WO2016180363A1

WO2016180363A1 - Chitosan citrate and composition containing same

Info

Publication number: WO2016180363A1
Application number: PCT/CN2016/082002
Authority: WO
Inventors: Ming Houng Chiu; Ming Shuen Hong; Chi Ming Chiu
Original assignee: Shin Era Technology Co., Ltd.
Priority date: 2015-05-13
Filing date: 2016-05-13
Publication date: 2016-11-17
Also published as: TW201703774A

Abstract

Provided is a phosphate-binding composition comprising chitosan citrate as the phosphate-binding agent. The chitosan citrate exhibits excellent phosphate-binding ability comparable to calcium carbonate, which is clinically used nowadays. Thus, the phosphate-binding composition of the present invention is valuable for treating hyperphosphatemia.

Description

CHITOSAN CITRATE AND COMPOSITION CONTAINING THE SAME

BACKGROUND

Description of Related Art

Hyperphosphatemia is a syndrome that the serum phosphate level of a subject is above a certain threshold causing deposits throughout the body and endangering circulation. There are many different disease may cause hyperphosphatemia, such as, renal disease, kidney failure, hypoparathyroidism, etc. In end-stage of the aforesaid diseases, the patient’s kidney function will be compromised and thereby the phosphate level in the blood becomes markedly elevated.

Patients suffering hypoparathyroidism are advised to have low phosphate diet. However, phosphate is very commonly present in food we eat everyday. That said, it is difficult to avoid foods containing phosphate. Since it is nearly impossible to eat only food containing low phosphate, others strategies shall be adopted for decreasing phosphate uptake. In this regard, phosphate binders are developed for this purpose. Typically, phosphate binders are substances taken orally and effective in the intestinal tract for binding with phosphate and preventing the same from being uptaken.

Conventional phosphate binders include, for example, various salts of aluminum and calcium, as well as some chemically synthesized cross-linked polymers. However, aluminum or calcium salts and cross-linked polymers may both cause undesirable results and therefore, are not ideal.

To sum up, although there are several medicines being used as phosphate binders exist, it is still continuous needs in the art for more options capable of relieving hyperphosphatemia symptom.

SUMMARY

One of the objectives of the present invention is to provide an alternative phosphate-binding agent that may exhibit better phosphate-binding ability and/or fewer side effects than the conventional medicine used clinically.

Another objective of the present invention is to provide a novel method for treating hyperphosphatemia by using a novel phosphate-binding agent.

In order to achieve the aforesaid objectives, the present invention provides a phosphate-binding composition, comprising 13 to 40 μg/μl of chitosan citrate； and a pharmaceutically acceptable carrier； wherein said μg/μl is based on the total volume of said composition.

Preferably, said composition comprises 13 to 26 μg/μl of chitosan citrate.

Preferably, said chitosan citrate comprises a chitosan moiety； wherein said chitosan moiety is of 50 to 95 ％of deacetylation. More preferably, said chitosan moiety is of 55 to 90 ％of deacetylation.

Preferably, said pharmaceutically acceptable carrier is water, alcohol, glycerol, chitosan, alginate, chondroitin, Vitamin E, mineral oil, dimethyl sulfoxide (DMSO) , or a combination thereof.

Preferably, said chitosan citrate comprises a chitosan moiety； wherein said chitosan moiety has a weight-average molecular weight of 15000 to 40000 dalton.

The present invention also provides a method for binding phosphate in an environment, comprising: contacting said environment with a chitosan citrate； wherein said chitosan citrate comprises a chitosan moiety； wherein said chitosan moiety is of 50 to 95 ％of deacetylation.

The present invention then provides a use of chitosan citrate for preparing a pharmaceutical composition for treating hyperphosphatemia in a subject； wherein said pharmaceutical composition is the composition above.

The present invention more provides a method for treating hyperphosphatemia in a subject, comprising administering said subject the composition above before, during, or after diet.

Preferably, said chitosan moiety is of 55 to 90 ％of deacetylation.

Preferably, said chitosan moiety has a weight-average molecular weight of 15000 to 40000 dalton.

Preferably, said chitosan citrate is formulated as a composition； wherein said composition comprises 13 to 40 μg/μl of said chitosan citrate； and a pharmaceutically acceptable carrier； wherein said μg/μl is based on the total volume of said composition. More preferably, said composition comprises 13 to 26 μg/μl of said chitosan citrate.

Preferably, said contacting is conducted by introducing said chitosan citrate into said environment.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the flow chart of preparing the chitosan citrate of the present invention.

Figure 2 shows the results of the phosphate-binding assay of experiment 1.

Figure 3 shows the results of the phosphate-binding assay of experiment 2.

DETAILED DESCRIPTION

The present invention provides a chitosan citrate for binding phosphate and thereby to relieve hyperphosphatemia symptom. In a preferable embodiment, said chitosan citrate is formulated as a composition comprising 13 to 40 μg/μl of chitosan citrate and a pharmaceutical carrier； wherein said μg/μl is based on the total volume of said composition. In a more preferable embodiment, said chitosan citrate is of 13 to 26 μg/μl, based on the total volume of said composition.

In an alternative embodiment, said chitosan citrate has a chitosan moiety； wherein said chitosan moiety is of 50 to 95 ％of deacetylation. The degree of deacetylation is a factor of the ability of phosphate binding of the present chitosan citrate. Without being bound by theory, the more deacetylation gives more active functional groups for phosphate binding and thereby contributes better phosphate-binding ability to the present chitosan citrate. In a preferable embodiment, said chitosan moiety is of 55 to 90 ％of deacetylation.

The present invention surprisedly proved that the present chitosan citrate exhibits phosphate-binding ability significantly better than chitosan or citric acid alone. That said, even if there is some publications implied the potential application of chitosan or citric acid in phosphate-binding, one can never foresee the significant improvement contributed by the present chitosan citrate in comparison with other chitosan salt (such as chtosan lactate) or citric acid alone.

In an alternative embodiment, said chitosan moiety has a weight-average molecular weight of 15000 to 40000 dalton； preferably, said weight-average molecular weight is of 20000 to 35000 dalton. In a preferable embodiment, said pharmaceutically acceptable carrier is water, alcohol, glycerol, chitosan, alginate, chondroitin, Vitamin E, mineral oil, dimethyl sulfoxide (DMSO) , or a combination thereof. Those having ordinary skill in the art can choose suitable pharmaceutically acceptable carrier in accordance with its need or administration route.

The present invention also provides a method for binding phosphate in an environment. Said method may be used for removing phosphate from an environment for any desired purposes. Case in point, the method can be conducted to remove phosphate from food containing phosphate so that the food can be edible for patients having feeble kidney function. The term “remove” used herein is referred to as “reducing the content” of an object but shall not necessary mean “removing” the object completely.

The present method for binding phosphate in an environment comprises contacting said environment with a chitosan citrate； wherein said chitosan citrate comprises a chitosan moiety； wherein said chitosan moiety is of 55 to 90 ％of deacetylation. Said environment may be a container containing a food. In an alternative embodiment, said environment may be the intestinal tract where the food ingested by a subject can be digested while reacting with the present chitosan citrate.

In an embodiment, said contacting can be achieved by introducing said chitosan citrate into said environment. Nevertheless, the way to achieve said contacting is not limited as long as said chitosan citrate can react with or in said environment to exhibit its phosphate-binding effect.

The present invention more provides a use of chitosan citrate for preparing a pharmaceutical composition for treating hyperphosphatemia in a subject； wherein said pharmaceutical composition is the composition described above.

The present invention also provides a method for treating hyperphosphatemia in a subject, comprising administering said subject the composition described above. In an embodiment of the preset invention, said composition is administered before, during or after diet of said subject.

The term of “treating” used herein is referred to as controlling, reducing, and/or avoiding from negative progression of a disease, illness, or symptoms at issue. Accordingly, said “treating hyperphosphatemia” may be construed as controlling, reducing, and/or avoiding from negative progression of hyperphosphatemia symptoms of a subject.

Experiment 1: Preparation of the present chitosan citrate.

In this example, an exemplary procedure of the present invention was exhibited for preparing the present chitosan citrate. The preparation was conducted through the following steps and described in Figure 1:

(1) Chitin extracted from crab (or shrimp) shells with molecular weight of more than 140,000 dalton was obtained and degraded by hydrogen peroxide (35 wt ％) at 20℃ for 72 hours. After the degradation, the weight-average molecular weight (according to standard measurement of CHS-E11-03 of Shin Era Technology Co., LTD. by using BROOKFIELD programmable DV-II +Viscometer) of the chitin was changed into about 23,000 dalton.

(2) After the chitin from step (1) was dried, the chitin was reacted with sodium hydroxide (NaOH； 42.5 wt％) for deacetylation. The solid/liquid ratio (ie. chitin/sodium hydroxide) was 1: 45. The reaction was maintain under 40 ℃. Mixtures containing chitosan of 60％, 87％, and 90 deacetylation were obtained respectively upon different reaction time (15 hours, 25 hours and 30 hours, respectively) .

(3) Then, chitosan of 60％deacetylation and chitosan of 90％deacetylation were processed respectively as follows:

3-1 Chitosan of 60％deacetylation was pressed to separate NaOH therein. Then, the pressed chitosan of 60％deacetylation was neutralized with 10 wt％H₂SO₄ and purified by dialysis (Spectrum Labs Hollow Fiber) to remove Na₂SO₄. Then citric acid (40 wt％) was added in to react with the chitosan. The reaction was conducted at 30 ℃ for 2 hours. After the reaction, chitosan citrate of 60％deacetylation was obtained and spray dried to provide powder thereof.

3-2 Chitosan of 90％deacetylation was pressed to separate NaOH therein. Then, the chitosan of 90％deacetylation was washed with water and neutralized with 10 wt％H₂SO₄. After that, the chitosan of 90％deacetylation was again pressed to separate from the liquid therein. Then citric acid (40 wt％) was added in to react with the chitosan. The reaction was conducted at 30 ℃ for 24 hours. After the reaction, chitosan citrate of 90％deacetylation was obtained and spray dried to provide powder thereof.

(4) The obtained chitosan citrates were formulated as aqueous solution of various concentrations for the subsequent phosphate-binding assay.

Experiment 2: Phosphate binding assay 1.

In this experiment, the chitosan citrate of 60％deacetylation and chitosan citrate of 90％deacetylation (hereinafter referred to as CHS60 and CHS90 respectively) obtained in the previous experiment 1 were tested for their ability of binding phosphate in comparison with calcium carbonate. Please see the experiment design in the following table 1. Co-incubation of KH₂PO₄ and the present chitosan citrate or calcium carbonate of indicated concentration was conducted for 2 hours under pH 7～8 and room temperature (about 28℃) .

Table 1: Experiment Design.

*after mixing with KH₂PO₄ the final concentration of the binding agent in the reaction is 6.6 μg/μl.

After the aforesaid incubation, 2 μl of the mixture of each group (and KH₂PO₄ without reaction with binding agents) was mixed with 120 μl of acetate buffer (0.1 N acetic acid and 0.025 N of CH₃COONa) , 12 μl of ascorbic acid (1 wt％) , and 12 ml of NH₄MoO₄ (1％, in 0.05 N H₂SO₄) at room temperature for 20 to 30 minutes. After that, the OD₇₀₀ of the mixture was detected by spectrometer. The experiments were repeated three times for determine the standard deviation. The binding ability was shown in the following table 2 and Figure 2.

Table 2

*The phosphate binding ability was calculated by the formula: (OD₇₀₀ of mixture after binding reaction) / (OD₇₀₀ of KH₂PO₄ without reaction with binding agents) *100％.

The data above showed that the present chitosan citrate can provide comparable effects in phosphate-binding in comparison with calcium carbonate. Calcium carbonate is clinically used as phosphate-binding agent in the field. Thus, the data above implied the potential of the present chitosan citrate to be an alternative option of calcium carbonate.

Experiment 3: Phosphate binding assay 2.

Experiment 2 set forth above has demonstrated the present chitosan citrate’s potential as phosphate-binding agent. The research proceeded to the next stage to compare the phosphate-binding ability of the present chitosan citrate and other phosphate-binding agents.

In this experiment, the chitosan citrate of 60％deacetylation and chitosan citrate of 90％deacetylation (hereinafter referred to as CHS60 abd CHS90 respectively) obtained in the previous experiment 1 were tested for their ability of binding phosphate at various concentration. Moreover, calcium carbonate, ferric citrate (Sigma F3388) , citric acid, and chitosan lactate of 87％deacetylation (CH87, purchased from Shin Era Technology Co., LTD. ) were respectively used as control groups in the trials. Please see the experiment design shown in the following table 3. Co-incubation of KH₂PO₄ and those binding agents respectively was conducted for 2 hours under pH 7～8 and room temperature (about 28℃) .

Table 3: Experiment Design.

1. after mixing with KH₂PO₄ the final concentration of the binding agent in the reaction is 6.6 μg/μl.

2. after mixing with KH₂PO₄ the final concentration of the binding agent in the reaction is 13.2 μg/μl.

*the concentration indicated is the final concentration in the reaction.

After the aforesaid incubation, 2 μl of the mixture of each group (and KH₂PO₄ without reaction with binding agents) was mixed with 120 μl of acetate buffer (0.1 N acetic acid and 0.025 N of CH₃COONa) , 12 μl of ascorbic acid (1 wt％) , and 12 ml of NH₄MoO₄ (1％, in 0.05 N H₂SO₄) at room temperature for 20 to 30 minutes. After that, the OD₇₀₀ of the mixture was detected by spectrometer. The experiments were repeated three time for determine the standard deviation. The binding ability was shown in the following table 4 and Figure 3.

Table 4

The data of experiment 3 confirmed the chitosan citrate’s potential in phosphate-binding. It was particularly noted that the phosphate-binding ability of chitosan citrate is much better than chitosan lactate or citric acid alone. In other words, even if chitosan and citric acid both showed some degree of phosphate-binding ability, it was unforeseeable that chitosan citrate is able to exhibit phosphate-binding ability that is significantly better than that of citric acid and other chitosan salts (ex. chitosan lactate) .

Claims

A phosphate-binding composition， comprising

13 to 40 μg/μl of chitosan citrate； and

a pharmaceutically acceptable carrier；

wherein said μg/μl is based on the total volume of said composition.
The composition of claim 1， wherein the composition comprises 13 to 26 μg/μl of chitosan citrate.
The composition of claim 1， wherein said chitosan citrate comprises a chitosan moiety； wherein said chitosan moiety is of 50 to 95 ％ of deacetylation.
The composition of claim 3， wherein said chitosan citrate comprises a chitosan moiety； wherein said chitosan moiety is of 55 to 90 ％ of deacetylation.
The composition of claim 1， wherein said pharmaceutically acceptable carrier is water， alcohol， glycerol， chitosan， alginate， chondroitin， Vitamin E， mineral oil， dimethyl sulfoxide (DMSO) ， or a combination thereof.
The composition of claim 1， wherein said chitosan citrate comprises a chitosan moiety； wherein said chitosan moiety has a weight-average molecular weight of 15000 to 40000 dalton.
A method for binding phosphate in an environment， comprising：

contacting said environment with a chitosan citrate； wherein said chitosan citrate comprises a chitosan moiety； wherein said chitosan moiety is of 50 to 95 ％ of deacetylation.
The method of claim 7， wherein said chitosan moiety is of 55 to 90 ％ of deacetylation.
The method of claim 7， wherein said chitosan moiety has a weight-average molecular weight of 15000 to 40000 dalton.
The method of claim 7， wherein said chitosan citrate is formulated as a composition； wherein said composition comprises 13 to 40 μg/μl of said chitosan citrate； and a pharmaceutically acceptable carrier； wherein said μg/μl is based on the total volume of said composition.
The method of claim 10， wherein said pharmaceutically acceptable carrier is water， alcohol， glycerol， chitosan， alginate， chondroitin， Vitamin E， mineral oil， dimethyl sulfoxide (DMSO) ， or a combination thereof.
The method of claim 7， wherein said contacting is conducted by introducing said chitosan citrate into said environment.
A use of chitosan citrate for preparing a pharmaceutical composition for treating hyperphosphatemia in a subject； wherein said pharmaceutical composition is of the composition of claim 1.
A method for treating hyperphosphatemia in a subject， comprising administering said subject a composition of claim 1 before， during， or after diet.