Patent Description:
Galactose is an epimer of glucose and one of the main components of lactose. Galactose is mainly metabolized in the body by the liver and can be converted into glucose or, in turn, synthesized to glycogen stored in the liver. As liver disease has a significant effect on the metabolism of galactose, maximum galactose elimination capacity (GEC) and galactose single point (GSP) blood concentration can be used as indicators of clinical quantitative liver function.

GEC has been performed as a quantitative human liver function test for years. However, GEC tests require a number of blood samples to establish a standard curve, which has difficulties in clinical application. There are many studies using GSP to assess human liver function. Liver function includes hepatic blood flow status, hepatic enzyme status, or metabolic ability of galactose.

<NPL>) have revealed that a variety of liver diseases such as chronic hepatitis, liver cirrhosis, and liver cancer can be accurately identified by the GSP method. Also, the GSP method can be used to assess residual liver function. The GSP method is recommended in the US Food and Drug Administration (FDA) benchmark, and has been proven to be an extremely simple and viable method to assess residual liver function, which has been verified by common liver diseases in our people. Furthermore, GSP has become one of the methods recommended for use in testing liver function in the Guidance for Industry published by the FDA.

In the past, a subject's blood galactose concentration was measured <NUM> minutes after rapid intravenous injection of <NUM>/kg of galactose for three minutes when in fasting, to obtain a GSP value (in µg/ml). However, the intravenous injection is invasive, which brings psychological stress to the subject and causes tissue damage, pain, or potential complications. In particular, multiple attempts are often needed to make a successful injection in children of primary school and preschool who are unable to cooperate with and have thinner blood vessels.

<CIT> discloses the use of <NUM>,<NUM>-anhydro-L-galactose for preventing dental caries. <CIT> discloses compositions comprising soluble fiber, which are effective in triggering the secretion of Glucagon-like-peptide-<NUM> (GLP-<NUM>). <CIT> discloses stable galactose injection solutions, which contain <NUM> to <NUM>% by weight of galactose, <NUM> to <NUM> of a buffer solution, and <NUM> to <NUM>% of an antioxidant. PCT Publication number discloses a method of blood sample preparation for liver function test and a sample paper, wherein the method comprises injecting a galactose composition into a subject. Thus, how to design a non-invasive galactose formula which can be administered orally to the subjects and can be detected accurately by the GSP method has become an important topic to be solved by the present invention.

The invention is defined in the claims. The invention provides a galactose oral composition comprising galactose, a buffer, and an antioxidant in an amount of from about <NUM> to about <NUM> percent by volume, wherein said galactose oral composition has a pH value ranging from <NUM> to <NUM>, and wherein said galactose includes at least one of D-(+)- galactose, L-(-)- galactose, stable isotope galactose, galactose ring, or galactose derivatives, wherein said antioxidant is selected from the group consisting of Vitamin A, Vitamin C, Vitamin E, sodium bisulfite, polyphenols, Ethylenediaminetetraacetic acid (EDTA), Diethylenetriaminepentaacetic acid (DTPA), flavonoids, and NTA-Nitrilotriacetate acid (NTA).

To achieve the above purpose of the present invention, said buffer is selected from a group including at least one of ascorbic acid buffer, citrate buffer, phosphate buffer, acetate buffer, carbonate buffer, and triethanolamine buffer.

To achieve the above purpose of the present invention, said galactose oral composition is a food composition and/or a pharmaceutical composition.

To achieve the above purpose of the present invention, said galactose oral composition further includes at least one of emulsifier, colorant, flavoring agents, sweetener, preservatives, excipients, extenders, stabilizers, and dispersants.

To achieve the above purpose of the present invention, said sweetener is selected from a group including at least one of D-Sorbitol, D-Sorbitol Solution <NUM>%, D-Xylitol, Glycyrrhizin, Trisodium Glycyrrhizinate, D-Mannitol, Saccharin, Saccharin Sodium, Sodium Cyclamate, Calcium Cyclamate, Aspartame, Steviol Glycoside, Licorice Extracts, Acesulfame Potassium, Ammoniated Glycyrrhizin, Monoammonium Glycyrrhizinate, Maltitol, Maltitol Syrup (Hydrogenated Glucose Syrup), Isomalt (Hydrogenated Palatinose), Lactitol, Monoglucuronyl Glycyrrhetic Acid, Thaumatin, Erythritol, Sucralose, and Neotame.

To achieve the above purpose of the present invention, said spice is selected from a group including at least one of cherry, lemon, lime, mandarin, orange, tangerine, mint, strawberry, banana, caramel, licorice, passion-fruit, peach, raspberry, tutti-frutti, grapefruit, vanilla, cream, chocolate, and grapes.

Another purpose of the present invention is to provide a kind of galactose oral composition mentioned above for the preparation of an agent for the detection of hepatic blood flow status, hepatic enzyme status, and metabolic ability of galactose.

To achieve the above purpose of the present invention, each oral dose of the galactose oral composition ranges from <NUM>. 0lg/kg to <NUM>/kg.

Another aspect of the present disclosure is to provide a galactose composition, comprising galactose, a buffer, and an antioxidant; wherein said galactose composition ranges the pH values from <NUM> to <NUM>; wherein said galactose composition keeps the original color under high temperature condition; wherein said galactose includes at least one of D-(+)- galactose, L-(-)- galactose, stable isotope galactose, galactose ring, and galactose derivatives.

To achieve the above aspect of the present disclosure, said high temperature is a temperature at <NUM> to <NUM>.

To achieve the above aspect of the present disclosure, said galactose composition is a nutritional sweetener for diabetes.

To achieve the above aspect of the present disclosure, said galactose composition can be put into a food, nourishment, and formula milk.

Another aspect of the present disclosure is to provide a kind of galactose composition mentioned above for the preparation of an agent for the detection of hepatic blood flow status, hepatic enzyme status, and metabolic ability of galactose.

To achieve the above aspect of the present disclosure, each dose of the galactose composition ranges from <NUM>. 0lg/kg to <NUM>/kg.

The present invention is exemplarily illustrated but not limited by the following embodiments. Any embodiments falling outside the scope of the claims are provided for comparative purposes.

In the present disclosure, various kinds and concentrations of buffer, and antioxidants, are added for adjustment into a galactose oral solution in different pH values, and then the stability tests are carried out to provide a good galactose oral formula. The invention is defined in the claims. The present invention refers to a galactose of the present invention including at least one of D-(+)- galactose, L-(-)-galactose, and stable isotope galactose.

The present invention refers to an antioxidant of the present invention including at least one of Vitamin C and/or sodium bisulfite, Vitamin A, Vitamin E, Ethylenediaminetetraacetic acid (EDTA), Diethylenetriaminepentaacetic acid (DTPA), flavonoids, polyphenols, and NTA-Nitrilotriacetate acid (NTA).

The present invention refers to a buffer of the present invention including at least one of citrate buffer, phosphate buffer, acetate buffer, carbonate buffer, ascorbic acid buffer, and triethanolamine buffer.

<NUM> liters of water at <NUM> are injected into a dispensing barrel and then <NUM> sodium citrate is added and agitated until completely dissolved. After adjusting the pH value to <NUM>±<NUM>, <NUM> sodium bisulfite is added and agitated until completely dissolved, and <NUM> D-Galactose is added and agitated until completely dissolved, and then <NUM> sodium citrate is added and agitated until completely dissolved. After adjusting the pH value to <NUM>±<NUM>, the water at <NUM> is filled until the volume of the whole mixture reaches <NUM> liters, then stirred for <NUM> minutes and filtered with a <NUM> pore size filter. After mixing evenly, the mixture is poured into a number of glass bottles each with a capacity of <NUM>, then sampled and sealed immediately. Finally the bottles are put into a high-pressure steam pot at <NUM> (<NUM>/cm<NUM>). After sterilization for <NUM> minutes, the bottles are taken out and sampled. Table <NUM> is the results of long-term stability tests for the galactose oral solution. It is shown that the formula has good stability after <NUM>-<NUM> months of placement.

The content of the galactose is <NUM>%-<NUM>%. The method is to configure the galactose at high temperature as <NUM>%-<NUM>% solutions, and then dilute it to the better content of <NUM>%-<NUM>% of the total weight. Do not add buffer or add buffer to the total weight of <NUM>%-<NUM>%. Do not add antioxidant or add antioxidant to the total weight of <NUM>%- <NUM>%. Appropriate oral solution formula can be prepared by selecting the buffer and antioxidant, and adding the content of the following ingredients: antioxidant of <NUM>-<NUM> sodium bisulfite, and/or Vitamin C, Vitamin A, Vitamin E, flavonoids, Ethylenediaminetetraacetic acid (EDTA), polyphenols,
Diethylenetriaminepentaacetic acid (DTPA), and/or NTA-Nitrilotriacetate acid (NTA); and/or buffer of one of seven solutions, namely <NUM>-<NUM> citrate buffer, phosphate buffer, acetate buffer, carbonate buffer, ascorbic acid buffer, and triethanolamine buffer with adjustment of the pH values ranged <NUM>-<NUM>. A stable oral solution formula can be obtained by adding <NUM>% citrate buffer and <NUM>% sodium bisulfite with the pH value of <NUM>.

According to the oral solution prepared by the above formula, the concentration of galactose is <NUM>/ml and the volume thereof is <NUM>. The galactose oral solution product is then tested and the results are shown in <FIG>. Based on the results in <FIG>, the color, main ingredients, pH value, volume for the galactose oral solution of the present invention are in line with the standard value. According to the regulations of USP-XXII edition, for Dextrose oral liquid main decomposition products of <NUM>-hydroxymethyl-furfural and other relevant regulations on the content of related substances, the absorbance value of the water as a blank control solution should not exceed <NUM> under the condition of Dextrose concentration <NUM>/<NUM>/ml at <NUM> wavelength. The content of <NUM>-hydroxymethyl-furfural in the galactose oral solution of the present invention after placing for a long time is only <NUM>. Thus, according to the results in <FIG> and Table <NUM>, the galactose oral solution of the present invention can be placed for a long time and maintain stability.

Preparing the galactose oral solution, the concentration of galactose is <NUM>% and the volume is <NUM>. In the stability study, according to the results in Table <NUM>, all the formulae of adding Vitamin C (<NUM>) as an antioxidant have changed the color of the oral solution after <NUM> hours under the condition of <NUM>. However, when sodium bisulfite (<NUM>, <NUM>) as the antioxidant and the pH value thereof is adjusted to <NUM>, there is no change in color after placing the formula under the condition of <NUM> for <NUM> hours.

Preparing the galactose oral solution, the concentration of galactose is <NUM>% and the volume is <NUM>. In the stability study, five different buffers with each having <NUM> concentration and antioxidants are added respectively for formation, wherein the buffer is selected from the group including citrate buffer, phosphate buffer, acetate buffer, carbonate buffer and triethanolamine buffer, and the antioxidants are still sodium bisulfite and Vitamin C of <NUM> in concentration. According to the results in Table <NUM>, all formulae of adding Vitamin C as the antioxidant have changed color after <NUM> hours, thus the stability is poor. However, all formulae of adding sodium bisulfite as the antioxidant, no matter in any buffer, there is no change in color under the condition of the pH value at <NUM> or <NUM>. By observing the galactose of the formulae <NUM>-<NUM> under the condition of the pH value at <NUM>, with citrate buffer and sodium bisulfite antioxidant, the stability is better in terms of the pH value and color change.

Preparing the galactose oral solution, the concentration of galactose is <NUM>% and the volume is <NUM>. In the stability study, five different buffers with each having <NUM> concentration and antioxidants are added respectively for formation, wherein the buffer is selected from a group including citrate buffer, phosphate buffer, carbonate buffer and triethanolamine buffer, and the antioxidants are still sodium bisulfite or Vitamin C of <NUM> in concentration. According to the results in Table <NUM>, all formulae of adding <NUM> Vitamin C as an antioxidant have changed color after <NUM> hours, so the stability is poor. And for the partial formulae in which <NUM> sodium bisulfite is added as an antioxidant, there are changes in color after <NUM> hours under the condition of <NUM>. Besides, when storing in <NUM> carbonate buffer under the condition of the pH value at <NUM> and in <NUM> triethanolamine buffer under the condition of the pH value at <NUM> for a week, there are changes in color. Therefore, it is known that the stability of the formula will be reduced when the concentration of the sodium bisulfite and the buffer increase together. When the remaining four buffers with high concentration, namely citrate buffer, phosphate buffer, carbonate buffer and triethanolamine buffer, are added into the antioxidant with high concentration of sodium bisulfite under the condition of the pH value at <NUM>, they are more stable, but better in the environment of citrate buffer and acetate buffer. After placing the formulae <NUM>-<NUM> under the condition of <NUM> for a week, there is no precipitation.

The stability tests of the galactose oral solution are carried out with the pH values ranging from <NUM> to <NUM>, wherein the galactose concentration is <NUM>%, volume is <NUM>, and does not contain any buffers
and antioxidants. Based on the regulations of USP-XXII edition, for Dextrose oral liquid main decomposition products of <NUM>-hydroxymethyl-furfural and other relevant regulations on the content of related substances, the absorbance value of the water as a blank control solution should not exceed <NUM> under the condition of Dextrose concentration <NUM>/<NUM>/ml at <NUM> wavelength. Accordingly, the corresponding absorbance value of the <NUM>% concentration of the galactose oral solution prepared by the invention should not exceed <NUM>. As a result, it is found that the variation of the formula is less before and after sterilization when the pH value is <NUM>. Thus, it is known that under the low pH environment, stability is better.

After observation on the stability of the galactose oral solution which is prepared with different antioxidant concentrations, it can be found that when the antioxidant concentration increases, both the pH value and the absorbance value have smallest changes before and after sterilization, and the stability in the situation where the antioxidant is added is better than that where the antioxidant is not added. In <NUM> citrate buffer, both the pH value of the oral solution and the antioxidant concentration of sodium bisulfite are adjusted, the results indicate that under acidic conditions and <NUM>% antioxidant concentration of sodium bisulfite, both the pH value and absorbance value have the smallest changes before and after sterilization.

In <NUM> phosphate buffer, both the pH value of the oral solution and the antioxidant concentration of sodium bisulfite are adjusted. The results indicate that, under acidic conditions and <NUM>% antioxidant concentration of sodium bisulfite, both the pH value and absorbance value have the smallest changes before and after sterilization, and the stability is better. In <NUM> acetate buffer, both the pH value of the oral solution and the antioxidant concentration of sodium bisulfite are adjusted, the results indicate that under the condition of <NUM>% antioxidant concentration of sodium bisulfite, both the pH value and the absorbance value have the smallest changes before and after sterilization, and the stability is better. In <NUM> triethanolamine buffer, both the pH value of the oral solution and the antioxidant concentration of sodium bisulfite are adjusted, the results indicate that under the condition of <NUM>% antioxidant concentration of sodium bisulfite, both the pH value and the absorbance value have the smallest changes before and after sterilization, and the stability is better.

According to the smallest change in the pH value and the least change in the color of the solution, the optimal formula with citrate buffer can be selected as an ideal model system. Further study on raising the concentration of the galactose to <NUM>%. In a pure water environment, the yellow color of the solution
increases with the increase of the galactose concentration. In acidic citrate buffer, adding different concentrations of sodium bisulfite has little effect on the pH value before and after sterilization. However, when the concentration of sodium bisulfite is greater than <NUM>%, the color of the oral solution is not changed, and the minimum changes of the absorbance value can also be obtained. In alkaline citrate buffer, no matter how much the concentration of sodium bisulfite is added, the color of the oral solution changes significantly after storing for a week at <NUM>.

General sugars such as sucrose or galactose will discolor at elevated temperatures of about <NUM>, and the galactose at high temperatures will discolor whether it is a solid or a solution. When the galactose oral solution formula of the present invention is placed at <NUM> for storage or <NUM> for high-temperature sterilization, there is no change in color. Accordingly, the galactose oral solution formula of the present invention can be used as a sweetness supplement in high temperature, such as in baking. Since the galactose has sweetness and calorie but does not affect blood sugar, it can also be a nutritional sweetener for diabetes or those who need to control the blood sugar.

The following further illustrates the feasibility study of applying the galactose oral composition formula to detect the concentration of the galactose in blood.

You must sign the subject consent prior to screening period and we will record your past medical history (including medication history) to assist the research physicians in qualifying you to participate in the study before using the studied medication and grouping your study.

Each subject will undergo two additional liver function tests, i.e., GSP and OGSP, each of which will be studied over a period of time. The time interval between these two tests should be at least <NUM> hours. One needs to be fasting for <NUM> hours before the day of testing.

OGSP examination: Subjects drank <NUM> of galactose solution per kg of body weight (<NUM> of galactose per ml), i.e. <NUM>/kg of galactose per kg body weight. After drinking galactose oral solution for <NUM> to <NUM> minutes, at least <NUM> of water is given. After <NUM> minutes after finishing oral administration, blood is taken from the fingers to take <NUM> of whole blood for slowly dropping on the galactose test strip, then the examination is completed.

The galactose contains <NUM>% to <NUM>% of the total amount by weight, preferably, <NUM>% to <NUM>%, contains <NUM>% to <NUM>% with or without addition of buffer, and <NUM>% to <NUM>% with or without addition of the antioxidant. The antioxidant therein is selected from a group including at least one of Vitamin C and/or sodium bisulfite, Vitamin A, Vitamin E, polyphenols, Ethylenediaminetetraacetic acid (EDTA), Diethylenetriaminepentaacetic acid (DTPA), flavonoids, and/or NTA-Nitrilotriacetate acid (NTA). The buffer is selected from a group including at least one of citrate buffer, phosphate buffer, acetate buffer, carbonate buffer, ascorbic acid buffer, and triethanolamine buffer.

GSP: Subjects are injected with <NUM> of galactose injection per kg of body weight (<NUM> of galactose per ml), i.e. <NUM>/kg of galactose per kg body weight. The galactose injection is completed within <NUM> to <NUM> minutes. After <NUM> minutes after finishing injection, blood is taken from the fingers to take <NUM> of whole blood for slowly dropping on the galactose test strip, then the examination is completed.

<FIG> and <FIG> are relative distribution views of the GSP results of the galactose intravenous injection and the OGSP results of the galactose oral administration for <NUM> subjects (<NUM> in normal liver function and <NUM> in impaired liver function). According to <NPL> recommendations, subjects were divided into <NUM> groups with GSP values (GSP) of intravenously administered galactose, wherein GSP < 280µg/ml is defined to indicate that the subject is in normal liver function, <NUM>< GSP <480µg/ml is defined to indicate that the subject is in moderately-impaired liver function, and GSP > 480µg/ml is defined to indicate that the subject is in severely-impaired liver function. According to results in <FIG> and <FIG>, the OGSP value for the oral galactose is higher than that for intravenous galactose and the OGSP value for oral galactose increases with severity of impaired liver function, and the OGSP and the GSP were positively correlated. Subjects in group of normal liver function have a galactose oral administration OGSP of <NUM>±27µg/ml (mean ± standard error SE), with a minimum of 18µg/ml and a maximum of 887µg/ml. Subjects with mild or moderate liver function impairment have a galactose oral administration OGSP of <NUM>±40pg/ml (mean ± standard error SE), with a minimum of 294µg/ml and a maximum of 1282µg/ml. Subjects with severe liver function impairment have a galactose oral administration OGSP of <NUM>±48µg/ml (mean ± standard error SE), with a minimum of 293µg/ml and a maximum of 1499µg/ml. <FIG> shows the OGSP results of the galactose oral administration and the GSP results of the galactose intravenous injection for three groups of subjects. As shown from the results, the OGSP value of the galactose oral administration increases with the severity of impaired liver function. In particular, the OGSP value for the oral galactose is higher than that for the intravenous galactose. According to results in <FIG>, <FIG> and Table <NUM>, it can be determined that the main range (mean ± <NUM> times standard error SE) of the OGSP galactose oral administration for the subjects in the group of normal liver function is about between <NUM> to 372µg/ml, the main range (mean ± <NUM> times standard error SE) of the OGSP galactose oral administration for the subjects in the group of mildly or moderately impaired liver function is about between <NUM> to 670µg/ml, and the main range (mean ± <NUM> times standard error SE) of the OGSP galactose oral administration for the subjects in the group of severely impaired liver function is about between <NUM> to 873µg/ml. Even if the subject's results differ from person to person, the OGSP galactose oral administration for the subjects in the group of normal liver function does not exceed 670µg/ml and the OGSP for subjects with impaired liver function may be larger than 370µg/ml. Thus the subjects who have the OGSP greater than 370µg/ml should acquire further liver function detection.

Table <NUM> The GSP results of the galactose intravenous injection and OGSP results of the galactose oral administration (mean±standard error SE).

<NUM> subjects (except diabetics) were divided into <NUM> groups with GSP values (GSP) of intravenously administered galactose, wherein GSP < 280µg/ml is defined to indicate that the subject is in normal liver function, <NUM>< GSP <480µg/ml is defined to indicate that the subject is in moderately-impaired liver function, and GSP > 480µg/ml is defined to indicate that the subject is in severely-impaired liver function. <FIG> shows the correlation between blood biochemical values and the OGSP results of the galactose oral administration, the GSP results of the galactose intravenous injection for three groups of subjects. The results show that GSP values of the intravenous galactose and OGSP values of the oral galactose are significantly correlated with a number of blood biochemical indexes, wherein AST (aspartate aminotransferase), ALT (alanine aminotransferase) are indicators of liver impairment, and the correlation between OGSP values of the oral galactose and AST, ALT is greater than that between GSP values of the intravenous galactose and AST, ALT.

GSP analysis is conducted on <NUM> patients with cirrhosis by galactose oral administration. <FIG> shows correlation between OGSP results of the oral galactose and cirrhosis. Typically, Child Pugh index (Child Pugh score) is clinically used to estimate the severity of cirrhosis. <FIG> shows that OGSP value of the oral galactose is positively correlated with Child Pugh index (r=<NUM> , p<<NUM>). Compared with GSP results of conventionally- intravenous galactose (<FIG>), the OGSP value of the oral galactose is higher. However, both OGSP value of the oral galactose and GSP value of the intravenous galactose are positively correlated with cirrhosis. Accordingly, both OGSP value of the oral galactose and GSP value of the intravenous galactose have the same ability of estimating cirrhosis.

MELD (Model for End-Stage Liver Disease) can predict the short-term and medium-term mortality of end-stage liver disease effectively, and its evaluation index is obtained simply, objective and easy to calculate. It is widely used in the diagnosis and treatment of liver diseases. OGSP analysis is conducted on <NUM> patients with cirrhosis by the galactose oral administration. <FIG> shows correlation between OGSP results of the oral galactose and MELD. <FIG> shows that OGSP value of the oral galactose is positively correlated with MELD (r=<NUM> , p<<NUM>). Compared with GSP results of the conventionally-intravenous galactose (<FIG>), OGSP value of the oral galactose is higher. However, both OGSP value of oral galactose and GSP value of the intravenous galactose are positively correlated with MELD. Accordingly, both OGSP value of the oral galactose and GSP value of the intravenous galactose have the same ability of estimating MELD.

<FIG> shows the OGSP results (OGSP) at each time point after the galactose oral administration. The mouse is led to non-alcoholic hepatitis (NASH) by being fed with high-fat diets. All animals with NASH have OGSP value that is higher than the value of normal individuals within <NUM> to <NUM> minutes after oral administration, wherein OGSP value changes a lot within <NUM> to <NUM> minutes after the oral administration. The results show that GSP value of the oral galactose has the ability of estimating NASH and is tested optimally from <NUM> to <NUM> minutes after the oral administration of the galactose.

The above-mentioned detailed description aims to specifically illustrate the practicable embodiments of the present invention, but the embodiments are not for limiting the patent scope of the present invention, which is defined in the claims.

Claim 1:
A galactose oral composition, comprising:
galactose;
a buffer; and
an antioxidant in an amount of from about <NUM> to about <NUM> percent by volume; wherein said galactose oral composition has a pH value ranging from <NUM> to <NUM>; and wherein said galactose includes at least one of D-(+)- galactose, L-(-)- galactose, stable isotope galactose, galactose ring, or galactose derivatives, and wherein said antioxidant is selected from the group consisting of Vitamin A, Vitamin C, Vitamin E, sodium bisulfite, polyphenols, Ethylenediaminetetraacetic acid (EDTA), flavonoids, Diethylenetriaminepentaacetic acid (DTPA), and NTA-Nitrilotriacetate acid (NTA).