indicate that ST-076 interferes with biological processes related to the regulation of the processes of response to food, appetite and nutrient levels. Leptin resistance is characterized by reduced satiety, over-consumption of nutrients, and increased total body mass (Izquierdo, et al., 2019).
  • Phosphatidylinositol 3-kinase (PI3K) inhibition could potentiate the effect of ⁇ -adrenergic agonists in the treatment of obesity (Huang et al., 2018; Araiz et al., 2019).
  • the catecholamine norepinephrine binds beta-adrenergic receptors on the plasma membrane of adipocytes by GPCR signaling pathway (Kim et al., 2005). These receptors are coupled with Gs-proteins that transmit a stimulatory signal to adenylyl cyclase to generate cAMP.
  • cAMP binds PKA, causing the regulatory subunits to dissociate from the catalytically active subunits, resulting in increased activity of the enzyme, decreased activity of AMPK ( Figure 25A and 27C) while increased PKA and Extracellular signal-regulated kinases (ERK1/2) in adipocytes ( Figure 25C), thus contributing to the regulation of lipolysis ([Fig.25] and 26) (Ducan et al., 2007; Gauthier et al. 2008).
  • test item ST-076 is highly effective in preventing body weight ( Figures 7 and 12) gain in diet-induced obesity mouse model. These results seem to be associated to an improvement in clinical biochemical parameters ( Figures 11, 13 and 14) related to obesity and metabolic diseases, such as, reduction of blood glucose levels, decreased insulin resistance and impairment of inflammatory response ([Fig. 30]).
  • the PCR-array results ( Figures 19 and 20) indicate that ST-076 interferes with biological processes related to the regulation of the processes of response to food, appetite and nutrient levels.
  • immunohistochemistry data Figures 25 – 29
  • cytokines quantification [Fig. 30] suggest that the underlying mechanism of action of ST-076 occurs through interaction with GPCR signaling pathways ([Fig. 31]).
  • Genotoxicity tests were developed to detect substances with the potential to induce damage to genetic material and are recommended by regulatory agencies worldwide as part of the safety assessment of chemicals. These tests identify risks related to DNA damage. Substances found positive in these tests that detect genetic modifications are potentially carcinogenic and / or mutagenic to humans. Thus, the bacterial mutagenicity test is widely used as an initial screening to assess possible genotoxic activity, in particular, for point mutation-inducing activity.
  • the reverse bacterial mutation assay was performed in accordance to OECD recommendations, guideline 471 - Guideline for Testing of Chemicals. Method 471 “Bacterial Reverse Mutation Test” (Adopted: 26 June 2020).
  • ST-076 showed no mutagenic activity in the reverse mutation test in Salmonella typhimirium bacteria, both in the absence and in the presence of metabolic activation (S9), in the TA 97a, TA 98, TA 100, TA 102 and TA 1535.
  • S9 metabolic activation
  • the micronucleus test detects genetic changes resulting from chromosomal lesions and/or damage to the mitotic system.
  • the formation of micronuclei is an indicative of irreversible losses to DNA and, its frequency can be used as an index of mutagenicity. It is already known that there is a positive correlation between the increase in the frequency of micronuclei and the appearance of tumors in rodents and humans.
  • the micronucleus test in mouse bone marrow was carried out in a GLP compliant condition in accordance to the recommendations of OECD guideline 474 - Guideline for Testing of Chemicals. Method 474 “Mammalian Erythrocyte Micronucleus Test” (Adopted: 29 July 2016). The results are shown in table 13.
  • mice treated with ST-076 Incidence of micronucleated polychromatic erythrocytes (MNPCE) and the ratio of polychromatic erythrocytes (PCE) to normochromatic erythrocytes in mice treated with ST-076.
  • Group Dose mg/Kg
  • Route MNPCE/4,000 PCE Mean ⁇ S.D.
  • Ratio PCE/NCE Mean ⁇ S.D.
  • Negative Control Water 0 p.o. 6.20 ⁇ 3.58 1.60 ⁇ 0.27 Test Item (ST-076) 2,000 p.o. 5.20 ⁇ 2.86 1.31 ⁇ 0.14 Positive Control (Cyclophosphamide) 25 i.p.
  • Phase I to determine the maximum tolerated dose (MTD) of ST-076, and Phase II) exploratory evaluation of the toxicity of repeated treatments with ST-076 for 7 days, as described below:
  • Phase I Determination of MTD by a single oral administration of ST-076 in an escalating dose scheme.
  • Animals were randomly distributed in five experimental groups (3 males and 3 females/group, Sprague Dawley rats). Animals from the vehicle-treated group were treated with physiologic solution (NaCl 0.9 %) and four additional groups were treated with different doses of ST-076 (175 mg/kg, 550 mg/kg, 1,750 mg/kg and 5,000 mg/kg). All animals were euthanized 14 days after the treatment for subsequent analyses.
  • Phase II 7-day repeated toxicity study.
  • the protocol consisted of two experimental groups (5 males and 5 females/group, Sprague Dawley rats). Group 1 was treated with the Vehicle and the group 2 was treated with ST-076 (1,000 mg/kg). All animals were treated by oral gavage once a day for 7 days. All animals were euthanized 14 days after the treatment for subsequent analyses. Clinical and behavior observations, as well as general macroscopic observations in the necropsy were carried out for animals from both phases as well as the hematological and biochemical analyzes for Phase II animals.
  • phase I of the study no adverse effects indicative of toxicity was observed in groups treated with different doses of ST-076.
  • the dose of ST-076 selected for phase II was 1,000 mg/kg, which is the recommended limit dose for repeated dose studies (ICH M3 (R2)).
  • ICH M3 (R2) the recommended limit dose for repeated dose studies
  • repeated treatment with ST-076 (1,000 mg/kg) did not cause death nor clinical signs toxicity.
  • no macroscopic and histopathological changes were observed in organs and tissues collected from animals treated with ST-076 (1,000 mg/kg).
  • the purpose of this study was to assess the potential toxicity after treatment with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis administered by oral route for 90 days in rats.
  • Male and female Sprague Dawley rats (7-9 weeks) were used. Animals were maintained under SPF (Specific Pathogen Free) animal conditions and were obtained from CIEnP facility, whose breeding colonies were purchased from Charles River Laboratories (USA).
  • SPF Specific Pathogen Free
  • necropsy On days 91 (animals of the main groups) and 105 (recovery groups), animals were subjected to necropsy. During necropsy, the outer surface of the body, orifices, cranial, thoracic and abdominal cavities, as well as their contents of each animal were examined. In the analysis of the body surface, a detailed evaluation was performed and the presence of lesions or deformities, size, color, texture, shape, severity, as well as weight and volume were noted and recorded.
  • Morbidity and mortality were evaluated twice daily throughout the study. There were deaths of animals from the main groups (200 mg/kg, 500 mg/kg and 1,000 mg/kg) and recovery group (1,000 mg/kg). The results of the histopathological evaluation of animals found dead did not indicate any relationship between treatment with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis and the death of the animals.
  • General and detailed clinical signs were evaluated daily. Detailed clinical signs were performed once before the beginning of the treatments to verify the health status of the animals, and once a week thereafter. Oral administration of the standardized aqueous extract obtained from the leaves of Ilex paraguariensis at doses of 200 mg/kg, 500 mg/kg and 1,000 mg/kg, did not result in any observable changes in general and detailed clinical signs. Moreover, any observable changes in general and detailed clinical signs were observed in recovery groups.
  • Body weight change s and food consumption Body weight and food consumption were measured once before the start of treatments (baseline) and then once a week.
  • Main groups no significant changes in body weight related to the treatment with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis (200 mg/kg, 500 mg/kg or 1,000 mg/kg) ([Fig. 32]) were observed.
  • food intake/week/animal was reduced in male animals treated with 200 mg/kg of the standardized aqueous extract obtained from the leaves of Ilex paraguariensis at weeks 9 and 10, when compared to Vehicle-treated males.
  • Recovery groups no changes in body weight or on the average feed intake/week/animal related to the treatment was observed in recovery groups when compared to the control.
  • Organs weight After the necropsy procedure, the absolute and relative weights (g) of the main organs (adrenal glands, spleen, brain, heart, kidney, thymus, liver, testicles, epididymis and ovary) were measured for each animal in all experimental groups.
  • Main groups treatment with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis (200 mg/kg, 500 mg/kg or 1,000 mg/kg) did not cause significant changes in either absolute or relative weight of main organs.
  • Hematology Main groups : there was a slight decrease in white blood cells in male animals, as well as a slight increase in white blood cells in female treated with standardized aqueous extract obtained from the leaves of Ilex paraguariensis at doses of 500 mg/kg or 1,000 mg/kg (males – Table 14) and 200 mg/kg (females – Table 15).
  • Recovery groups an increase in platelets and neutrophil count, as well as a slight reduction in lymphocytes count in males rats treated with standardized aqueous extract obtained from the leaves of Ilex paraguariensis (1,000 mg/kg) were observed when compared to the vehicle group.
  • Table 14 Analysis of hematological parameters of male rats after 90 days of daily treatment with ST-076.
  • Clinical chemistry Main groups : increased cholesterol serum levels (1,000 mg/kg) in males (Table 16) as well as increased ALT serum levels (500 mg/kg), cholesterol and alkaline phosphatase (1,000 mg/kg) in females (Table 17) treated with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis when compared to the vehicle group were observed.
  • Recovery groups a reduction in urea serum levels in males and an increase in glucose serum levels as well as a reduction in calcium and urea levels in females treated with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis were observed when compared to the vehicle group.
  • Table 16 Analysis of biochemical parameters of male rats after 90 days of daily treatment with ST-076.
  • GRUPO VEHICLE DOSE 1 200 mg/kg
  • DOSE 2 500 mg/kg
  • DOSE 3 1,000 mg/kg
  • Units Mean SD& Mean SD& Mean SD& Mean SD& ALT (U/L) 91.130 50.181 65.025 16.235 88.244 34.775 56.413 10.867
  • GGT U/L
  • 0.600 1.075 0.000 0.000 5.889 9.714 0.375 0.744
  • TRI (mg/dL) 71.100 27.045 74.000 11.414 85.444 19.717 63.250 12.453
  • PT 9.681 0.406 9.691 0.342 9.711 0.461 9.406 0.379
  • CRE mg/dL
  • 0.276 0.022 0.258 0.018 0.211 0.162 0.246 0.049
  • AST U/L 152.570 75.203 115.638 20.968 141.367 41.531 96.088 6.994 C ⁇ LCIO (mg/dL) 6.389
  • Urinalysis No significant changes in urine parameters (volume, specific gravity, pH, protein) of the main groups treated with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis (200 mg/kg, 500 mg/kg and 1,000 mg/kg) were observed when compared to the vehicle. Moreover, any visible changes were observed in urine parameters of the recovery group (1,000 mg/kg) when compared to the vehicle group.
  • Ophthalmology Ophthalmological evaluations were performed in male and female rats treated with the Vehicle or with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis (1,000 mg/kg). Since no changes were observed in the ophthalmological health of both sexes in the highest dose (1,000 mg/kg), the analysis was not extended to the other experimental groups (main and recovery).
  • Macroscopy did not reveal any significant changes related to the treatment with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis for 90 days at doses of 200 mg/kg, 500 mg/kg and 1,000 mg/kg. Moreover, no significant macroscopic changes were observed in the recovery groups when compared to the vehicle.
  • Histopathology There were no effects related to the treatment with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis (1,000 mg/kg) in any of the organs and tissues examined, when compared to animals from the Vehicle control group. Due to the absence of treatment-related effects, organs and tissues from groups treated with 200 and 500 mg/kg as well as recovery groups were not analyzed, as recommended by the OECD 408 guideline.
  • the Irwin Test integrates the core battery for evaluation of Central Nervous System (CNS) Safety Pharmacology preconized by ICH S7A guideline (2000).
  • CNS Central Nervous System
  • Data generated by the Irwin Test can be grouped in categories related to effects on general activity (excitation/sedation), motor/coordination behavior, response to stimulus and autonomic signs.
  • the aim of this assay was to evaluate the potential neurotoxic effect of acute administration of the extract according to the present invention in rats comparatively to the well-established effects of Reference Items on the CNS.
  • rats were treated orally with Bromazepam (10 mg/kg), Caffeine (24 mg/kg) or with the extract according to the present invention (50, 1,000 and 2,500 mg/kg) and evaluated in 0-15; 30; 60; 120 and 240 minutes and 24 hours after treatment. Behavior modifications, physiological and neurotoxicity symptoms as well as temperature were recorded according to a standardized observation grid derived from that previously described by Irwin (1968) for mice and adapted for rats (modified Irwin Test).
  • Results show that the extract according to the present invention induced clinical signals and behavior alterations indicative of excitatory effects in all tested doses including increased general activity (excitation) in different levels as well as in reactivity to touch stimuli and exacerbation of the fear/scare behavior in comparison to the control group (Vehicle). Moreover, treatment with the extract in accordance to the present invention increased respiratory rate and muscular tonus.
  • results of the present assay demonstrate that the ST-076 according to the present invention administered orally after 10 days in rats, caused a middle excitatory effect, such as increased general activity, reactivity to the touch stimulus, respiratory rate and exacerbation of behavior of fear/fright when compared to animals treated with Vehicle.
  • OECD 2018 Aug 6;14(11):1483-1496.
  • OECD (2020), Test No. 471: Bacterial Reverse Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264071247-en.
  • OECD (2018), Test No.
  • ICH S7A Safety Pharmacology Studies for Human Pharmaceuticals, 2000.
  • ICH S7B The Non-Clinical Evaluation of the Potential for Delayed Ventricular Repolarization

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Abstract

The present invention is a method of preparation of standardized ethanolic and aqueous plant extract powder from Ilex paraguariensis leaves for human or veterinary use. The present invention also provides the identification of compounds including rutin, caffeine, theobromine, theophylline and caffeoylquinic acid and its isomers quantified as chlorogenic acids, particularly obtained from a standardized aqueous or ethanolic extract from Ilex paraguariensis plant. The extract of Ilex paraguariensis plant is rich in methylxanthines such as caffeine, theobromine and theophylline which presented good oral bioavailability. Moreover, the present invention describes the use of Ilex paraguariensis plant extract for the preventive and/or therapeutic treatment of obesity and related metabolic diseases, including type 2 diabetes, hypertension, heart disease, lipid dysfunction, non-alcoholic fatty liver disease (steatosis), inflammatory diseases and other related diseases associated with metabolic syndrome. Moreover, the extract of Ilex paraguariensis plant was safe (no toxic) and not mutagenic when tested in vivo. The mechanism of action by which extract of Ilex paraguariensis plant improved obesity and metabolic syndrome seems to be related with the up or down regulation of most relevant genes in peripheral organs and in the central nervous system, altering cellular singling pathways involved in such diseases.

Description

PHARMACEUTICAL PRODUCT FOR THE TREAMENT OF OBESITY AND ASSOCIATED METABOLIC SYNDROME DISEASES, USES AND METHODS OF TREATMENT. Field of the invention
The present invention generally relates to pharmaceutical product for human or veterinary use for the preventive or therapeutic treatment of obesity and related metabolic syndrome diseases, comprising a standardized aqueous or ethanolic extracts containing a mixture of natural compounds such as rutin, caffeine, theobromine, theophylline and chlorogenic acids, particularly obtained from the leaves of the plants from genus Magnoliopsida, family Aquifoliaceae, especially from Ilex paraguariensis, as well its preventive and therapeutic uses, methods of preparation, in vitro and in vivo pharmacological effects, pharmacokinetic, safety and its molecular mechanism of action.
Background of the invention
The prevalence of obesity has dramatically increased worldwide in the past 50 years, reaching pandemic levels in most occidental countries. Obesity currently represents a major health burden as it substantially increases the risk of many chronic diseases such as type 2 diabetes mellitus, fatty liver disease (steatosis), hypertension, myocardial infarction, stroke, dementia, osteoarthritis, obstructive sleep apnea and several cancers, among others, thereby contributing to a decline in both quality of life and life expectancy.
Obesity and metabolic diseases can be interrelated and the prevention and treatment strategies for both have not been successful in a long term. Lifestyle and behavioral interventions aimed at reducing calorie intake and increasing energy expenditure have limited effectiveness because the complex and persistent hormonal, metabolic and neurochemical adaptations defend against weight loss and promote weight regain. Reducing the obesity burden requires approaches that combine appropriated drug intervention combined with changes in the environment and society.
Despite the great scientific advances that have taken place in the last few decades, few safe and effective therapies are currently available for the treatment of diseases associated with obesity and the metabolic syndrome. The main available medicines to treat obesity and metabolite syndrome include orlistat, lorcaserin, liraglutide, semaglutide and some combination of drugs, such as diethylpropion-phentermine-phendimetrazine-benzphetamine, phentermine-topiramate, or naltrexone-bupropion sustained release. Nevertheless, most of clinical availably medicines to treat obesity and metabolite diseases cause severe side effects that can be directly associated with these drugs themselves or by their combination. Thus, the development of a safer and a more efficacious therapy is urgently needed. Furthermore, the metabolite syndrome associated with obesity is an immense burden for public health system and for quality of life of the patients and constitute an unmet medical need.
 Summary of the invention
The present invention provides the development method for preparation of aqueous and ethanolic standardized extracts and presents the use of such combination of natural compounds, particularly the standardized aqueous extract obtained from the leaves of the plants of genus Magnoliopsida, preferably Ilex paraguariensis , in the manufacture of a pharmaceutical product. Standardized aqueous and ethanolic extract obtained from the leaves of the plant of the genus Magnoliopsida, family Aquifoliaceae, more especially from Ilex paraguariensis (also called in this invention as ST-076, ST-076.1, ST-076.2, ST-076.3, ST-076.4, ST-076.7 or ST-076.8), contain a mixture of compounds including rutin, caffeine, theobromine, theophylline, and caffeoylquinic acid and its isomers quantified as chlorogenic acid, among others.
The present invention provides the development of a pharmaceutical product for human or veterinary use, composed of standardized and very stable aqueous extracts from the leaves of Ilex paraguariensis . The extract given orally was found to be very effective given either in a preventive or in a therapeutic manner for the treatment of obesity and related metabolic syndrome diseases, such as type 2 diabetes, hypertension, dyslipidemia, steatosis, inflammation, among others. Furthermore, the standardized aqueous extract obtained from the leaves of Ilex paraguariensis can be considered safe as it was not genotoxic nor toxic according to acute and repeated dose 90-day oral toxicity assessments.
The mechanism by which the standardized aqueous extract obtained from the leaves of Ilex paraguariensis improves the obesity and metabolic syndrome, seems to be related with up or down regulation of most relevant genes in both peripheral organs and central nervous system with changing of cellular singling pathways which are involved in regulation of obesity, metabolic diseases and others related diseases.
Brief description of the figures
[Figures 1]. Chromatogram showing the phytochemical constituents found in the dried extract prepared from the standardized aqueous extract from the leaves of Ilex paraguariensis, also called in this invention as ST-076.
[Fig.2]. Pharmacokinetic for oral (p.o.) administration of ST-076 and evaluation of plasma concentration profile of caffeine, theobromine and theophylline after acute administration according to the present invention (1,000 mg/Kg, p.o.). The blood was collected at 0.083, 0.5, 1, 2, 4, 8, 12, 16 and 24 hours after ST-076 oral administration for the determination of pharmacokinetic profiles (AUClast, Tmax, Cmax, T1/2 and Ke). Data are represented as mean ± standard error of mean (S.E.M.) of 5 animals for each time point.
[Fig.3]. Plasma concentration profile of caffeine, theobromine and theophylline following a 7-day treatment with ST-076 according to the present invention (1,000 mg/Kg, p.o.). The blood was collected at 0.083, 0.5, 1, 2, 4, 8, 12, 16 and 24 hours after ST-076 administration for the determination of pharmacokinetic profiles (AUClast, Tmax, Cmax, T1/2 and Ke). Data are represented as mean ± S.E.M. of 5 animals for each time point.
[Fig.4]. In vivo CYP3A4-mediated possible interaction with ST-076. The animals were treated with vehicle (1 ml/kg; p.o.) or with ST-076 (1,000 mg/kg; p.o.) for 7 consecutive days. On the seventh day, 10 mg/kg of midazolam, a CYP3A4 substrate, was administered and blood samples were collected at 0.083, 0.25, 0.5, 1, 2 e 4 hours after drug administration for the determination of pharmacokinetic profiles (AUClast, Tmax, Cmax e T1/2). Data are represented as mean ± S.E.M. of 3 animals for each time point.
[Fig.5]. Effect of ST-076 on body weight gain induced by high-fat diet in mice. Body weight gain was evaluated from week 1 to week 16. Animals were fed with normal chow diet (NCD) or with high-fat diet (HFD) during 16 weeks. From week 8 until week 16, mice that were fed with HFD began to be daily treated with vehicle (10 mL/Kg, p.o.), ST-076 (500 or 1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c. used as a positive control drug). Body weight was measured once a week. Data are presented as mean ± S.E.M. Two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test were performed. # indicates significant statistical difference (p < 0.05) when compared to NCD group. * indicates significant statistical difference (p < 0.05) when compared to vehicle HFD. NCD: Normal Chow Diet; HFD: High-fat Diet. 9-10 animals per group.
[Fig.6]. Effect of ST-076 on food intake. Animals were fed with NCD or HFD during 16 weeks. From week 8 until week 16, mice that were fed with HFD began to be daily treated with vehicle (10 mL/Kg, p.o.), ST-076 (500 or 1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.). Food intake was measured once a week until the end of protocol. Data are presented as mean ± S.E.M. Two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test were performed. # indicates significant statistical difference (p < 0.05) when compared to NCD group. NCD: Normal Chow Diet; HFD: High-fat Diet. 9-10 animals per group.
[Fig.7]. Effect of ST-076 on serum glucose levels in an Oral Glucose Tolerance Test (OGTT). Animals were fed with NCD or HFD during 16 weeks. From week 8 until week 16, mice that were fed with HFD began to be daily treated with vehicle (10 mL/Kg, p.o.), ST-076 (500 or 1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.). On week 15, an OGTT were performed. Data are presented as mean ± S.E.M. Two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test were performed. # indicates significant statistical difference (p < 0.05) when compared to NCD group. * indicates significant statistical difference (p < 0.05) when compared to vehicle HFD. NCD: Normal Chow Diet; HFD: High-fat Diet. 9-10 animals per group.
[Fig.8]. Representative images of mice phenotype in the end of the study of high-fat diet induced obesity. Animals were fed with NCD or HFD during 16 weeks. From week 8 until week 16, mice that were fed with HFD began to be daily treated with vehicle (10 mL/Kg, p.o.), ST-076 (500 or 1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.). NCD: Normal Chow Diet; HFD: High-fat Diet.
[Fig.9]. Effect of ST-076 on high-fat diet induced fat accumulation in mice. Animals were fed with normal chow diet (NCD) or high-fat diet (HFD) during 16 weeks. From week 8 to week 16, mice that were been fed with HFD began to be daily treated with vehicle (10 mL/Kg, p.o.), ST-076 (1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.). On week 16 animals were euthanized and epididymal white fat tissues were removed. H&E staining of epididymal white fat tissue was performed in all groups. A ×100; B ×400.
[Fig.10]. Histological micrograph showing the effect of ST-076 on high-fat diet induced liver fat accumulation in mice. Animals were fed with NCD or HFD during 16 weeks. From week 8 to week 16, mice that have been fed with HFD began to be daily treated with vehicle (10 mL/Kg, p.o.), ST-076 (1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.). On week 16 animals were euthanized and liver were removed. H&E staining of liver tissue was performed in all groups. A ×100; B ×400. NCD: Normal Chow Diet; HFD: High-fat Diet.
[Figure 11A and 11B]. Effect of ST-076 on clinical chemistry parameters associated to metabolic diseases. (A) Total cholesterol serum levels and (B) triglycerides serum levels. Animals were fed with NCD or HFD during 16 weeks. From week 8 until week 16, mice that were fed with HFD were treated daily with vehicle (10 mL/Kg, p.o.), ST-076 (1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.). On week 16, animals were euthanized, and blood samples were obtained for clinical chemistry analysis of total cholesterol and triglycerides. Data are presented as mean ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. # indicates significant statistical difference (p < 0.05) when compared to NCD group. * indicates significant statistical difference (p < 0.05) when compared to vehicle HFD. NCD: Normal Chow Diet; HFD: High-fat Diet. 9-10 animals per group.
[Figure 12A and 12B]. Effect of ST-076 on liver and inguinal (ingWAT) white tissue weights. (A) Liver weight and (B) Inguinal white adipose tissue (IngWAT) weight. Animals were fed with NCD or HFD during 16 weeks. From week 8 to week 16, mice that have been fed with HFD began to be daily treated with vehicle (10 mL/Kg, p.o.), ST-076 (1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.). On week 16, animals were euthanized, liver and IngWAT were removed and weighed. Data are presented as mean ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. # indicates significant statistical difference (p < 0.05) when compared to NCD group. * indicates significant statistical difference (p < 0.05) when compared to vehicle HFD. NCD: Normal Chow Diet; HFD: High-fat Diet. 9-10 animals per group.
[Figure 13A and 13B]. Effect of ST-076 on biochemistry parameters associated to metabolic diseases. (A) Glycemia serum levels and (B) Insulin serum levels. Animals were fed with NCD or HFD during 16 weeks. From week 8 until week 16, mice that were fed with HFD were treated daily with vehicle (10 mL/Kg, p.o.) or ST-076 (1,000 mg/Kg, p.o.). On week 16, animals were euthanized and blood was obtained for clinical chemistry analysis. Data are presented as mean ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. # indicates significant statistical difference (p < 0.05) when compared to NCD group. * indicates significant statistical difference (p < 0.05) when compared to vehicle HFD. NCD: Normal Chow Diet; HFD: High-fat Diet. 7-9 animals per group.
[Figure 14A and 14B]. Effect of ST-076 on biochemistry parameters associated to metabolic diseases. (A) Glucagon Like Peptide 1 (GLP-1) and (B) Leptin serum levels. Animals were fed with NCD or HFD during 16 weeks. From week 8 until week 16, mice that were fed with HFD were treated daily with vehicle (10 mL/Kg, p.o.) or ST-076 (1,000 mg/Kg, p.o.). On week 16, animals were euthanized and blood was obtained for clinical chemistry analysis. Data are presented as mean ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. # indicates significant statistical difference (p < 0.05) when compared to NCD group. * indicates significant statistical difference (p < 0.05) when compared to vehicle HFD. NCD: Normal Chow Diet; HFD: High-fat Diet. 7-9 animals per group.
[Fig.15]. Effect of ST-076 on body weight gain induced by high-fat diet in rats. Body weight gain from week 1 to week 11. Animals were fed with NCD or HFD during 11 weeks and were treated daily with vehicle (10 mL/Kg, p.o.), ST-076 (1,000 mg/Kg, p.o.) or with liraglutide (0.2 mg/Kg, s.c.). Body weight was measured once a week. Data are presented as mean ± S.E.M. Two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test were performed. # indicates significant statistical difference (p < 0.05) when compared to NCD group. * indicates significant statistical difference (p < 0.05) when compared to vehicle HFD. NCD: Normal Chow Diet; HFD: High-fat Diet. 7-9 animals per group.
[Figure 16A and 16B]. Effect of ST-076 on liver and epididymal white adipose tissue (EpiWAT) weights in rats fed with high-fat diet. Liver weight (A) and EpiWAT (B). Animals were fed with normal chow or high-fat diet during 11 weeks and were daily treated with vehicle (10 mL/Kg, p.o.), ST-076 (1,000 mg/Kg, p.o.) or Liraglutide (0.2 mg/Kg, s.c.). Liver and EpiWAT were removed at the end of experimental protocol. Data are represented as Dunnett post hoc test were performed. * indicates significant statistical difference (p < 0.05) when compared to HFD vehicle group. NCD: Normal Chow Diet; HFD: High-fat Diet. 7-9 animals per group.
[Figure 17A and 17B]. Effect of ST-076 on blood glucose in Type 2 Diabetes Model (T2DM). One experimental group received saline (non-diabetics, ND), while the other groups received nicotinamide (180 mg/kg, i.p.) and then streptozotocin (65 mg/kg, i.v.) (DB). ND animals were treated with vehicle; DB animals were treated with vehicle, Liraglutide (0.2 mg/kg; s.c.), ST-076 (500 mg/kg; p.o.) or ST-076 (1,000 mg/kg; p.o.). (A) Glycemia assessment was performed once a week throughout the experimental period; (B) Area Under Curve from graph A (1st to the 4th week: AUC1-4 week). Data are expressed as mean ± S.E.M of 8-18 animals per group. Two-way analysis of variance (ANOVA) (A) and one-way ANOVA (B) followed by the Bonferroni post hoc test were performed. #p <0.05 when compared to the ND + Vehicle group; * p <0.05 when compared to the DB + Vehicle group.
[Figure 18A, 18B and 18C]. Effect of ST-076 on cardiovascular parameters of animals with Type 2 Diabetes Model (DMT2). One experimental group received saline (non-diabetics, ND), while the other groups received nicotinamide (180 mg/kg, i.p.) and then streptozotocin (65 mg/kg, i.v.) (DB). ND animals were treated with vehicle, DB animals were treated with vehicle, Liraglutide (0.2 mg/kg; s.c.), ST-076 (500 mg/kg; p.o.) or ST-076 (1,000 mg/kg; p.o.). Two weeks after treatments, Systolic Blood Pressure (A), Diastolic Blood Pressure (B) and Mean Arterial Pressure (C) were measured in conscious animals. Data are expressed as mean ± S.E.M of 7-13 animals per group. One-way analysis of variance (ANOVA) was performed.
[Fig.19]. Evaluation of ST-076 mechanism of action in the hypothalamus of mice through PCR-array data. mRNA expression levels were normalized to the expression level of 18S housekeeping gene using the 2−ΔΔCt formula. The results were expressed as fold change. Heat map represents the fold gene in mRNA expression compared to NCD + Vehicle group for every identified gene. NCD: Normal Chow Diet; HFD: High-fat Diet. NCD + Vehicle (p.o.); 2) HFD + Vehicle (p.o.); 3) HFD + ST-076 (1,000 mg/kg, p.o.). n = 9-10 animals per group.
[Fig.20]. Evaluation of ST-076 mechanism of action in white adipose tissue through PCR-array data. mRNA expression levels were normalized to the expression level of 18S housekeeping gene using the 2−ΔΔCt formula. The results were expressed as fold change. Heat map represents the fold gene in mRNA expression compared to NCD+Vehicle group for every identified gene. NCD: Normal Chow Diet; HFD: High-fat Diet. NCD + Vehicle (p.o.); 2) HFD + Vehicle (p.o.); 3) HFD + ST-076 (1,000 mg/kg, p.o.). n = 9-10 animals per group.
[Figure 21A, 21B and 21C]. PCR-array validation using RT-qPCR in white adipose tissue. RT q-PCR analysis of gene expression in white adipose tissue for the following genes: (A) Cocaine-And Amphetamine-Regulated Transcript Protein - Cardpt, (B) Fatty Acid Amide Hydrolase - Faah, and (C) Glucagon Receptor - Gcgr. The mRNA expression levels were normalized to the expression level of 18S housekeeping gene using the 2−ΔΔCt formula. The results were expressed as the means ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. # indicates significant statistical difference (p < 0.05) when compared to NCD + Vehicle group. NCD: Normal Chow Diet; HFD: High-fat Diet. n = 3-4 animals per group.
[Figure 22A, 22B and 22C]. PCR-array validation using RT-qPCR in white adipose tissue. RT q-PCR analysis of gene expression in white adipose tissue was performed for the following genes: (A) Monoacylgliceride Lipase - Mgll, (B) NLR Family Pyrin Domain Containing 3 – Nlrp3, (C) Neuropeptide Y – Npy. The mRNA expression levels were normalized to the expression level of 18S housekeeping gene using the 2−ΔΔCt formula. The results were expressed as the means ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. # indicates significant statistical difference (p < 0.05) when compared to NCD + Vehicle group. * indicates significant statistical difference (p < 0.05) when compared to HFD + vehicle. NCD: Normal Chow Diet; HFD: High-fat Diet. n = 3-4 animals per group.
[Figure 23A, 23B and 23C]. PCR-array validation using RT-qPCR in white adipose tissue. RT q-PCR analysis of gene expression in white adipose tissue was performed for the following genes: (A) the peroxisome proliferator activated receptor alpha - Ppara, (B) peroxisome proliferator activated receptor gamma - Pparg and (C) Receptor Activity-Modifying Protein 3 - Ramp3. The mRNA expression levels were normalized to the expression level of 18S housekeeping gene using the 2−ΔΔCt formula. The results were expressed as the means ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. * indicates significant statistical difference (p < 0.05) when compared to HFD + vehicle. NCD: Normal Chow Diet; HFD: High-fat Diet. n = 3-4 animals per group.
[Figure 24A, 24B and 24C]. PCR-array validation using RT-qPCR in liver tissue. RT q-PCR analysis of gene expression in liver tissue was performed for the following genes: (A) Fatty Acid Amide Hydrolase - Faah, (B) Monoacylgliceride Lipase - Mgll, and (C) peroxisome proliferator activated receptor alpha. The mRNA expression levels were normalized to the expression level of 18S housekeeping gene using the 2−ΔΔCt formula. The results were expressed as the means ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. NCD: Normal Chow Diet; HFD: High-fat Diet. n = 3-4 animals per group.
[Figure 25A, 25B and 25C]. Immunohistochemistry in white adipose tissue for (A) p-AMPK, (B) p-CREB and (C) p-ERK. The area quantification of the images was performed by the ImageJ software. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. * indicates significant statistical difference (p < 0.05) when compared to vehicle HFD. NCD: Normal Chow Diet; HFD: High-fat Diet. n = 3-4 animals per group.
[Figure 26A and 26B]. Immunohistochemistry carried out in white adipose tissue for (A) p-JNK and (B) p-PI3K. Quantification of the image area was performed using the ImageJ software. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. # indicates significant statistical difference (p < 0.05) when compared to NCD group. * indicates significant statistical difference (p < 0.05) when compared to vehicle HFD. NCD: Normal Chow Diet; HFD: High-fat Diet. n = 3-4 animals per group.
[Figure 27A, 27B and 27C]. Immunohistochemistry carried out in liver tissue for (A) Insulin, (B) IRS-1 and (C) p-AMPK. Quantification of the image area was performed using the ImageJ software. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. * indicates significant statistical difference (p < 0.05) when compared to vehicle HFD. NCD: Normal Chow Diet; HFD: High-fat Diet. n = 3-4 animals per group.
[Figure 28A, 28B and 28C]. Immunohistochemistry carried out in liver tissue for (A) p-GSK3β, (B) p-JNK and (C) p-mTOR. Quantification of the image area was performed using the ImageJ software. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. * indicates significant statistical difference (p < 0.05) when compared to vehicle HFD. NCD: Normal Chow Diet; HFD: High-fat Diet. n = 3-4 animals per group.
[Figure 29A and 29B]. Immunohistochemistry carried out in liver tissue for (A) p-PI3K and (B) p-PPAR. Quantification of the image area was performed using the ImageJ software. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. * indicates significant statistical difference (p < 0.05) when compared to vehicle HFD. NCD: Normal Chow Diet; HFD: High-fat Diet. n = 3-4 animals per group.
[Fig.30]. Effect of Test Item ST-076 on cytokine levels. (A) IFN-γ; (B) MCP-1 and (C) TNF-α. Animals were fed with normal chow diet (NCD) or high-fat diet (HFD) during 16 weeks. From week 8 until week 16, mice that were fed with HFD were treated daily with vehicle (10 mL/Kg, p.o.) or ST-076 (1,000 mg/Kg, p.o.). On week 16, animals were euthanized and blood was obtained for cytokines response analysis. Data are presented as mean ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett post hoc test were performed. # indicates significant statistical difference (p < 0.05) when compared to NCD group. * indicates significant statistical difference (p < 0.05) when compared to vehicle HFD. n = 7-9 animals per group.
[Fig.31]. Representative scheme of the ST-076 mechanism of action in adipose tissue obtained from high-fat diet-induced obesity in mice. ST-076 administration showed a decrease in AMPK activity following the increase of ERK1/2 signaling and increase of the lipolysis. In the liver, ST-076 induces activation of PI3K and downstream stimulation of JNK and PPAR pathways, resulting in tissue lipolysis. In addition, the effect of ST-076 seems to be related with release of GLP-1 in serum, which reduces the food intake, changes feeding behavior and tissue lipolysis. NCD: Normal Chow Diet; HFD: High-fat Diet. n = 7-9 animals per group.
[Figure 32A and 32B]. Effect of ST-076 on body weight in repeated dose 90-day oral toxicity study in rats. Body weight gain (A) in males and (B) in females throughout the 13-week treatment period. Animals were treated daily with vehicle (10 mL/Kg, p.o.) or ST-076 (200, 500 or 1,000 mg/Kg, p.o.). Body weight was measured weekly. Data are presented as mean ± S.E.M. Two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test were performed. n = 10 animals per group.
[Figure 33A and 33B]. Effect of ST-076 on hemodynamic parameters in conscious rats. Animals received 2,500 mg/kg of ST-076, by oral route, and were evaluated at 0.25; 0.5; 1; 2; 4 and 24 hours after administration in comparison to basal values (pretreatment, time 0). The hemodynamic parameters recorded were: (A) mean arterial pressure (MAP) and (B) systolic arterial pressure (SAP). The results are represented as the variation between the values obtained before (baseline) and after Acetylcholine administration (Δ – delta). Each column represents the mean ± S.E.M. One-way analysis of variance (ANOVA) followed by Dunnett's post hoc test were performed. n = 11 animals per group.
[Figure 34A and 34B]. Effect of ST-076 on hemodynamic parameters in conscious rats. Animals received 2,500 mg/kg of ST-076, by oral route, and were evaluated at 0.25; 0.5; 1; 2; 4 and 24 hours after administration in comparison to basal values (pretreatment, time 0). The hemodynamic parameters recorded were: (A) diastolic arterial pressure (DAP) (mmHg) and (B) heart rate (HR) (bpm). The results are represented as the variation between the values obtained before (baseline) and after Acetylcholine administration (Δ – delta). One-way analysis of variance (ANOVA) followed by Dunnett's post hoc test were performed. n = 11 animals per group.
Detailed description of the invention
The present invention provides a method of preparation of a very stable pharmaceutical product useful for human and veterinary health for the preventive or therapeutic treatment of obesity and associated metabolic syndrome diseases comprising a combination of selected phytochemical compounds obtained from the standardized aqueous extract obtained from the leaves of Ilex paraguariensis plant including: rutin, caffeine, theobromine, theophylline, and chlorogenic acids.
In one aspect, the method of preparing an Ilex paraguariensis extract provided herein comprises:
(a) contacting Ilex paraguariensis material with water or a solvent at a temperature below boiling to produce a mixture, and milling the mixture to obtain a crude extract;
(b) filtering the crude extract to obtain a purified extract; and
(c) drying and standardizing the alcoholic or aqueous extract to produce the Ilex paraguariensis extract.
Described herein, in some embodiments an Ilex paraguariensis extract prepared according to methods provided herein, the standardized extract preferentially comprising:
At least rutin: about 100 mg to 1,000 mg per 100 grams of standardized extract of Ilex paraguariensis;
At least caffeine: about 100 mg to 3,000 mg per 100 grams of standardized extract of Ilex paraguariensis;
At least theobromine: about 100 mg to 500 mg per 100 grams of standardized extract of Ilex paraguariensis;
At least theophylline: about 2 to 10 mg per 100 grams of standardized extract of Ilex paraguariensis;
At least chlorogenic acids: about 100 mg to 10,000 mg per 100 grams of standardized extract of Ilex paraguariensis;
In a particular embodiment, without limitations, the present invention is a combination of compounds such as Rutin (861.01 mg per 100 grams of ST-076), Caffeine (2,219.43 mg per 100 grams of ST-076), Theobromine (370.92 mg per 100 grams of ST-076), Theophylline (6.89 mg per 100 grams of ST-076) and Chlorogenic acids (6,450.22 mg per 100 grams of ST-076), preferably obtained from a standardized aqueous extract of Ilex paraguariensis.
In a particular embodiment, the phytochemical constituents of the standardized aqueous extract obtained from the leaves of Ilex paraguariensis according to the present invention were quite stable for up to 20 months at a temperature of 20 to 30 °C since Rutin, Chlorogenic Acid, Theophylline, Theobromine and Caffeine were measured by HPLC (High Performance Liquid Chromatography).
The standardized aqueous extract obtained from the leaves of Ilex paraguariensis according to the present invention is useful as it can be given either preventively or therapeutically to a patient in need via oral route, or to be used in the preparation of a pharmaceutical product in the form of pills, liquid, soft capsule, among others, for the treatment of obesity and associated metabolic syndrome diseases.
The pharmaceutical product according to the present invention additionally comprises pharmaceutically acceptable excipients (for human or veterinary use). Suitable excipients for the invention are, for example, and without any limitation, those cited in the book Remington's Pharmaceutical Sciences, by the American publisher Mack Publishing, European Pharmacopoeia or Brazilian Pharmacopoeia. The excipients are selected according to the pharmaceutical form to be composed. Suitable pharmaceutical forms according to the present invention include, without limitations, pill, tablet, capsule, syrup, etc. Such forms are adapted according to the standard route of administration, for instance as approved by The Food and Drug Administration (FDA) at the following website: https://www.fda.gov/drugs/data-standards-manual-monographs/route-administration.
In a second object, the present invention includes the use of the combination of compounds or the standardized aqueous of the plant Ilex paraguariensis in the manufacturing of a pharmaceutical stable product for the treatment of obesity and associated metabolic syndrome diseases. In an alternative embodiment, the standardized aqueous extract obtained from the leaves of Ilex paraguariensis according to the present invention can be administered directly to the patient in need via oral route.
In a third object, the present invention includes methods for the treatment of obesity and/or metabolic diseases comprising the administration of an effective dosage of the pharmaceutical product of the standardized aqueous extract obtained from the leaves of Ilex paraguariensis to a patient in need. The dosage of the standardized aqueous/ethanolic extracts range from about 1 to about 5,000 mg/kg/day, one or at different times a day.
In another aspect, the present invention deals with a method for preventing manifestations involved in the obesity and diseases associated with the metabolic syndrome, as well as in the prophylaxis or treatment of diseases resulting from the syndrome, such as heart disease, lipid problems (steatosis), hypertension, type 2 diabetes, dementia, cancer, polycystic ovarian syndrome, non-alcoholic fatty liver disease, among others.
In the scope of the present invention the standardized aqueous/ethanolic extract is manufactured according to a specific method of preparing comprising the steps of 1) material cleaning; 2) solvent used, 3) time of maceration, 4) filtration, 5) evaporation, 6) drying and 7) stability and storage.
The step “1” (material cleaning) covers the use of different parts of the plant, preferably leaves and branches, separated into “fresh”, “aged” and “mix of fresh and aged leaves”. In a particular embodiment, the process according to the present invention uses a “mix of fresh and aged leaves” and “pulverized plant in natura”. The plant material composed by leaves and branches was washed in running water to eliminate impurities and kept for 1-3 days drying at room temperature. Then, leaves were weighed and placed in an incubator at 50-70 °C for approximately 2-4 days.
The steps “2” and “3” (solvent used and time of maceration) cover preferably the use of 1 part of plant material to 10 parts of Extractor Liquid, which was a mixture of ethanol and water in 50% ratio (v/v) or pure water heated at 90 °C. The extract is left macerating at room temperature (approximately 20 °C) for 3 to 10 days, preferably in the dark, with occasional agitation.
The step “4” (filtration) comprises the filtration of the extracts by gravity with the aid of “voil” fabric filter to remove the thicker part of the plant material (larger particles), followed by vacuum filtration using filter paper of 80.0 g/m², porosity of 3 microns, to remove smaller particulate material.
The step “5” (evaporation) includes the evaporation of ethanol only from hydroalcoholic extracts, particularly by rotary evaporation.
The steps “6” and “7” comprise the drying/storage of aqueous or hydroalcoholic extracts. The aqueous and hydroalcoholic extracts that would be dried in the incubator were distributed into containers previously weighed and placed in the incubator at 50-70 °C; then the extracts were weighed and stored in a freezer at -20 °C until further analysis. The extracts that would be lyophilized were light protected and stored in a freezer at -20 °C.
The lyophilization process took between 7 - 10 days, depending on the volume of extract in each container. After completion of lyophilization, the extracts were homogenized and aliquoted in flasks wrapped with light protection and stored in a freezer at -20 °C until further analysis.
The present invention is described in detail through the examples presented below. It is necessary to emphasize that the invention is not limited to these examples, but also includes variations and modifications within the limits in which it can be developed.
Examples
In order to fully understand the invention described herein, the following examples are set forth. The methods, conditions, and results showed in these examples are not meant to be limiting, but are meant to demonstrate the effects of the present invention. Starting materials and reagents used in these examples, when not prepared by the procedure described herein, are generally either commercially available, or are reported in the chemical literature, or may be prepared by using procedures described in the chemical literature.
Example 1 – Production process and characterization of Ilex paraguariensis extract
In the invention described herein, the first aim was to produce standardized aqueous or hydroalcoholic extracts from plant leaves (ST-076: plant material of Ilex paraguariensis, same as ST-076.1, ST-076.2, ST-076.3, ST-076.4; ST-076.7: an extract of Ilex paraguariensis, same as ST-076.8) on a small scale, but viable for large scale production, and free of potentially toxic substances, such as organic solvents (e.g. methanol, hexane, among others). All extracts yielded an average of 17% in mass. The chemical characterization of the extracts resulting from this invention was performed by identifying the major phytochemical components using selective and sensitive analytical techniques, such as liquid chromatography coupled to mass spectrometry. For this purpose, an Ultra Performance Liquid Chromatography (UPLC) equipment coupled to a mass spectrometer with a triple-quadrupole analyzer (Xevo™ TQ-S, Waters) was used. Theobromine, theophylline, caffeine, rutin and caffeoylquinic acid and its isomers are among the compounds identified. The procedures involved in the extraction process were: 1) material cleaning; 2) maceration, 3) filtration, 4) evaporation, 5) drying and 6) storage.
Cleaning Stage : The leaves and branches were selected and damaged leaves and/or stems were discarded. The remaining leaves were separated into “fresh” (ST-076.1), “aged” (ST-076.2) and “mix of fresh and aged leaves” (ST-076.3). After this separation, the plant material was washed in running water to eliminate impurities and was kept for two days drying at room temperature. Then, the leaves were weighed and placed to dry further in an incubator at 60 °C, for approximately three days. Subsequently, the dried plant material was packed in clean plastic bags and stored at room temperature and protected from light, until further analysis.
The purchased plant in natura (ST-076.4) was already in conditions suitable for consumption. Therefore, it was not necessary to carry out any cleaning procedure with this plant material. After the arrival at Centro de Inovação e Ensaios Pré-Clínicos (CIEnP), the vacuum packaging was stored at room temperature and protected from light, until further use.
Maceration Stage : The extraction tests with leaves (ST-076.1, ST-076.2 and ST-076.3) were accomplished using portions of approximately 500 grams of manually crushed material. The extraction proportion was of 1 part of plant material to 10 parts of Extractor Liquid, which was a mixture of absolute ethanol and water in 50% ratio (v/v). The extract was left to macerate at room temperature (approximately 20 °C) for 5-10 days in the dark, with occasional agitation.
The extraction tests with Pulverized plant in natura (ST-076.4) were performed in three optimization tests:
i. Selection of the extractor liquid: two types of extracts were produced: a heated aqueous extract, and a hydroalcoholic extract in which the extractor liquid was a mixture of deionized water and absolute Ethanol in 1:1 ratio (v/v);
ii. Maceration time: periods of three and six days were tested;
iii. Drying procedure: incubator at 60 °C or Lyophilization.
The extraction tests with pulverized plant material were carried out using 25 grams portions (approximately), in the proportion of 1 part of plant material to 10 parts of extractor liquid. This proportion was maintained for both extractor liquid tests (heated aqueous and hydroalcoholic).
Therefore, to produce the heated aqueous extract, the procedure was the following: approximately 25 grams of the plant material was weighed in a glass beaker wrapped in light protection, and 250 mL of deionized water, heated in a microwave at approximately 90 °C, was added to it. The beaker with aqueous extract was allowed to cool until room temperature was reached with occasional agitation. Finally, after cooling, the beaker containing the aqueous extract was sealed and was left macerating by the stipulated time in a refrigerator at 4 °C.
The hydroalcoholic extract was produced as described above, except that instead of 250 mL of hot water, 125 mL of deionized water at room temperature with 125 mL of absolute ethanol were used. For both extracts, two replicates were made, one for each drying process (incubation or lyophilization), and thus periods of 3-6 days of maceration were tested.
Filtration Stage : After the maceration period, all extracts, regardless of the type of raw material, were filtered by gravity using "voil" fabric filter to remove the thicker part of the plant material (larger particles). Subsequently, the extracts were vacuum filtered using filter paper of 80.0 g/m², porosity of 3 microns, to remove smaller particulate material.
All filtered extracts were placed in amber glass bottles, properly labeled, and kept in a refrigerator at 4 °C, until time for removal of solvent through rotary evaporation.
At this point in the extraction process, it was considered that in industry scale process there would be no separation of fresh leaves (ST-076.1) from the aged (ST-076.2) ones. Hence, the extracts of ST-076.1 and ST-076.2 were discarded after the filtration step. Only the extract containing a mix of fresh and aged leaves (ST-076.3) remained for the subsequent phases of extraction.
Rotary Evaporation Stage : Following the filtration process, both hydroalcoholic extracts (ST-076.3 and ST-076.4) were submitted to the rotary evaporation process in order to eliminate the organic solvent. The volume of solution placed in the evaporation flask was always less than half of the total volume of the flask. A rotary evaporator (model R-100, Buchi) with heating bath, chiller for cooling and vacuum pump was used for evaporating ethanol. Heating bath and chiller temperatures were set at 60 °C and 10 °C, respectively.
After finishing evaporation of ethanol from the extract, the resulting solution was fractionated into borosilicate glass containers protected of light. The amount of extract placed in each container corresponded to at most half its volume (for example, approximately 125 ml of extract was placed in a 250 mL beaker). After fractioning, extracts were frozen at -20 ºC for at least 24 hours before the Lyophilization process.
Drying/Lyophilization/Storage Stage : The aqueous and hydroalcoholic extracts that would be dried in the incubator were weighed and placed in the incubator for drying at 60 °C. After completely dried, the extracts were left at room temperature to cool. Next, the extracts were weighed and stored in a freezer at -20 °C until further analysis.
The extracts that were lyophilized were distributed into beakers, protected from light and placed in a freezer at -20 °C until Lyophilization. However, the aqueous extracts were frozen right after the filtration step, while the hydroalcoholic extracts were placed in the freezer following the rotary evaporation step.
The transformation of frozen extracts into dry extracts occurred through lyophilization, which is a process were water changes from a solid state (ice) to a gaseous state. This change in physical state only happens with water at very low temperatures, approximately -55 °C, and under vacuum conditions. A lyophilizer (model LS3000, Terroni) with a capacity for sixteen bottles (or 3 kg of ice, counting the containers) was used for this purpose. The procedure consisted of freezing the extract for at least 24 hours and placing the container in the machine, with the temperature stabilized beforehand. The Lyophilization process took between 7 - 10 days until the extract was completely dry, depending on the volume of extract in each container. After completion of lyophilization, the amount of resulting dried extract in each (previously weighed) container was quantified. The extracts were homogenized and aliquoted in flasks protected of light, properly identified and stored in a freezer at -20 °C until further use.
After lyophilization, 27.84 grammas of dried extract of ST-076.3 (Mix of fresh and aged hand crushed leaves) were obtained from the starting amount of 472.19 grammas of dried and grounded leaves, with a resulting yield of 5.90%. Nevertheless, 1,368.00 grams of mixed fresh and aged leaves, selected and clean, were required in order to obtain the initial mass of 472.19 grams.
The yields of aqueous and hydroalcoholic extracts of ST-076.4 (Pulverized plant in natura) dried in the incubator were the following: ST-076.4-hidroalcholic started with 12.71 grammas of pulverized plant, resulted in 2.32 grammas of dry extract, yielding 18.25%; ST-076.4-aqueous used 26.60 grammas of pulverized plant produced 4.88 grams of dried extract, which yielded 18.34%. Table 1 presents the amount of dried extract obtained in each extraction of ST-076.
Table 1. Yield as percentage of dried extract mass of ST-076.
Identification Plant material source Extractor liquid Drying Dried extract (g) % of Mass
ST-076.3-HA Mix of fresh and aged hand crushed leaves Ethanol 50% (v/v) Lyophilization 27.84 5.90
ST-076.4-HA Pulverized plant in natura Ethanol 50% (v/v) Lyophilization 2.15 16.92
ST-076.4-HA Pulverized plant in natura Ethanol 50% (v/v) Incubator 2.32 18.25
ST-076.4-AQ Pulverized plant in natura Aqueous heated Lyophilization 4.28 16.46
ST-076.4-AQ Pulverized plant in natura Aqueous heated Incubator 4.88 18.34
AQ: Aqueous extract; HA: Hydroalcoholic Extract.
Phytochemical Extract Characterization: An investigative method was applied for identifying the phytochemical constituents, such as alkaloids and other constituents present in the dried extract. The method was built from the compilation of literature data referring to precursor and product ions, both in positive and negative ionization modes, of compounds commonly found in plant material. Thus, initially, samples of the extract were submitted to a screening analysis.
Lyophilized extracts of ST-076.4 (pulverized plant in natura), both aqueous and hydroalcoholic, were also not considered for phytochemical characterization. In order for the extract to be produced on a larger scale, the lyophilization step would not be optimal for industrial use.
Phytochemical characterization was performed for aqueous and hydroalcoholic extracts prepared with the pulverized plant in natura and dried in the incubator (ST-076.4).
Through investigative methods, 26 compounds were detected in ST-076.4 extract (Table 2). Yet, the majority of them are isomers of caffeoylquinic acid, di-caffeoylquinic acid, and feruloylquinic acid, most of which do not have commercial reference standards available for purchase. Therefore, the identified substances are those that have had their confirmation through the test of analytical standard addition. Nevertheless, caffeoylquinic acid and its isomers were quantified as chlorogenic acid.
Chromatographic peak areas greater than or equal to 5,000,000 arbitrary units were stipulated towards determining the major constituents. Accordingly, table 2 displays the extracts’ major constituents.
Table 2. Chromatographic area of compounds identified in aqueous and hydroalcoholic extracts prepared with pulverized plant in natura, dried in an incubator (ST-076.4) using Liquid Chromatography Coupled to Mass Spectrometry.
Compound ST-076.4-AQ ST-076.4-HA
Rutin 8.114.598 10.560.230
Caffeine 40.153.866 24.658.958
Theophylline 743.551 661.145
Theobromine 5.968.946 7.554.462
Chlorogenic acid 15.051.548 16.558.842
Caffeic Acid 5.156.240 6.299.108
Quinic acid 5.262.927 4.846.805
Cis-4,5-diferuloylquinic acid 25.304.791 31.302.624
4-feruloylquinic acid 3.707.131 3.601.669
Cis-5-feruloylquinic acid 15.403.986 17.064.631
5-feruloylquinic acid 15.569.107 17.262.743
Cis-3-feruloylquinic acid 15.478.619 17.192.555
4-feruloylquinic acid 11.734.195 14.097.731
3-feruloylquinic acid 15.450.429 17.000.771
4-p-coumaroylquinic 760.759 734.315
4,5-diferuloylquinic acid 24.947.057 30.981.442
diferuloylquinic acid 24.975.907 31.210.684
3,5-diferuloylquinic acid 25.048.131 31.141.365
3,4-diferuloylquinic acid 25.157.920 31.061.052
3-feruloylquinic acid 1.702.412 1.965.340
4-feruloyl-5-feruloylquinic acid 2.066.582 3.044.400
Cis-3-caffeoyl-4-feruloylquinic acid 2.027.600 3.137.933
3-caffeoyl-5-feruloylquinic acid 738.860 1.154.273
4-caffeoyl-5-feruloylquinic acid 721.964 1.121.830
caffeoyl-feruloylquinic acid 1.947.301 3.025.953
3-feruloyl-5-feruloylquinic acid 1.898.476 2.777.626
AQ: Aqueous extract; HA: Hydroalcoholic Extract.
After analyzing the dried extract using chromatographic methods with multiple reaction monitoring scanning mode, it was possible to confirm the following constituents: Rutin, Caffeine, Theobromine, Theophylline and Chlorogenic acid (Table 3).
Table 3. Multiple Reaction Monitoring, Transitions and Retention Time (tR) of major components detected in dried extract samples.

Phytochemical Constituents		 Precursor ion Product ion tR (min)
Rutin 611.2 303.0 6.68
Caffeine 195.5 138.0 5.50
Theobromine 181.1 138.0 3.55
Theophylline 181.2 124.0 4.45
Caffeoylquinic acid and its isomers (quantified as chlorogenic acid) 355.1 163.1 4.43/5.59/5.75
Results based on the chromatographic analysis of the incubator-dried extracts showed the same chemical composition towards the different analytical methods used. However, the hydroalcoholic extract 50% (v/v) showed higher content of compounds. For comparison purposes, it has been produced a large scale of hydroalcoholic extract and an aqueous extract. These extracts were identified as ST-076.7 and ST-076.8, respectively, when first arrived at CIEnP.
After the characterization of these extracts (Table 4), it was decided to produce a larger quantity of the aqueous extract. Therefore, approximately 3 kilograms of this extract were produced to be used in non-clinical studies.
Table 4. Chromatographic area of compounds identified in the aqueous and hydroalcoholic extracts of ST-076.7 and ST-076.8, using the technique of Liquid Chromatography Coupled to Mass Spectrometry.
Compound ST-076.7-HA ST-076.8-AQ
Rutin 7.324.456 6.736.698
Caffeine 13.677.366 14.062.357
Theophylline 369.243 487.388
Theobromine 5.100.243 5.452.255
Chlorogenic acid 24.347.392 26.354.295
Caffeic Acid 6.047.217 7.557.288
Quinic acid 1.929.614 2.014.283
Cis-4,5-diferuloylquinic acid 22.905.184 28.005.148
4-feruloylquinic acid 3.056.903 3.953.144
Cis-5-feruloylquinic acid 14.057.188 12.123.997
5-feruloylquinic acid 14.166.146 15.193.463
Cis-3-feruloylquinic acid 14.349.163 15.624.020
4-feruloylquinic acid 10.294.309 11.072.024
3-feruloylquinic acid 14.444.474 15.376.769
4-p-coumaroylquinic 616.823 9.522.511
4,5-diferuloylquinic acid 23.055.001 23.663.495
diferuloylquinic acid 22.922.191 23.770.778
3,5-diferuloylquinic acid 22.869.206 23.775.283
3,4-diferuloylquinic acid 23.014.976 23.527.499
3-feruloylquinic acid 1.372.255 1.478.596
4-feruloyl-5-feruloylquinic acid 1.973.751 2.258.044
Cis-3-caffeoyl-4-feruloylquinic acid 1.970.342 2.265.106
3-caffeoyl-5-feruloylquinic acid 620.183 799.553
4-caffeoyl-5-feruloylquinic acid 611.882 737.717
caffeoyl-feruloylquinic acid 1.828.002 2.064.571
3-feruloyl-5-feruloylquinic acid 1.847.295 2.133.606
AQ: Aqueous extract; HA: Hydroalcoholic Extract.
The samples were analyzed by using UPLC-MSMS type equipment (Ultra Performance Liquid Chromatography; MSMS, Mass Spectrometry in tandem) whose system is composed of a Xevo TQS mass spectrometer with a Waters (United Kingdom) triquadrupole mass analyzer. The mass spectrometer is coupled to a high performance liquid chromatograph (Acquity H-Class) equipped with a degasser, quaternary pump system, chromatographic column oven, temperature-controlled sampler and automatic injector, also branded Waters. The data acquisition and treatment were performed with the MassLynx software, version 4.1.
The mass spectrometer was calibrated in both positive and negative ion modes with a standard phosphoric acid calibration solution. Nitrogen was used as nebulizer gas (flow rate of 150 L/hour) and drying gas (flow rate of 1,000 L/hour). Other parameters: ionization source, electrospray; capillary voltage, 4,000 V; source temperature, 500 °C; cone voltage, 40 V. The parameters of the collision cell were: collision gas flow (argon), 0.15 mL/min; collision energy, 28 eV.
Analytical method for the determination of the phytochemical constituents of the extract: For the quantification of the major compounds identified in the extract (Rutin, Chlorogenic Acid, Theophylline, Theobromine and Caffeine), a C18 reverse phase chromatographic column (Kinetex 50 mm x 2.1 mm, particle size 2.6 µm, brand Phenomenex, USA) was used. The constituent solvents of the mobile phase were 0.1% formic acid in deionized water (solvent A) and acetonitrile (solvent C), respectively. The linear gradient elution mode provided the best separation of components from the matrix. The mobile phase flow rate was set at 400 µL min-1. In all runs, the injected volume was 1 µL. The column oven temperature was set at 30 °C for all analytical runs, as well as the sampler temperature was maintained at 10 °C whenever samples, quality controls or calibrators remained inside the equipment. Table 5 presents the gradient programming used in the method.
Table 5. Composition of mobile phase and gradient used in the analytical method for identifying the constituents of ST-076 extracts.
Time (min) % Acetonitrile
0,00 1
3,50 5
8,50 40
9,00 1
10,00 1
Quantification of the extract constituents: Sample preparation for injection in UPLC-MSMS consisted in weighing approximately 10 mg of dried extract, and solubilize it in sufficient volume of deionized water to obtain 10 mg/mL. After dissolution, the samples previously prepared in duplicate, were centrifuged for 5 minutes at 14,000 rpm and the supernatant was injected into the equipment. This procedure was necessary to prevent non-soluble extract particles from clogging the equipment injector. Table 6 shows quantification of compounds detected in the extract. The content of caffeoylquinic acid and its isomers is equivalent to the sum of their quantities measured as chlorogenic acid. shows the phytochemical constituents found in the dried extract prepared from the aqueous extract (ST076.8).
Table 6. Results for the quantification of compounds detected in aqueous extract (ST-076.8).
Class/Compound Content (mg/100 g of extract)
Alkaloids
Theobromine 370.92
Theophylline 6.89
Caffeine 2,219.43
Total alkaloids 2,597.25
Phenolic Acids
Caffeoylquinic acid and its isomers 6,450.22
Flavonoids
Rutin 861.01
Validation of analytical method: The proposed analytical method was validated only for the compounds identified in the extract by the standard addition test, which were Rutin, Chlorogenic acid, Theophylline, Theobromine and Caffeine.
Linearity/Calibration curve: For the linearity evaluation, a methanol calibration curve was constructed in the application range of 50 a 1,000 ng mL-1. The calibrators were prepared as follows: first, individual stock solutions 1 mg mL- 1 were made with each reference substance; second, a working solution 50 μg mL-1 with five compounds together was made by diluting of the stock solutions with methanol; lastly, the calibrators were prepared in methanol in the desired range of application.
Long term stability: In the invention described herein, the stability of the ST-076 was monitored from the moment of arrival at CIEnP until the end of non-clinical studies. The evaluation was carried out by monitoring the peak chromatographic area of the compounds identified in the extract: chlorogenic acid, rutin, caffeine, theophylline and theobromine. According to recommendations from national and international regulatory agencies (ICH, FDA and ANVISA), samples must be analyzed in a time defined as initial (time zero) and every 3, 6, 9, 12, 18 and 24 months. Therefore, since the ST-076 is a plant extract, samples were analyzed monthly during the entire non-clinical study period performed at CIEnP (Table 7).
Table 7. Schedule for the analysis of samples for the evaluation of the long-term stability of the standardized extract of ST-076.
STAGE DATE
Start = zero time: May/2018
1) June/2018
2) July/2018
3) August/2018
4) September/2018
5) October/2018
6) November/2018
7) December/2018
8) January/2019
9) February/2019
10) March/2019
11) April/2019
12) May/2019
14) July /2019
15) August/2019
17) October/2019
18) November/2019
19) December/2019
20) January/2020
21) February/2020
The procedure adopted for analyzing the samples was the following: at each selected time point, an aliquot of approximately 20 mg of the extract was weighed. After weighing, this amount of material was solubilized in enough volume of deionized water to result in a concentration of approximately 20 mg/mL. Then, the sample was centrifuged for 5 min at 14,000 rpm and 200 µL of the supernatant were removed, placed in insert vials and injected into the LC-MSMS equipment on the same preparation day. Stability was assessed by monitoring variation of the chromatographic peak areas of compounds (rutin, chlorogenic acid, caffeine, theobromine and theophylline) from May 2018 to February 2020. May 2018 was considered as zero time (Table 8).
Table 8. Chromatographic peak area of the monitored compounds to assess the long-term stability of the standardized extract from ST-076.
Sample Theobromine Theophylline Caffeine Chlorogenic
Acid		 Rutin
Zero time
(May/2018)		 142935 13445 1849409 1496874 568486
June/2018 139129 13292 1717691 1430715 544072
July/2018 153363 14627 1919334 1559279 611853
August/2018 135656 12346 1796716 1397604 558193
September/2018 161068 15056 2038376 1634040 622844
October/2018 167342 15726 2058804 1662161 654893
November/2018 142602 13221 1803976 1440346 566627
December/2018 163359 15710 2071176 1659953 645270
January/2019 141661 13522 1815644 1436989 552151
February/2019 146126 13607 1817772 1493436 563641
March/2019 156650 14593 1931284 1597324 624450
April/2019 178620 23031 1686541 2566647 847223
May/2019 154178 13939 1477702 1762673 526065
July/2019 133277 10207 1552494 1365479 335006
August/2019 303494 15797 1962389 2944821 605501
October/2019 180780 12106 1166278 3121710 887338
November/2019 114193 11451 1433283 1334435 421269
December/2019 151484 8592 879051 1909278 316673
January/2020 132048 10763 1527852 4555561 813423
February/2020 209058 20112 1419329 4420992 833099
The evaluation of long-term stability was performed by analyzing the percentage variation between the analytical signals (peak chromatographic area) of the compounds at zero time compared to the analytical signal in the months of evaluation. According to RDC Nº 26 of the National Health Surveillance Agency (ANVISA), a Brazilian FDA-like agency, and the sample is considered stable when the variability between analytical markers is not greater than 20%.
According to data presented in Table 9, the five compounds monitored proved to be stable during the entire investigation period, with minimal difference from time zero. The percentage values relative to zero time were: 96.48% for caffeine, 98.53% for rutin, 99.11% for theophylline, 101.24% for chlorogenic acid, and 110.06% for theobromine, considering the average of the months. Based on these results, it can be concluded that the standardized extract from the ST-076 has remained stable over the 20- month evaluation period.
Table 9. Evaluation of the long-term stability of ST-076. Data obtained by comparing the analytical signal (peak chromatographic area) of the monitored compounds at zero time and over 20 months. Results are expressed as percentage of variation.
Month Theobromine Theophylline Caffeine Chlorogenic Acid Rutin
June/2018 97.34 98.86 92.88 95.58 95.71
July/2018 107.30 108.79 103.78 104.17 107.63
August/2018 94.91 91.83 97.15 93.37 98.19
September/2018 112.69 111.98 110.22 109.16 109.56
October/2018 117.08 116.97 111.32 111.04 115.20
November/2018 99.77 98.33 97.54 96.22 99.67
December/2018 114.29 116.85 111.99 110.89 113.51
January/2019 99.11 100.57 98.17 96.00 97.13
February/2019 102.23 101.20 98.29 99.77 99.15
March/2019 109.60 108.54 104.43 106.71 109.84
April/2019 107.77 125.09 72.39 82.92 82.40
May/2019 93.03 75.71 63.43 56.95 51.17
July /2019 86.47 80.03 83.46 132.99 101.55
August/2019 135.15 93.89 75.80 119.70 106.19
October/2019 122.32 107.41 110.71 114.22 124.60
November/2019 117.77 97.11 102.74 124.69 105.96
December/2019 140.10 69.16 99.85 75.25 50.83
January/2020 111.20 81.28 108.36 84.29 96.66
February/2020 122.99 99.44 90.57 109.62 107.07
Average 110.06 99.11 96.48 101.24 98.53
Example 2 – Pharmacokinetic profile after acute and repeated oral treatment for 7 th days.
The pharmacokinetic profile was evaluated after acute and repeated oral administration with ST-076 for 7 days in Sprague Dawley rats. Animals were treated orally (p.o.) by gavage with the extract according to the present invention (1,000 mg/Kg). Plasma was collected by caudal vein puncture at the following time points: 0.083, 0.5, 1, 2, 4, 8, 12, 16 and 24 hours after administration of the extract according to the present invention. The ST-076 was prepared and quantified using an analytical method previously developed and validated by means of UPLC-MSMS. Caffeine, theobromine and theophylline were assessed. All compounds were very well orally absorbed and reach micromolar plasma concentration (Table 10). No significant differences in the pharmacokinetic parameters assessed for the 3 compounds when comparing acute ([Fig.2]) and repeated 7-day treatments ([Fig.3]. with the extract according to the present invention (1,000 mg/kg, p.o.). The pharmacokinetics parameters are presented in table 10.
It can be concluded that methylxanthines do not seem to accumulate in the organism following the 7-day daily treatment as the AUClast obtained for caffeine, theobromine and theophylline on day 0, did not changed when compared to the AUClast on the 7th day.
Table 10 . Pharmacokinetic parameters for caffeine, theobromine and theophylline after acute treatment (day 0 or following daily treatment for 7 days) (day 7) with ST-076 (1,000 mg/Kg, p.o).
Experimental group Day AUClast
(min*µg/mL)		 Cmax
(µg/mL)		 Tmax
(min)		 T1/2
(min)		 Ke
(min)
Caffeine 0 4,003.1 8.65 120 116.1 0.0059
Caffeine 7 3,456.3 9.72 120 114.2 0.0060
Theobromine 0 2,489.5 3.89 480 97.05 0.0071
Theobromine 7 2,817.6 4.34 480 130.7 0.0053
Theophylline 0 2,714.8 5.28 480 84.3 0.0082
Theophylline 7 3,197.3 5.50 480 146.1 0.0047
Mean Concentration Area Under the Curve (AUClast), Maximum plasma concentration (Cmax), Time to reach the observed maximum (peak) concentration (Tmax), Elimination half-life (T1/2), and Elimination constant (Ke).
Example 3 – E valuation of the possible CYP3A4 -mediated interaction with the extract according to the present invention test item in vivo .
This study aimed to evaluate the possible CYP3A4-mediated interaction with ST-076 in vivo. The study was performed using rats treated with vehicle or with ST-076 (1,000 mg/kg, p.o) for 7 consecutive days. On the last day, animals were treated with midazolam (10 mg/kg, p.o. a compound that is metabolized mainly by CYP3A4) and the animal’s blood was collected by caudal vein puncture at the following time points: 0.083, 0.25, 0.5, 1, 2 and 4 hours. Midazolam plasma concentration was quantified using a previously validated UPLC-MS/MS analytical method. The pharmacokinetics parameters obtained for the animals treated with ST-076 are presented in and table 11.
Table 11. Pharmacokinetics of midazolam (CYP3A4 substrate)
EXPERIMENTAL GROUP AUClast
(min*µg/ml)		 Cmax
(µg/ml)		 Tmax
(min)		 T1/2
(min)
Vehicle 24,264.9 278.5 30 85.1
ST-076 28,291.6 466.3 5 44.6
Thus, by comparing Midazolam AUClast in the two experimental groups, no significant differences between animals treated for 7 days with ST-076 according to the present invention or vehicle were observed. It can be concluded that the extract according to the present invention did not show CYP3A4 enzyme interaction under the experimental conditions tested.
Efficacy studies of ST-076
Example 4 – Evaluation of the efficacy of ST-076 according to the present invention in high-f at diet-induced obesity mice
The efficacy of the ST-076 according to the present invention on body weight gain in a high-fat diet-induced obesity mouse model was investigated, and its efficacy was compared with Liraglutide, a reference drug used in type 2 diabetes and obesity treatments. Animals received normal chow diet (NCD) or high-fat diet (HFD) for 8 consecutive weeks. From week 8 to week 16, animals fed with HFD were treated with ST-076 according to the present invention (500 or 1,000 mg/Kg, p.o.), Liraglutide (0.2 mg/Kg, s.c. - subcutaneously), a reference item, or with vehicle (10 ml/Kg, p.o.), once a day, until week 16. The evaluation of body weight change was assessed weekly from week 8 to week 16. On week 15, an oral glucose tolerance test (OGTT) was performed, and on week 16 blood was collected for chemistry analysis of total cholesterol, triglyceride, glucose, insulin, GLP-1 and leptin. In addition, liver, inguinal (IngWAT) and epididymal white adipose tissue (EpiWAT) were removed and weighed. The ST-076 according to the present invention (500 or 1,000 mg/Kg, p.o.) or Liraglutide (0.2 mg/Kg, s.c.) significantly prevented body weight gain induced by HFD when compared to the HFD vehicle group (p < 0.05) ([Fig. 5]). Interestingly, in animals fed with HFD, the extract according to the present invention (1,000 mg/Kg, p.o.) or Liraglutide (0.2 mg/Kg, s.c.) were able to significantly reduce food intake from week 8 to week 16 ([Fig. 6]). Also, daily treatment with the extract according to the present invention (500 or 1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.), improved glucose oral tolerance ([Fig. 7]). The effect of ST-076 was evident through images of animals in the end of study ([Fig. 8]) and histology of white adipose tissue ([Fig. 9]) and liver ([Fig. 10]). Daily treatment with ST-076 according to the present invention (500 or 1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.) also significantly prevented HFD-induced increase in total cholesterol (Figure 11A) and triglyceride (Figure 11B) serum levels (p < 0.05). Moreover, daily treatment with ST-076 (500 or 1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.) prevented the increase in liver (Figure 12A) and IngWAT (Figura 12B) sizes induced by HFD. Furthermore, daily treatment with ST-076 (500 or 1,000 mg/Kg, p.o.) or with Liraglutide (0.2 mg/Kg, s.c.) improved blood glucose levels (Figure 13A) and insulin (Figure 13B) resistance of mice fed with HFD when compared to the vehicle treated group. Interestingly, the ST-076 increased GLP-1 levels (Figure 14A) and reduced leptin serum level (14B) when compared to NCD mice. According to the results obtained, it is possible to conclude that the extract according to the present invention (500 or 1,000 mg/Kg, p.o.), as well as liraglutide, is highly effective in preventing body weight gain in a rodent model of obesity. This effect is associated to a reduction of fat deposits in IngWAT and to an improvement in clinical chemistry parameters related to metabolic diseases, such as, high blood glucose levels, insulin resistance and total cholesterol, triglyceride, leptin and GLP-1 serum levels (see figures 5-14).
Example 5 - Evaluation of the efficacy of ST-076 according to the present invention in a high-fat diet-induced obesity rat model
According to the present invention the effect of ST-076 on body weight gain in a high-fat diet (HFD) model in rats also was studied, and the activity compared with liraglutide, a reference drug used for clinical obesity treatment. Animals received normal chow diet (NCD) or HFD, during 11 consecutive weeks. Animals fed with HDF were treated with ST-076 according to the present invention (1,000 mg/Kg, p.o.), liraglutide (0.2 mg/Kg, s.c.), a reference item, or with vehicle (10 ml/Kg, v.o.), once a day, for 11 consecutive weeks. Body weight changes were evaluated weekly until the 11th week. At the end of study, liver and epididymal white adipose tissue (EpiWAT) were removed and weighed. The extract according to the present invention (1,000 mg/Kg, p.o.) and liraglutide (0.2 mg/Kg, s.c.) prevented body weight gain induced by HFD when compared to the HFD vehicle group (p < 0.05) ([Fig. 15]). This effect was evident in the 5th week and lasted until the 11th week. Also, rats fed with HFD and daily treated with ST-076 (1,000 mg/Kg, p.o.) or liraglutide (0.2 mg/Kg, s.c.), showed a reduced liver (Figure 16A) and EpiWAT (Figure 16B) weights when compared to rats fed with HFD plus vehicle (p < 0.05). Considering the results obtained in the present study, it is possible to confirm that the extract according to the present invention (1,000 mg/Kg, p.o.) is highly effective in preventing body weight gain in a rodent model of obesity.
Example 6 - Evaluation of the efficacy of the extract according to the present invention in streptozotocin-nicotinamide-induced rat model of type 2 diabetes
According to the present invention, the effect of ST-076 was evaluated in a rat model of Type 2 Diabetes Mellitus (T2DM). The hypoglycemic activity, changes in body weight, water intake, diuresis and possible interactions with blood pressure were investigated. For this, fasted Sprague Dawley rats were treated orally with nicotinamide and, 15 minutes later, streptozotocin was administered through caudal vein for induction of experimental T2DM. After 4 days, glucose levels were evaluated to confirm the induction of T2DM, and animals considered diabetic (blood glucose above 150 mg/dL) were randomly assigned to different experimental groups and treated for 4 weeks. Weekly assessments of body weight and fasting blood glucose as well as urine volume and water consumption were performed. Since T2DM leads to cardiovascular changes, parameters such as blood pressure were evaluated in animals with T2DM and the effect of the extract according to the present invention on these parameters was observed. Over the 4 weeks of the study, the maintenance of fasting hyperglycemia was observed. These data indicate that animals with T2DM remained diabetic for the entire experimental period.
Evaluation of glycemia in the Type 2 Diabetes Model (T2DM): Non-diabetics animals presented normal blood glucose levels, a value that was maintained until the end of the study, according to the average (105.9 ± 7.3 mg/dL) of six glycemic assessments. One group of diabetic animals presented basal glycemic values (before T2DM induction) of 97.4 ± 5.6 mg/dL. After diabetes induction, values reached 232.7±18.5 mg/dL. After this measure, animals were treated daily with the Vehicle and presented glycemic levels of 217.1 ± 27.4, 198.1 ± 25.0, 185.6 ± 24.2 and 230.2 ± 26.6 mg/dL after the 1st, 2nd, 3rd and 4th weeks of treatment, respectively. Blood glycemia in T2DM animals treated with the vehicle was higher than the group of non-diabetic animals (p<0.05), in all evaluations after induction of T2DM, indicating that animals remained diabetic until the end of the experimental protocol. The second group of diabetic animals showed basal glycemia (before T2DM induction) of 94.6 ± 3.6 mg/dL, and after diabetes induction, glycemic levels reached 230.9 ± 21.6 mg/dL. After this measure, animals were treated daily with the Test Article according to the present invention (500 mg/kg) and presented glycemic levels of 145.6 ± 27.7, 164.0 ± 29.5, 131.8 ± 22.7 and 221.8 ± 39.3 mg/dL, following the 1st, 2nd, 3rd and 4th week of treatment, respectively. The third group of diabetic animals showed basal glycemia (before T2DM induction) of 93.0 ± 4.1 mg/dL, and after diabetes induction the glycemia was 234.7 ± 26.2 mg/dL. After this measure, animals were treated daily with the ST-076 extract according to the present invention (1,000 mg/kg) and presented glycemic levels of 114.6 ± 24.2, 125.0 ± 22.9, 101.7 ± 9.1 and 252.0 ± 59.6 mg/dL, following the 1st, 2nd, 3rd and 4th week of treatment, respectively (Figure 17A). The fourth group of diabetic animals showed basal glycemic values (before T2DM induction) of 101.0 ± 4.2 mg/dL, and after diabetes induction the glycemia was 263.3 ± 26.5 mg/dL. After this measure, animals were treated daily with Liraglutide (0.2 mg/kg, s.c.) and presented glycemic levels of 113.4 ± 27.6, 113.6 ± 11.6, 116.4 ± 15.4 and 166.8 ± 2011 mg/dL, after the 1st, 2nd, 3rd and 4th week of treatment, respectively (Figure 17A). The reduction of glycemia in general was more evident in the area under the curve (Figure 17B).
Evaluation of cardiovascular parameters: Two weeks after the induction of experimental diabetes, the Systolic Blood Pressure (SBP) (Figure 18A), Diastolic Blood Pressure (DBP) (Figure 18B) and the Mean Arterial Pressure (MAP) (Figure 18C) of diabetic animals treated with the vehicle (DB + Vehicle) were higher when compared to non-diabetic animals (ND + Vehicle) (p>0.05). In diabetic animals treated with the ST-076 according to the present invention (500 or 1,000 mg/kg, p.o.), a reduction in SBP, DBP and MAP (p>0.05) (Figure 18A-C) was observed. The same was observed in animals treated with Liraglutide (p>0.05), although the differences did not reach statistical significance.
Considering the results obtained in the present assay, is possible to conclude that the ST-076 according to the present invention was effective in reducing glycemia in DMT2 animal models. A possible improvement was also observed in events related to the cardiovascular system that are associated with metabolic syndrome. Taken together, the present results show the potential of the ST-076 for the treatment of DMT2 and associated elevation of blood pressure.
Example - 7 Evaluation of the mechanisms of action of the ST-076 according to the present invention in mice with high-fat diet-induced obesity
According to the present invention, was evaluated the possible mechanism underlying ST-076 potential for reducing body weight and changing biochemical parameters in a mouse model of obesity ( -14). On week 16 (see example 4), animals were euthanized and liver, hypothalamus, inguinal (IngWAT) and epididimal (EpiWAT) white tissue were dissected and evaluated by RT-qPCR and immunohistochemistry.
In order to identify ST-076 underlying mechanisms of action in the experimental obesity model, RT-qPCR was performed in liver, hypothalamus and adipose tissue, which are important tissues in regulation of energy homeostasis and feeding behavior. PCR-array allowed the evaluation of the expression of most genes related to lipid metabolism and its possible modulation with the daily use of ST-076. Results obtained with PCR-array in liver and adipose tissue were validated using RT-qPCR. For these analyzes, total RNA from tissues was isolated and transcribed in cDNA using random primers and probes. Normalized gene expression was calculated as a ratio of Ct of target gene vs housekeeping gene (18S) transcripts (ΔCt), changes of expression vs control (2−ΔΔCt). For PCR-array analysis, genes in the HFD + vehicle group and in the HFD + ST-076 group presented an increase close to or greater than four folds when compared to the NCD + vehicle group, which were considered relevant. To identify the possible underlying mechanism of action of ST-076, scientific publications and online databases containing information about signaling pathway interactions were used (in silico analysis).
The PCR-assay data in the hypothalamus tissue ([Fig. 19]) showed that the invention ST-076 promoted an increase of the expression of the following genes: Cocaine-And Amphetamine-Regulated Transcript Protein gene ( CartptI), Leptin (Lep) and Receptor Activity-Modifying Protein 3 (Ramp3). It is important to mention that these genes are related to feeding behavior and reduction of food intake ([Fig. 19]).
The PCR-assay data in the adipocyte tissue ([Fig. 20]) showed an increase in the expression of Growth Hormone Secretagogue Receptor ( Ghsr) gene promoted by ST-076. This gene is related to energy homeostasis and body weight regulation. According to results depicted in [Fig. 20], treatment with ST-076 induced an increase in transcription of the following genes: Ramp3 (Receptor activity-modifying protein 3), Insulin-2 (Ins2), Peptide YY (Pyy), Somatostatin Receptor 2 (Sstr2) and Urocortin (Ucn). These genes are related to amylin modulation, glycemia control and satiety induction. Zinc Finger Protein Atypical E3 Ubiquitin Ligase (Zfp91) have functions as regulator of the non-canonical Nuclear Factor kappa B (NFkB), which has a critical role in regulating inflammation.
The results obtained from PCR-array (Figures 19 and 20) suggest that ST-076 mechanism of action in an experimental obesity model seems to be related to upregulation of genes involved in food response process, appetite and nutrient levels. To validate the results obtained with PCR-array, RT-qPCR in adipose tissue and liver, important tissues in regulation of energy homeostasis and metabolism, were performed. In adipose tissue, RT-qPCR revealed an increase in the expression of Cocaine-And Amphetamine-Regulated Transcript Protein (Cartpt) (Figure 21A), Fatty Acid Amide Hydrolase ( Faah) (Figure 21B), Glucagon Receptor (Gcgr) (Figure 21C), Neuropeptide Y (Npy) (Figure 21C), Monoacylgliceride Lipase (Mgll) (Figure 22A), Peroxisome Proliferator Activated Receptor alpha (Ppara) (Figure 23A), Peroxisome Proliferator Activated Receptor gamma (Pparg) (Figure 23B) and Receptor Activity-Modifying Protein 3 (Ramp3) (Figure 23C), genes expression showed an increase by ST-076. The NLR Family Pyrin Domain Containing 3 (Nlrp3) (Figure 22B) gene expression showed a significant reduction in Nlrp3 gene expression promoted by ST-076.
In liver tissue, RT-qPCR revealed that the invention ST-076 promoted increased Fatty Acid Amide Hydrolase (Faah) (Figure 24A), Monoglyceride Lipase ( Mgll) (Figure 24B) and Peroxisome Proliferator-Activated Receptor alpha (Ppara) (figure 24C) gene expression.
The ST-076 treatment decreased inflammation and NFκB plays a critical role in regulating inflammation. Many NFκB target genes ([Fig. 30]), including TNF-α and MCP-1, are also implicated in the development of obesity-induced insulin resistance (Lee and Lee, 2013). In fact, treatment with ST-076 reduces plasma levels of TNF-α and MCP-1. Furthermore, the PCR-array (Figures 19 and 20) and PCR results (Figures 21 – 24) indicate that ST-076 interferes with biological processes related to the regulation of the processes of response to food, appetite and nutrient levels. Leptin resistance is characterized by reduced satiety, over-consumption of nutrients, and increased total body mass (Izquierdo, et al., 2019). In obese HFD + vehicle group was observed high levels of leptin which was reduced after ST-076 treatment (Figure 14B). It has been suggested that the role for GLP-1 in stimulating adipocytes lipolysis via elevation of cAMP and activation of protein kinase A (PKA), which in turn may activate hormone-sensitive lipase (HSL), contributes to fatty acid generation (FFA) from intracellular triglyceride stores (Winzell and Ahrén, 2004). ST-076 treatment of obese mice increase GLP-1 plasma levels (Figure 14A). Daily treatment with ST-076 decreased glycemia (Figure 13A) and insulin levels (Figure 13B) in a mice model of obesity when compared to vehicle HFD.
Phosphatidylinositol 3-kinase (PI3K) inhibition (Figure 26B and 29A) could potentiate the effect of β-adrenergic agonists in the treatment of obesity (Huang et al., 2018; Araiz et al., 2019). The catecholamine norepinephrine binds beta-adrenergic receptors on the plasma membrane of adipocytes by GPCR signaling pathway (Kim et al., 2005). These receptors are coupled with Gs-proteins that transmit a stimulatory signal to adenylyl cyclase to generate cAMP. cAMP binds PKA, causing the regulatory subunits to dissociate from the catalytically active subunits, resulting in increased activity of the enzyme, decreased activity of AMPK (Figure 25A and 27C) while increased PKA and Extracellular signal-regulated kinases (ERK1/2) in adipocytes (Figure 25C), thus contributing to the regulation of lipolysis ([Fig.25] and 26) (Ducan et al., 2007; Gauthier et al. 2008).
It is well known that lipolysis products increase in circulating FFA and glycerol. Upregulation of serum concentration of FFA derivatives can bind to and activate members of the nuclear receptor family of transcription factors that control the expression of genes involved in lipid and energy homeostasis and inflammation. The best-studied FA-activated nuclear receptors are the PPARs. PPAR family is highly expressed in oxidative tissues and regulate genes involved in substrate delivery, substrate oxidation, and oxidative phosphorylation (Zechner et al. 2012). Likewise, increased PI3K, PPAR and JNK pathways were showed in liver tissue ([Fig. 27] - 29) suggesting regulation of the mechanism of lipolysis by ST-076 and up stimulation of FFA and nonesterified fatty acids (NEFA) intakes ([Fig. 31]).
In the light of the results obtained, it is possible to conclude that the test item ST-076 is highly effective in preventing body weight (Figures 7 and 12) gain in diet-induced obesity mouse model. These results seem to be associated to an improvement in clinical biochemical parameters (Figures 11, 13 and 14) related to obesity and metabolic diseases, such as, reduction of blood glucose levels, decreased insulin resistance and impairment of inflammatory response ([Fig. 30]). The PCR-array results (Figures 19 and 20) indicate that ST-076 interferes with biological processes related to the regulation of the processes of response to food, appetite and nutrient levels. In conjunction with PCR validation (Figures 21 – 24), immunohistochemistry data (Figures 25 – 29) and cytokines quantification ([Fig. 30]) suggest that the underlying mechanism of action of ST-076 occurs through interaction with GPCR signaling pathways ([Fig. 31]).
Safety studies of ST-076
Example 8 – Genotoxicity evaluation of ST-076 – AMES test
Genotoxicity tests were developed to detect substances with the potential to induce damage to genetic material and are recommended by regulatory agencies worldwide as part of the safety assessment of chemicals. These tests identify risks related to DNA damage. Substances found positive in these tests that detect genetic modifications are potentially carcinogenic and / or mutagenic to humans. Thus, the bacterial mutagenicity test is widely used as an initial screening to assess possible genotoxic activity, in particular, for point mutation-inducing activity. The reverse bacterial mutation assay was performed in accordance to OECD recommendations, guideline 471 - Guideline for Testing of Chemicals. Method 471 “Bacterial Reverse Mutation Test” (Adopted: 26 June 2020). The preliminary test with the strain TA 100, in the absence or presence of metabolic activation (S9) was conducted aiming at selecting adequate concentrations of the ST-076 for the definitive test. The results are presented in table 12.
Table 12: Evaluation of possible Mutagenicity effect of ST-076 tested in the Ames tester strains TA 97a, TA 98, TA100, TA102 and TA 1535. The test was performed in the absence and presence of the metabolic activation system (8% of S9 in the mixture with required co-factors).
Treatments Concentration
(µg/mL)		 TA 97a TA 98 TA 100 TA 102 TA 1535
- S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9 - S9 + S9
ST-076 8 - - - - - - - - - -
40 - - - - - - - - - -
200 - - - - - - - - - -
1,000 - - - - - - - - - -
5,000 - - - - - - - - - -
Positive Control # + + + + + + + + + +
(-S9) = absence of the metabolic activation system; (+S9) = presence of the metabolic activation system (-) = negative; (+) = positive. # Positive controls = 4-nitroquinoline-N-oxide (4NQO) 0.5 µg/plate: TA97a, TA98 and TA102 (-S9); sodium azide (AZS) 1.5 µg/plate: TA100 and TA 1535 (-S9); 2-aminofluorene (2-AF) 50 µg/plate: TA97a, TA98 and TA100 (+S9); 2-aminoanthracene (2-AA): 2.5 and 5 µg/plate: TA 1535 and TA102, respectively (+S9).
ST-076 showed no mutagenic activity in the reverse mutation test in Salmonella typhimirium bacteria, both in the absence and in the presence of metabolic activation (S9), in the TA 97a, TA 98, TA 100, TA 102 and TA 1535.
Example 9 – Genotoxicity evaluation of ST-076 – Micronucleous test
The micronucleus test detects genetic changes resulting from chromosomal lesions and/or damage to the mitotic system. The formation of micronuclei is an indicative of irreversible losses to DNA and, its frequency can be used as an index of mutagenicity. It is already known that there is a positive correlation between the increase in the frequency of micronuclei and the appearance of tumors in rodents and humans. The micronucleus test in mouse bone marrow was carried out in a GLP compliant condition in accordance to the recommendations of OECD guideline 474 - Guideline for Testing of Chemicals. Method 474 “Mammalian Erythrocyte Micronucleus Test” (Adopted: 29 July 2016). The results are shown in table 13.
Table 13: Incidence of micronucleated polychromatic erythrocytes (MNPCE) and the ratio of polychromatic erythrocytes (PCE) to normochromatic erythrocytes in mice treated with ST-076.
Group Dose
(mg/Kg)		 Route MNPCE/4,000 PCE
(Mean ± S.D.) 		Ratio PCE/NCE
(Mean ± S.D.)
Negative Control
(Water)		 0 p.o. 6.20 ± 3.58 1.60 ± 0.27
Test Item
(ST-076)		 2,000 p.o. 5.20 ± 2.86
 1.31 ± 0.14
Positive Control
(Cyclophosphamide)		 25 i.p. 25.80 ± 10.12 * 1.62 ± 0.34
p.o. = per os; i.p. = intraperitoneal; PCE = polychromatic erythrocytes; NCE = normochromatic erythrocytes; MNPCE = micronucleated polychromatic erythrocytes; S.D. = standard deviation. * Significant difference from negative control by Kruskal-Wallis test: *p < 0.05.
It can be concluded that, under the conditions evaluated and, in the doses tested, ST-076 did not increase the number of micronucleated PCEs in mice and therefore has no genotoxic action.
Example 10 – Maximum tolerated dose and dose selection in rats
This assay was designed to investigate the safety and tolerability of ST-076. For this, two different phases were conducted: Phase I: to determine the maximum tolerated dose (MTD) of ST-076, and Phase II) exploratory evaluation of the toxicity of repeated treatments with ST-076 for 7 days, as described below:
Phase I: Determination of MTD by a single oral administration of ST-076 in an escalating dose scheme. Animals were randomly distributed in five experimental groups (3 males and 3 females/group, Sprague Dawley rats). Animals from the vehicle-treated group were treated with physiologic solution (NaCl 0.9 %) and four additional groups were treated with different doses of ST-076 (175 mg/kg, 550 mg/kg, 1,750 mg/kg and 5,000 mg/kg). All animals were euthanized 14 days after the treatment for subsequent analyses.
Phase II: 7-day repeated toxicity study. The protocol consisted of two experimental groups (5 males and 5 females/group, Sprague Dawley rats). Group 1 was treated with the Vehicle and the group 2 was treated with ST-076 (1,000 mg/kg). All animals were treated by oral gavage once a day for 7 days. All animals were euthanized 14 days after the treatment for subsequent analyses. Clinical and behavior observations, as well as general macroscopic observations in the necropsy were carried out for animals from both phases as well as the hematological and biochemical analyzes for Phase II animals.
In phase I of the study, no adverse effects indicative of toxicity was observed in groups treated with different doses of ST-076. Thus, the dose of ST-076 selected for phase II was 1,000 mg/kg, which is the recommended limit dose for repeated dose studies (ICH M3 (R2)). In phase II, repeated treatment with ST-076 (1,000 mg/kg) did not cause death nor clinical signs toxicity. In addition, no macroscopic and histopathological changes were observed in organs and tissues collected from animals treated with ST-076 (1,000 mg/kg). Only a slight reduction in serum levels of calcium (male rats) and gamma glutamyltransferase (GGT) (female rats) was observed, as well as an increase in the absolute weight of the testes (male rats) and a reduction in the absolute weight of the brain (female rats) when compared to the Vehicle group. Thus, the dose of 1,000 mg/kg is the maximum dose to be tested to assess the subchronic and chronic toxicity of ST-076.
Example 11 – Repeated dose 90-day toxicity study of ST-076 in rats
The purpose of this study was to assess the potential toxicity after treatment with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis administered by oral route for 90 days in rats. Male and female Sprague Dawley rats (7-9 weeks) were used. Animals were maintained under SPF (Specific Pathogen Free) animal conditions and were obtained from CIEnP facility, whose breeding colonies were purchased from Charles River Laboratories (USA).
Necropsy and postmortem analysis : On days 91 (animals of the main groups) and 105 (recovery groups), animals were subjected to necropsy. During necropsy, the outer surface of the body, orifices, cranial, thoracic and abdominal cavities, as well as their contents of each animal were examined. In the analysis of the body surface, a detailed evaluation was performed and the presence of lesions or deformities, size, color, texture, shape, severity, as well as weight and volume were noted and recorded.
Morbidity and mortality – Morbidity and mortality were evaluated twice daily throughout the study. There were deaths of animals from the main groups (200 mg/kg, 500 mg/kg and 1,000 mg/kg) and recovery group (1,000 mg/kg). The results of the histopathological evaluation of animals found dead did not indicate any relationship between treatment with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis and the death of the animals.
General and detailed clinical signs : General clinical signs were evaluated daily. Detailed clinical signs were performed once before the beginning of the treatments to verify the health status of the animals, and once a week thereafter. Oral administration of the standardized aqueous extract obtained from the leaves of Ilex paraguariensis at doses of 200 mg/kg, 500 mg/kg and 1,000 mg/kg, did not result in any observable changes in general and detailed clinical signs. Moreover, any observable changes in general and detailed clinical signs were observed in recovery groups.
Body weight change s and food consumption : Body weight and food consumption were measured once before the start of treatments (baseline) and then once a week. Main groups: no significant changes in body weight related to the treatment with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis (200 mg/kg, 500 mg/kg or 1,000 mg/kg) ([Fig. 32]) were observed. On the other hand, food intake/week/animal was reduced in male animals treated with 200 mg/kg of the standardized aqueous extract obtained from the leaves of Ilex paraguariensis at weeks 9 and 10, when compared to Vehicle-treated males. Recovery groups: no changes in body weight or on the average feed intake/week/animal related to the treatment was observed in recovery groups when compared to the control.
Organs weight : After the necropsy procedure, the absolute and relative weights (g) of the main organs (adrenal glands, spleen, brain, heart, kidney, thymus, liver, testicles, epididymis and ovary) were measured for each animal in all experimental groups. Main groups: treatment with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis (200 mg/kg, 500 mg/kg or 1,000 mg/kg) did not cause significant changes in either absolute or relative weight of main organs. Recovery groups: reduction in the absolute weight of the liver, kidneys and testicles and a slight increase in the relative weight of the brain, epididymis and testicles of male rats treated with standardized aqueous extract obtained from the leaves of Ilex paraguariensis (1,000 mg/kg) in comparison to the Vehicle group were observed.
Hematology : Main groups: there was a slight decrease in white blood cells in male animals, as well as a slight increase in white blood cells in female treated with standardized aqueous extract obtained from the leaves of Ilex paraguariensis at doses of 500 mg/kg or 1,000 mg/kg (males – Table 14) and 200 mg/kg (females – Table 15). Recovery groups: an increase in platelets and neutrophil count, as well as a slight reduction in lymphocytes count in males rats treated with standardized aqueous extract obtained from the leaves of Ilex paraguariensis (1,000 mg/kg) were observed when compared to the vehicle group.
Table 14 Analysis of hematological parameters of male rats after 90 days of daily treatment with ST-076.
GROUPS VEHICLE DOSE 1
(200 mg/kg) 		DOSE 2
(500 mg/kg) 		DOSE 3
(1,000 mg/kg)
Units Mean SD& Mean SD& Mean SD& Mean SD&
WBC (x103/µL) 12.960 2.789 12.944 2.478 10.002* 1.932 9.710* 1.472
RBC (x106/µL) 7.538 0.802 7.784 0.806 7.333 0.550 7.505 0.640
HGB (g/dL) 13.590 1.347 14.113 1.131 13.544 0.723 13.638 0.981
HCT (%) 41.560 4.103 42.738 3.383 41.733 2.461 42.213 3.232
MCV (fL) 55.190 1.977 55.000 1.806 56.978 1.648 56.275 1.537
MCH (pg) 18.060 0.638 18.175 0.639 18.500 0.626 18.188 0.494
MCHC (g/dL) 32.710 0.407 33.013 0.387 32.478 0.447 32.325 0.480
PLT (x103/µL) 710.80 109.57 613.62 213.82 723.44 52.53 708.12 74.98
W-SCR (%) 80.810 4.131 81.625 5.016 79.756 5.875 82.625 2.426
W-MCR (%) 6.010 1.809 6.875 2.637 6.300 1.606 7.313 3.195
W-LCR (%) 12.220 3.285 10.275 2.954 13.267 4.377 9.413 2.338
& Standard deviation; N – number of animals; *Differs significantly in relation to the Vehicle group; WBC – Total Cells; RBC – Red blood cells; HGB – Hemoglobin; HCT – Hematocrit; MCV – Corpuscular Volume; MCH – Mean corpuscular hemoglobin; MCHC – Mean corpuscular hemoglobin concentration; PLT – Platelet count; W-SCR – Small leukocyte (lymphocyte) index; W-MCR – Mean leukocyte (monocyte) index; W-LCR – Large leukocyte (neutrophil) index.
Table 15 Analysis of hematological parameters of female rats after 90 days of daily treatment with ST-076.
GROUPS VEHICLE DOSE 1
(200 mg/kg) 		DOSE 2
(500 mg/kg) 		DOSE 3
(1,000 mg/kg)
Units Mean SD& Mean SD& Mean SD& Mean SD&
WBC (x103/µL) 7.005 1.988 9.328* 1.398 7.592 2.191 7.846 1.386
RBC (x106/µL) 7.100 0.625 6.898 0.293 6.880 0.476 6.644 0.267
HGB (g/dL) 13.570 1.218 13.111 0.504 13.122 0.771 12.780 0.259
HCT (%) 41.000 3.283 39.656 1.482 40.144 2.359 37.920 0.867
MCV (fL) 57.840 1.391 57.556 1.640 58.400 1.664 57.140 1.932
MCH (pg) 19.130 0.287 19.011 0.333 19.089 0.465 19.240 0.422
MCHC (g/dL) 33.070 0.735 33.059 0.682 32.689 0.478 33.720 0.581
PLT (x103/µL) 749.50 205.00 714.66 199.28 790.22 70.655 768.80 473.48
W-SCR (%) 85.040 2.752 85.756 2.441 87.656 2.711 83.740 4.653
W-MCR (%) 4.020 0.996 4.078 1.109 3.878 0.858 5.320 1.921
W-LCR (%) 9.810 2.181 8.956 2.818 8.056 2.045 9.620 2.566
& Standard deviation; N – number of animals; *Differs significantly in relation to the Vehicle group; WBC – Total Cells; RBC – Red blood cells; HGB – Hemoglobin; HCT – Hematocrit; MCV – Corpuscular Volume; MCH – Mean corpuscular hemoglobin; MCHC – Mean corpuscular hemoglobin concentration; PLT – Platelet count; W-SCR – Small leukocyte (lymphocyte) index; W-MCR – Mean leukocyte (monocyte) index; W-LCR – Large leukocyte (neutrophil) index.
Clinical chemistry : Main groups: increased cholesterol serum levels (1,000 mg/kg) in males (Table 16) as well as increased ALT serum levels (500 mg/kg), cholesterol and alkaline phosphatase (1,000 mg/kg) in females (Table 17) treated with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis when compared to the vehicle group were observed. Recovery groups: a reduction in urea serum levels in males and an increase in glucose serum levels as well as a reduction in calcium and urea levels in females treated with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis were observed when compared to the vehicle group.
Table 16 Analysis of biochemical parameters of male rats after 90 days of daily treatment with ST-076.
GRUPO VEHICLE DOSE 1
(200 mg/kg) 		DOSE 2
(500 mg/kg) 		DOSE 3
(1,000 mg/kg)
Units Mean SD& Mean SD& Mean SD& Mean SD&
ALT (U/L) 91.130 50.181 65.025 16.235 88.244 34.775 56.413 10.867
GGT (U/L) 0.600 1.075 0.000 0.000 5.889 9.714 0.375 0.744
TRI (mg/dL) 71.100 27.045 74.000 11.414 85.444 19.717 63.250 12.453
PT (g/dL) 9.681 0.406 9.691 0.342 9.711 0.461 9.406 0.379
CRE (mg/dL) 0.276 0.022 0.258 0.018 0.211 0.162 0.246 0.049
AST (U/L) 152.570 75.203 115.638 20.968 141.367 41.531 96.088 6.994
CÁLCIO (mg/dL) 6.389 0.284 5.469 2.255 6.356 0.214 6.516 0.331
GLICOSE (mg/dL) 238.960 51.538 317.275 66.860 353.089 123.107 317.613 100.155
BT (mg/dL) 0.061 0.058 0.024 0.045 0.076 0.085 0.053 0.042
CT (mg/dL) 74.500 13.443 88.875 9.296 87.111 14.555 91.750* 12.870
FA (U/L) 171.300 40.064 177.875 34.602 190.111 57.209 125.625 19.863
P (mg/dL) 1.493 0.119 1.391 0.098 1.408 0.138 1.540 0.123
UREIA (mg/dL) 31.270 9.256 31.063 2.405 32.811 14.082 33.975 2.757
Na (nmol/L) 141.90 3.41 145.50 8.09 125.78 41.66 140.38 2.67
K (nmol/L) 7.15 1.21 6.68 1.31 8.28 1.16 7.89 0.77
Standard deviation; N – number of animals; *Differs significantly in relation to the Vehicle group; ALT - Alanine aminotransferase; GGT - Gamma-glutamyltransferase; TRI - Triglycerides; EN - Total protein; CRE - Creatinine; ALB - Albumin; AST - Aspartate Aminotransferase, BT - Total Bilirubin; TC - Total cholesterol; FA - Alkaline phosphatase; P - Phosphorus; Na - Sodium; K – Potassium.
Table 17 Analysis of biochemical parameters of female rats after 90 days of daily treatment with ST-076.
GRUPO VEHICLE DOSE 1
(200 mg/kg) 		DOSE 2
(500 mg/kg) 		DOSE 3
(1,000 mg/kg)
Units Mean SD& Mean SD& Mean SD& Mean SD&
ALT (U/L) 51.270 7.405 45.656 10.835 63.867* 8.691 54.380 9.731
GGT (U/L) 2.200 5.922 1.222 2.587 1.889 5.667 0.600 0.894
TRI (mg/dL) 78.300 20.177 83.222 22.879 65.000 9.798 78.400 29.645
PT (g/dL) 10.637 0.440 10.443 0.841 10.707 0.425 10.530 0.879
CRE (mg/dL) 0.299 0.022 0.246 0.057 0.246 0.159 0.192 0.039
AST (U/L) 110.870 16.315 99.744 18.536 114.389 22.281 115.980 47.043
CÁLCIO (mg/dL) 6.521 0.422 6.512 0.138 6.163 0.315 7.194 2.718
GLICOSE (mg/dL) 195.290 58.995 217.356 82.475 275.022 66.272 255.800 107.526
BT (mg/dL) 0.113 0.126 0.079 0.056 0.082 0.117 0.020 0.039
CT (mg/dL) 90.700 18.172 99.000 20.809 101.222 8.348 117.800* 15.090
FA (U/L) 118.100 37.981 87.444 33.731 109.111 46.657 131.400* 86.786
P (mg/dL) 1.454 0.093 1.170 0.150 1.347 0.145 1.314 0.097
UREIA (mg/dL) 32.500 5.294 29.533 9.143 32.956 6.439 32.660 2.273
Na (nmol/L) 141.100 6.967 139.00 2.872 142.00 7.348 137.40 4.04
K (nmol/L) 8.990 2.224 8.356 1.808 9.556 1.721 8.88 2.02
Standard deviation; N – number of animals; *Differs significantly in relation to the Vehicle group; ALT - Alanine aminotransferase; GGT - Gamma-glutamyltransferase; TRI - Triglycerides; EN - Total protein; CRE - Creatinine; ALB - Albumin; AST - Aspartate Aminotransferase, BT - Total Bilirubin; TC - Total cholesterol; FA - Alkaline phosphatase; P - Phosphorus; Na - Sodium; K – Potassium.
Urinalysis: No significant changes in urine parameters (volume, specific gravity, pH, protein) of the main groups treated with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis (200 mg/kg, 500 mg/kg and 1,000 mg/kg) were observed when compared to the vehicle. Moreover, any visible changes were observed in urine parameters of the recovery group (1,000 mg/kg) when compared to the vehicle group.
Ophthalmology : Ophthalmological evaluations were performed in male and female rats treated with the Vehicle or with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis (1,000 mg/kg). Since no changes were observed in the ophthalmological health of both sexes in the highest dose (1,000 mg/kg), the analysis was not extended to the other experimental groups (main and recovery).
Macroscopy: Macroscopy did not reveal any significant changes related to the treatment with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis for 90 days at doses of 200 mg/kg, 500 mg/kg and 1,000 mg/kg. Moreover, no significant macroscopic changes were observed in the recovery groups when compared to the vehicle.
Histopathology: There were no effects related to the treatment with the standardized aqueous extract obtained from the leaves of Ilex paraguariensis (1,000 mg/kg) in any of the organs and tissues examined, when compared to animals from the Vehicle control group. Due to the absence of treatment-related effects, organs and tissues from groups treated with 200 and 500 mg/kg as well as recovery groups were not analyzed, as recommended by the OECD 408 guideline.
The results of the present study demonstrate that ST-076 administered orally once daily for 90 days did not cause any relevant toxic effects in rats.
Example 12 – Evaluation of safety of the extract according to the present invention on cardiovascular system of ST-076:
Blood pressure, heart rate and electrocardiogram evaluations are part of the battery of fundamental tests for determining the cardiovascular safety of new chemical entities, as recommended by ICH S7A (2000) and ICH S7B (2005) guidelines. The acute oral administration of ST-076, at a dose of 2,500 mg/kg, did not cause any significant changes in the mean arterial pressure (MAP) (Figure 33A), systolic arterial pressure (SAP) (Figure 33B), diastolic arterial pressure (DAP) (Figure 34A) and heart rate (HR) (Figure 34B) in the evaluated periods, showing no acute hypertensive nor hypotensive effect on the cardiovascular system. Therefore, ST-076 at the dose and route tested as well as in the periods assessed did not cause relevant changes on the cardiovascular hemodynamic.
Example 13 – Central nervous system safety pharmacology: evaluation of potential neurotoxic effect of ST-076
The Irwin Test integrates the core battery for evaluation of Central Nervous System (CNS) Safety Pharmacology preconized by ICH S7A guideline (2000). By means of core battery, it is possible to investigate the potential neurotoxic effect of substances through behavior evaluations (observational and interactive).
Data generated by the Irwin Test can be grouped in categories related to effects on general activity (excitation/sedation), motor/coordination behavior, response to stimulus and autonomic signs. The aim of this assay was to evaluate the potential neurotoxic effect of acute administration of the extract according to the present invention in rats comparatively to the well-established effects of Reference Items on the CNS. For this, rats were treated orally with Bromazepam (10 mg/kg), Caffeine (24 mg/kg) or with the extract according to the present invention (50, 1,000 and 2,500 mg/kg) and evaluated in 0-15; 30; 60; 120 and 240 minutes and 24 hours after treatment. Behavior modifications, physiological and neurotoxicity symptoms as well as temperature were recorded according to a standardized observation grid derived from that previously described by Irwin (1968) for mice and adapted for rats (modified Irwin Test).
Results show that the extract according to the present invention induced clinical signals and behavior alterations indicative of excitatory effects in all tested doses including increased general activity (excitation) in different levels as well as in reactivity to touch stimuli and exacerbation of the fear/scare behavior in comparison to the control group (Vehicle). Moreover, treatment with the extract in accordance to the present invention increased respiratory rate and muscular tonus.
The Reference Items Bromazepam and Caffeine according to the tested doses induced behavior modifications, physiological and neurotoxicity symptoms according to previous data from the literature. In conclusion, the results show that the extract according to the present invention administrated orally in rats, caused a slight excitatory effect such as increased general activity, reactivity to touch stimuli, respiratory rate and muscular tonus as well as exacerbated fear/scare behavior.
The results of the present assay demonstrate that the ST-076 according to the present invention administered orally after 10 days in rats, caused a middle excitatory effect, such as increased general activity, reactivity to the touch stimulus, respiratory rate and exacerbation of behavior of fear/fright when compared to animals treated with Vehicle.
Reference
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Duncan RE, Ahmadian M, Jaworski K, Sarkadi-Nagy E, Sul HS. Regulation of Lipolysis in Adipocytes. Annu Rev Nutr. 2007; 27: 79–101.
Gauthier MS, Miyoshi H, Souza SC, Cacicedo JM, Saha AK, Greenberg AS, Ruderman NB. AMP-activated protein kinase is activated as a consequence of lipolysis in the adipocyte: potential mechanism and physiological relevance. J. Biol. Chem. 2008. 283, 16514–16524.
Lee B, Lee J. Cellular and molecular players in adipose tissue inflammation in the development of obesity-induced insulin resistance. Biochim Biophys Acta. 2014 Mar;1842(3):446-62. subjects. Nat. Med 1995;1:950–53.
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Winzell MS, Ahrén B. Glucagon-like peptide-1 and islet lipolysis. Horm Metab Res. Nov-Dec 2004;36(11-12):795-803.
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OECD (2020), Test No. 471: Bacterial Reverse Mutation Test, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264071247-en.
OECD (2014), Test No. 474: Mammalian Erythrocyte Micronucleus Test, OECD Publishing, Paris, https://doi.org/10.1787/9789264224292-en.
OECD (2018), Test No. 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents, OECD Guidelines for the Testing of Chemicals, Section 4, OECD Publishing, Paris, https://doi.org/10.1787/9789264070707-en.
ICH S7A: Safety Pharmacology Studies for Human Pharmaceuticals, 2000.
ICH S7B: The Non-Clinical Evaluation of the Potential for Delayed Ventricular Repolarization

Claims (45)

  1. A method of preparing a standardized, safe and orally bioavailable Ilex paraguariensis extract comprising:
    (a) Contacting Ilex paraguariensis material with a solvent at a temperature below boiling to produce a mixture, and milling the mixture to obtain a crude extract;
    (b) Filtering the crude extract to obtain a purified extract; and
    (c) Drying the purified extract to produce the Ilex paraguariensis extract.
  2. The method of claim 1, wherein, in step (a), the solvent is water and the temperature ranging from 20 °C to about 90 °C.
  3. The method of claim 1 or 2, wherein the Ilex paraguariensis material is washed in running water and dried at a temperature ranging from 20 °C to about 30 °C prior to step (a).
  4. The method of any one of claims 1-3, wherein, in step (a), the extractor liquid comprises ethanol and the temperature ranging from 20 °C to about 60 °C.
  5. The method of any one of claims 1-4 comprising maintaining the Ilex paraguariensis material contacted with an extractor liquid at a temperature ranging from 1°C to about 5 °C for a period of about 1 day to about 3 days.
  6. The method of any one of claims 1-4 comprising maintaining the Ilex paraguariensis material contacted with an extractor liquid at a temperature ranging from 1°C to about 5 °C for a period of about 1 day to about 6 days.
  7. The method of any one of claims 1-4 comprising maintaining the Ilex paraguariensis material contacted with an extractor liquid at a temperature ranging from 1°C to about 5 °C for a period of about 1 day to about 10 days.
  8. The method of any one of claims 1-7, wherein, in step (a), the ratio of plant material to the extractor liquid is about 1:10 weight/volume.
  9. The method of any one of claims 1-8, wherein the extractor liquid comprises ethanol and/or water.
  10. The method according to claim 9 wherein the percent (v/v) of ethanol to water in step (a) is 50% ethanol and 50% water.
  11. The method according to claim 9 wherein the percent (v/v) of ethanol to water in step (a) is 0% ethanol and 100% water.
  12. The method of claim 9, wherein the ratio of ethanol to water (v/v) in step (a) is 25% ethanol and 75% water.
  13. The method of any one of claims 1-12, wherein, in step (b), filtering the crude extract comprising:
    (i) Passing the crude extract through a first filter, wherein the first filter is comprised of a voile-type fabric; and
    (ii) Passing the crude extract through a second filter, wherein the second filter has a basis weight of about 50-80.0 g/m2.
  14. The method of claim 13, wherein the first filter comprises paper.
  15. The method of claim 13 or 14, wherein the second filter comprises paper weighing from 50 g/m2 to about 80 g/m2.
  16. The method of any one of claims 1-15, wherein, in step (c), drying the standardized extract comprises:
    (i) Removing the extractor liquid (ethanol) from the standardized extract via rotary evaporation to obtain a concentrated extract;
    (ii) Lyophilizing the concentrated extract to produce an Ilex paraguariensis extract; and
    (iii) Drying the aqueous or hydroalcoholic extracts in the incubator at 60 °C.
  17. The method of claim 16, wherein removing the extractor liquid from the crude extract via rotary evaporation is conducted at a temperature ranging from 30 °C to about 70 °C.
  18. The method of claim 16, wherein lyophilization of the concentrated extract is conducted at a temperature ranging from -35 °C to about -55 °C and a pressure of about 200 µm Hg to about 300 µm Hg.
  19. The method of claim 16, wherein drying the aqueous or hydroalcoholic extracts in the incubator is conducted at a temperature ranging from 50 °C to about 70 °C.
  20. The method of any one of claims 1-19, wherein the Ilex paraguariensis extract comprises one or more phenolic compounds selected from the group consisting of rutin, caffeine, theobromine, theophylline, and caffeoylquinic acid and its isomers quantified as chlorogenic acid, and combinations thereof.
  21. An Ilex paraguariensis extract composition prepared according to any one of claims 1-20 comprising:
    (i) Rutin: about 100 mg -1,000 mg/100 grammas of standardized extract of Ilex paraguariensis;
    (ii) Caffeine: about 100 mg - 3,000 mg/100 g of standardized extract of Ilex paraguariensis;
    (iii) Theobromine: about 100 mg - 500 mg/100 g of standardized extract of Ilex paraguariensis;
    (iiii) Theophylline: about 2 mg - 10 mg/100 g of standardized extract of Ilex paraguariensis;
    (iiiii) Caffeoylquinic acid and its isomers quantified as chlorogenic acids: about 100 mg - 10,000 mg/100 g of standardized extract of Ilex paraguariensis;
  22. Pharmaceutical composition comprising the extract composition according to claim 21, wherein it is a standardized, stable, non-toxic, non-mutagenic and orally absorbed extract containing a mixture of phytochemical constituents suitable for human or veterinary use, which was stable to up 20 months.
  23. The pharmaceutical composition of claim 22 which is formed into a formulation of capsules, tablets, granules, powders or drinks for the prevention and treatment of human or veterinary health.
  24. The standardized extract obtained from the leaves Ilex paraguariensis plant, according to one of claim 21, wherein it is orally absorbed and used in preventive and/or therapeutic treatment for obesity and related metabolic diseases, including type 2 diabetes, hypertension, heart disease, lipid dysfuction, non-alcoholic fatty liver disease (steatosis), inflammatory diseases and other related diseases associated with metabolic syndrome in humans or animals.
  25. The standardized extract obtained from the leaves Ilex paraguariensis plant, according to one of claim 21, wherein the orally bioavailability of compounds caffeine, theobromine and theophylline present in the standardized aqueous extract obtained from the leaves of Ilex paraguariensis remain in the plasma of animals for up to 12-16 hours after a single treatment and does not accumulate after 7 days of treatment.
  26. Pharmaceutical product comprising the pharmaceutical composition according to claim 22 comprising the administration of an orally effective dosage (1 to 5,000 mg/kg).
  27. Use of the standardized extract obtained from the leaves Ilex paraguariensis plant, as defined in claim 21, wherein it is for the preparation of a pharmaceutical composition to be used in preventive and/or therapeutic treatment for obesity and related metabolic diseases, including type 2 diabetes, hypertension, heart disease, lipid dysfuction, non-alcoholic fatty liver disease (steatosis), inflammatory diseases and other related diseases associated with metabolic syndrome in humans or animals.
  28. Use, according to claim 24, characterized by the fact that standardized aqueous extract obtained from the leaves of Ilex paraguariensis reduces the body weight and regulate the insulin levels, plasma glucose, leptin, cholesterol and triglycerides and increase GLP-1 level, thus can be used for treatment of obesity and related metabolites diseases.
  29. Use, according to claim 24, characterized by the fact that the standardized aqueous extract obtained from the leaves of Ilex paraguariensis efficiently reduces high plasma glucose levels and can be used for the treatment of diabetes.
  30. Use, according to claim 24, characterized by the fact that the standardized aqueous extract obtained from the leaves of Ilex paraguariensis efficiently reduces high blood pressure and can be used for the treatment of hypertension associated with metabolic syndrome.
  31. Use, according to claim 24, characterized by the fact that the standardized aqueous extract obtained from the leaves of Ilex paraguariensis efficiently reduces inflammatory cytokine levels of TNF-α and MCP-1 and can be used for treatment of inflammation.
  32. Pharmaceutical product, according to claim 26, characterized by the standardized aqueous extract obtained from the leaves of Ilex paraguariensis revealed its safety (not toxic) according to the acute (MTD) and repeated dose 90-days oral toxicity studies.
  33. Pharmaceutical product, according to claim 26, characterized by the standardized aqueous extract obtained from the leaves of Ilex paraguariensis proved to be safe (nontoxic nor genotoxic) to the cardiovascular and central nervous system.
  34. Use, according to claim 27, wherein the metabolic disorders, mainly obesity, seem be related with up or down regulation of most relevant genes in peripheral organs and central nervous system with changes in cellular singling pathways which are involved in regulation of obesity, metabolic diseases, and other related diseases.
  35. Use of claim 34, wherein the mechanism of action of standardized aqueous extract obtained from the leaves of Ilex paraguariensis is related to the activity of modulating peroxisome proliferator-activated receptor (PPAR alpha and gamma) in adipocytes and liver.
  36. Use of claim 34, wherein the mechanism of action of standardized aqueous extract obtained from the leaves of Ilex paraguariensis is related to the increase in PI3K/AKT signaling pathway that plays a central role in cellular physiology by mediating growth factor signals during organismal growth and critical cellular processes, such as glucose homeostasis, lipid metabolism, protein synthesis and cell proliferation.
  37. Use of claim 34, wherein the mechanism of action of standardized aqueous extract obtained from the leaves of Ilex paraguariensis is related to the increase in Cocaine and amphetamine regulated transcript (Cardpt) gene expression that promotes anorexic effects, reduction of  food intake and induces satiety.
  38. Use of claim 34, wherein the mechanism of action of standardized aqueous extract obtained from the leaves of Ilex paraguariensis is related to the increase in monoacylglycerol lipase ( Mgll) gene in adipocytes tissue, that promotes fat storage in gonadal white fat with increased lipogenesis and unchanged lipolysis.
  39. Use of claim 34, wherein the mechanism of action of standardized aqueous extract obtained from the leaves of Ilex paraguariensis is related to the increase in Insulin-2 (Ins2) gene that plays a vital role in the regulation of carbohydrate and lipid metabolism.
  40. Use of claim 34, wherein the mechanism of action of standardized aqueous extract obtained from the leaves of Ilex paraguariensis is related to the increase in Glucagon Receptor (Gcgr) gene that regulates blood glucose levels and glucose homeostasis.
  41. Use of claim 34, wherein the mechanism of action of standardized aqueous extract obtained from the leaves of Ilex paraguariensis is related to the reduction in inflammasome (NLRP3) in adipocytes, that is responsible for the apoptosis-associated speck-like protein PYCARD/ASC, which contains a caspase recruitment domain, and is a member of the NALP3 inflammasome complex.
  42. Use of claim 34, wherein the mechanism of action of standardized aqueous extract obtained from the leaves of Ilex paraguariensis is related to the increase of Ramp3 (Receptor activity modifying protein 3) gene expression that modulates amylin and controls glycemia and induces satiety.
  43. Use of claim 34, wherein the mechanism of action of standardized aqueous extract obtained from the leaves of Ilex paraguariensis is related to the increase in mRNA expression of Neuropeptide Y (Npy) gene that modulate, stress response, food intake and cardiovascular function.
  44. Use of claim 34, wherein the impairment of AMP-activated protein kinase (AMPK) is a highly conserved master regulator of metabolism, whose activation has been proposed to be therapeutically beneficial for the treatment of several metabolic diseases, including nonalcoholic fatty liver disease (steatosis).
  45. Use of claim 34, wherein the mechanism of action of standardized aqueous extract obtained from the leaves of Ilex paraguariensis is related to the reduction of cAMP Response Element Binding protein (CREB, transcription factor) which is activated in the adipose tissue under obese conditions, where it promotes insulin resistance.
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