WO2020105070A1

WO2020105070A1 - Dietary supplement used to reduce the release of inflammatory agents and related use

Info

Publication number: WO2020105070A1
Application number: PCT/IT2019/000106
Authority: WO
Inventors: Massimo Defilippi
Original assignee: Biohealth Italia S.R.L.
Priority date: 2018-11-19
Filing date: 2019-11-18
Publication date: 2020-05-28
Also published as: EP3883587A1; IT201800010393A1

Abstract

A dietary supplement is described, to be used to reduce a release of inflammatory cytokines in patients in whose organism there are leukemic cells of a TPH-1 type, such release being a consequence of a response of the TPH-1 cells to a stimulation with Lipo-polysaccharides (LPS), the dietary supplement comprising the following active ingredients: Buglossoides arvensis seed oil (L.,I.M. Johnst) titrated in essential fatty acids; Ananas (Ananas comosus, L. MERR, stipites) dry extract titrated in bromeline; Boswellia (Boswellia sacra Flueck. (syn. (Boswellia carteri Birdw.), rubber resin) fluid extract, titrated 4% in acetyl-11-keto-beta-boswellic acid (AKBA); Myrrh (Commiphora myrra, Engl., oil-rubber-resin) fluid extract, titrated 4% in furan-dienes.

Description

DIETARY SUPPLEMENT USED TO REDUCE THE RELEASE OF INFLAM4AT0RY AGENTS AND RELATED USE

The present invention refers to a dietary supplement designed to drastically reduce the inflammatory effects in people affected by leukemia with infections from Gram-negative bacteria.

It is known that monocytes and macrophages synthetize and release in their environment a variety of cytokines and other proteins which perform a central role in the development of acute and chronic inflammations.

THP-1 is a human monocyte leukemic cell line; it is known from scientific literature that these cells are capable of producing pro- inflammatory cytokines (THE- 1 , THE-6 and TNF) and chemokines (THE-8 and MCP-1) in reply to a stimulation by

Lipo-polysaccharides (LPS) .

LPS is a polysaccharide deemed as the main component of the external membrane of Gram-negative bacteria. Recent studies have clarified how LPS is recognized by monocytes and macrophages of the innate immune system; in particular, human monocytes are extremely sensitive to LPS and their response is translated into the expression and the release of many inflammatory cytokines.

From what is stated above, it results that, in a person affected by leukemia, in whose organism there are THP-1 cells, in case of infection by

Gram-negative bacteria, many inflammatory cytokines are released.

EP-A1-3 130 336, US-A1-2008/213357, WO-A1-

2017/029643 and WO-A1-2010/037213 disclose the various components which make the dietary supplement of the present invention; none of them, however, can be used individually to reduce a release of inflammatory cytokines in patients in whose organism there are leukemic cells of a TPH-1 type.

The main object of the present invention is reducing, till almost cancelling, the release of inflammatory cytokines, in patients in whose organism there are leukemic cells of the TPH-1 type, said release being a consequence of the response of said TPH-1 cells to a stimulation with

Lipo-polysaccharides (LPS) .

The above object and the advantages of the invention, as will appear from the following description, are obtained with a dietary supplement as claimed in claim 1, namely comprising the following active ingredients :

Buglossoides arvensis seed oil (L., I.M.

Johnst) titrated in essential fatty acids , preferably Omega 3 and Omega 6;

Ananas (Ananas comosus (L. MERR, stipites) dry extract titrated in bromeline;

Boswellia (Boswellia sacra Flueck . (syn.

Boswellia carteri Birdw . ) , rubber resin) fluid extract , titrated 4% in acetyl - 11-keto-beta- boswellic acid (AKBA) ;

Myrrh (Commiphora myrra, oil-rubber resin) fluid extract , titrated 4% in furan-dienes .

According to a preferred embodiment of the invention, said dietary supplement comprises the above four active ingredients, with the following relative concentrations :

Buglossoides arvensis seed oil (L., I.M.

Johnst ) titrated in essential fatty acids, preferably Omega 3 and Omega 6: 14%;

Ananas (Ananas comosus, L. MERR, stipites) dry extract titrated in bromeline: 16%;

Boswellia (Boswellia sacra Flueck . (syn. (Boswellia carteri Birdw. ) , rubber resin) fluid extract , titrated 4% in acetyl -11-keto-beta- boswellic acid (AKBA) : 30%;

Myrrh (Commiphora myrra, Engl • 9 oil -rubber- resin) fluid extract , titrated 4% in furan-dienes :

40%.

preferred embodiments and non-trivial variations of the present invention are the subject matter of the dependent claims.

It is intended that all enclosed claims are an integral part of the present description.

It will be immediately obvious that numerous variations and modifications (for example replacements of one or more active ingredients and/or excipients with other of recognized equivalence) can be made to what is described, without departing from the scope off the invention, as appears in the enclosed claims .

The present invention will be better described by some preferred embodiments thereof , provided as a non-limiting example , with reference to the enclosed drawings , in which:

Figure 1 shows the release of inflammatory cytokines TNF-a, due to the stimulation with LPS, of THP-1 cells as such and pre-treated with a formulation according to the invention;

Figure 2 shows a Table with a formulation according to the invention subjected to experimentations ;

Figure 3 shows a Table with the results of experimentations performed on the formulation of

Figure 2.

The dietary supplement of the invention

(formulated below) has been subjected to experimentations to verify its efficacy in inhibiting the release of inflammatory cytokines, simulating in vitro the effect of the cellular membrane of Gram-negative bacteria on THP-1 cells.

The invention therefore deals with a dietary supplement, to be used to reduce the release of inflammatory cytokines in patients in whose organism there are leukemic cells of the TPH-1 type, such release being a consequence of the response of said TPH-1 cells to a stimulation with Lipo-polysaccharides (LPS) ; the dietary supplement of the invention comprises the following active ingredients :

Buglossoides arvensis seed oil (L., I.M.

Johnst ) titrated in essential fatty acids, preferably Omega 3 and Omega 6; Ananas (Ananas comosus (L. MERR, stipites) dry extract titrated in bromeline;

Boswellia (Boswellia sacra Flueck. (syn.

Boswellia carter! Birdw . ) , rubber resin) fluid extract , titrated 4% in acetyl-ll-keto-beta- boswellic acid (AKBA) ;

Myrrh (Commiphora myrra, oil -rubber resin) fluid extract, titrated 4% in furan-dienes.

According to the preferred embodiment , to obtain the best effect, the dietary supplement of the invention comprises the four above active ingredients, with the following relative concentratios :

Buglossoides arvensis seed oil (L. , I.M. Johnst) titrated in essential fatty acids, preferably Omega 3 and Omega 6: 14%;

Boswellia (Boswellia sacra Flueck. (syn. (Boswellia carter! Birdw. ) , rubber resin) fluid extract , titrated 4% in acetyl- 11-keto-beta- boswellic acid (AKBA) : 30%;

Myrrh (Commiphora myrra, Engl., oil-rubber- resin) fluid extract, titrated 4% in furan-dienes : 40%. The above described formulation can be integrated with the addition of:

silicon dioxide ,- hydroxyl-propyl -methylcelluloae ;

DL-or Tocopherol;

ethyl cellulose;

extra-virgin olive oil (Olea europaea L., fructus) ;

glycerol ;

sodium alginate;

medium-chain triglycerides;

rameic chlorophyllin complexes ;

titanium dioxide ,- oleic acid;

stearic acid.

In particular, the above described supplement can be packaged in capsules and have the following global composition:

Buglossoides arvensis seed oil (L., I.M. Johnst) titrated in essential fatty acids, preferably Omega 3 and Omega 6: 9.764%;

Ananas (Ananas comosus, L. MERR, stipites) dry extract titrated in bromeline: 11.159%;

Boswellia (BosweIlia sacra Plueck. (syn. (Boswellia carteri Birdw.), rubber resin) fluid extract , titrated 4% in acetyl -11-keto-beta- boswellic acid (AKBA) : 20.923%;

Myrrh (Commiphora myrra, Engl., oil -rubber- resin) fluid extract , titrated 4% in furan-dienes : 28.456%;

hydroxyl -propyl-methylcellulose (capsule ingredient) : 12.554%;

silicon dioxide : 0.139%;

DL-a Tocopherol : 1.395%;

ethyl cellulose: 2.664%;

extra-virgin olive oil: 6.138%;

hydroxyl -propyl-methylcellulose : 3.613%;

glycerol: 0.753%;

sodium alginate : 0.621%;

medium-chain triglycerides : 0.711%;

rameic chiorophyl1in complexes : 0.446%;

titanium dioxide: 0.349%;

oleic acid: 0.307%;

stearic acid: 0.006%.

The above described dietary supplement is then used as anti-inflammatory agent.

As such, it is administered in a dosage from

500 mg to 2000 mg.

The composition of the formulation subjected to experimentations is also included in the Table in Figure 2.

THP-1 cells have been kept in a RPMI-1640 ground free from endotoxins and containing: 5,5 mmol/1 glucose, 50 mihoΐ/ΐ mercapto-ethanol, 10* bovine fetal serum, 2 mmol/1 glutamine, 1 mmol/1 sodium pyruvate, 10 mmol/1 HEPES.

Experimentations in vitro have been performed by stimulating the TPH-1 cells with LPS, since it is the major component of the external membrane of Gram-negative bacteria. In this way, it is possible to evaluate the amount of inflammatory cytokines released and, therefore, repeat the experiment by adding the formulation and evaluating the differences in the release of said inflammatory cytokines .

The stimulation in vitro has been performe through the addition of LPS [100 ^g/l] for 4 hours to THP-1 cells seeded in a 12 -well plate (~lxl0⁶ cells/ml) and incubated at 37°C and 5* C0₂ grown in 75-mm² flasks in RPMI-1640 ground enriched with bovine fetal serum and taken to 70* of confluence.

The Tumor Necrosis Factor (TNF-a) is an inflammatory cytokine and is a 17-kDa polypeptide mainly produced by activated monocytes and macrophages. This polypeptide mediates some immune and inflammatory responses, including activation and differentiation of monocytes and macrophages, expression of MHC of classes I and II and expression of adhesion molecules on endothelial cells.

The performed experimentation provided for the dosage of TNF-a released following the stimulation of THP-1 cells with LPS. The determination of the concentration of TNF-a in the colture ground has been performed through ELISA (Enzyme-Linked

Immunosorbent Assay) (Cayman Chemical, Michigan

48108 USA) , based on a double-antibody sandwich technique .

The anti-inflammatory effects of the treatment with the formulation has been evaluated by using

THP-1 cells (lxlO⁶ cells/ml) stimulated with LPS

[100 jzg/l] for 4 hours; afterwards, the concentration of TNF-a possibly present in the cellular culture ground has been detected and quantified.

The experimentations have been performed in three steps :

1 pre-treatment of the TPH-1 cells with the formulation;

2 stimulation with LPS of the cells pre-treated with the formulation;

3 qualitative and quantitative analysis of the experimental results .

Step 1 - the THP-1 cells (lxlO⁶ cells/ml) have been pre-treated for 1 hour with two different formulation concentrations:

a [0.2 mg/ml] ;

b [67 mg/ml] .

The choice of the concentration [0.2 mg/ml] has been determined by the fact that the daily posology is 2 capsules/day for an adult; every capsule contains 500 mg of active ingredients : therefore, 1 g/day, which in vivo is distributed in about 5 1 of blood . In vitro this corresponds to [0.2 mg/ml] of formulation.

The choice of the concentration [67 mg/ml] has been determined both by the compliance with the accurate experimental conditions of the model in vitro (lxlO⁶ monocytes/ml of culture ground + LPS

100 pg/l) , and by the consideration that, in vivo, under normal conditions, there are about 10^e monocytes per liter of blood; therefore, 0.2 g/1 of formulation potentially have effect on 10⁶ monocytes .

Step 2 - The pre-treated TPH-1 cells have been stimulated with LPS [100 pg/l] for 4 hours . The culture ground with the cells has been recovered at the end of the experimental time and the cells have been lysed by using a sonicator . The thereby- obtained supernatant has then been used for the following qualitative and quantitative analyses .

Step 3 The supernatant has been used to quantify the TNF-or through ELISA test (Tumor Necrosis

Factor-a human EIA Kit, Cayman Chemical, Michigan 48108 USA) . The results for the release of cytokines are included per ml of supernatant .

All experiments have been performed in triplicate . The Student's "T* test for unpaired data has been applied for a comparison between the means, accepting a significance of 5%.

Experimentation results

Figure 1 shows the results of the stimulation of THP-l cells with LPS, as such and pre-treated with the formulation.

The maximum stimulation level of THP-l cells has been obtained by treating them with LPS [100 pg/l] after a 4 -hour incubation.

Columns (A) and (D) show the releases of cytokines from TPH-1 cells not stimulated with LPS

(CTR-) , while columns (B) and (E) show the releases of cytokines from two samples of cells stimulated with LPS, namely the dosage of TNF-a detected on a first and on a second sample of TPH-1 cells, respectively.

It can be noted how the concentration of TNF-a released by the not stimulated cells (CTR-) is greatly different from the concentration of TNF-a released from cells stimulated with LPS .

The columns designated as (C) and (F) include the TNF-a released after stimulation with LPS of the THP-1 cells pre-treated with the formulation at the concentration of [0.2 mg/ml] and [67 mg/ml] respectively .

As can be seen in Figure 1 and in the Table in Figure 3, the formulation at the concentration of

[0.2 mg/ml] has inhibited the release of TNF-a by

6.82%, while at the concentration of [67 mg/ml] has inhibited the release of TNF-a by 99.85%.

The results are included in the Table in Figure 3, as mean ± Standard Deviation (DS) from experiments performed in triplicate.

In the same Table:

D is the percentage variation computed between the sample of TPH-1 cells stimulated with LPS and

25 pre-treated with the formulation vs the sample of TPH-1 cells only stimulated with LPS;

p is the p value , according to the Student's statistics, computed between sample stimulated with

LPS and treated with the formulation with respect to the sample stimulated with LPS;

CTR is the control only of the TPH-1 cells not treated with LPS.

The in vitro model which has been applied has allowed to test two different concentrations [0.2 mg/ml] and [67 mg/ml] of the formulation according to the invention.

Both concentrations has been chosen by taking into account: 1) daily posology; 2) total volume of blood which is present in an adult individual; 3) number of cells (monocytes) present both in vivo in

1 1 of blood, and in vitro in the experimental model followed.

The smaller concentration [0.2 mg/ml] has reduced the measured concentration of TNF-a by 6.82% and the bigger concentration [67 mg/ml] has caused a strong reduction of TNF-a (p=3,4*10^"10) (D

= 99.85%) .

In compliance with the test and the experimental conditions used, it can be concluded that the formulation according to the invention, whose composition in included in the Table in

Figure 2, is capable of inhibiting the release of

TNF-a, known mediator of several immune and inflammatory responses, suggesting an anti- inflammatory activity of the tested sample.

The Table in Figure 2 shows a composition comprising the above mentioned four active ingredients, plus various ingredients having other functions.

Other tests performed on similar compositions to the one included in the Table in Figure 2, and comprising one or more of the above active ingredients, have shown a validity in inhibiting the release of TNF-a, even with a lower efficacy than the one shown by the composition included in the Table in Figure 2.

The formulation of the invention can be conveniently packaged in capsules, for a practical up-take by a patient.

Claims

1. Dietary supplement to be used to reduce a release of inflammatory cytokines in patients in whose organism there are leukemic cells of a TPH-1 type, said release being a consequence of a response of said TPH-1 cells to a stimulation with

Lipo-polysaccharides (LPS) , the dietary supplement comprising the following active ingredients:

Buglossoides arvensis seed oil (L., I.M. Johnst) titrated in essential fatty acids;

Ananas (Ananas comosus, L. MERR, stipites) dry extract titrated in bromeline;

Boswellia (Boswellia sacra Flueck. (syn.

(Boswellia carteri Birdw.), rubber resin) fluid extract, titrated 4% in acetyl-11-keto-beta- boswellic acid (AKBA) ;

Myrrh (Commiphora myrra, Engl., oil-rubber- resin) fluid extract, titrated 4% in furan-dienes .

2. Dietary supplement according to claim 1, characterized in that it comprises said four active ingredients, with the following relative concentrations :

Buglossoides arvensis seed oil (L., I.M.

Johnst) titrated in essential fatty acids: 14%;

Boswellia (Boswellia sacra Flueck. (syn.

(Boswellia carter! Birdw. ) , rubber resin) fluid extract , titrated 4* in acetyl -11 -keto-beta- boswellic acid (AKBΆ) : 30%;

Myrrh (Commiphora myrra, Engl., oil -rubber- resin) fluid extract, titrated 4% in furan-dienes :

40%.

3. Dietary supplement according to claim 2, with an addition of :

silicon dioxide;

hydroxyl -propyl -methylcellulose ;

DL-ot Tocopherol;

ethyl cellulose;

extra-virgin olive oil;

glycerol ;

sodium alginate ;

medium-chain triglycerides ;

rameic chlorophyllin complexes ;

titanium dioxide;

oleic acid;

stearic acid.

4. Dietary supplement according to any one of the previous claims, characterized in that it is packaged in capsules and it has the following global composition:

Buglossoides arvensis seed oil (L. , I.M. Johnst) titrated in essential fatty acids : 9.764%;

Boswellia (Boswellia sacra Flueck. (syn.

(Boswellia carteri Birdw. ) , rubber resin) fluid extract , titrated 4% in acetyl -11-keto-beta- boswellic acid (AKBA) : 20.923%;

Myrrh (Commiphora myrra, Engl., oil-rubber- resin) fluid extract , titrated 4% in furan-dienes :

28.456%;

hydroxyl -propyl -methylcellulose (capsule ingredient) : 12.554% ;

silicon dioxide : 0.139%

DL-ct Tocopherol: 1.395%

ethyl cellulose : 2.664%

extra-virgin olive oil: 6.138%;

hydroxyl -propyl -methylcellulose : 3.613%;

glycerol: 0.753%;

sodium alginate : 0.621%;

medium-chain triglycerides : 0.711%;

rameic chlorophyl1in complexes : 0.446%;

titanium dioxide: 0.349%;

oleic acid: 0.307%; stearic acid: 0.006%.

5. Use of the dietary supplement according to any one of the previous claims as anti-inflammatory agent .

6. Use according to claim 5, wherein said dietary supplement is administered in a dosage ranging from

500 mg to 2000 mg.