WO2022007790A1

WO2022007790A1 - Enzyme composition and use thereof

Info

Publication number: WO2022007790A1
Application number: PCT/CN2021/104728
Authority: WO
Inventors: Chi-Ting Horng; Po-Chuen Shieh
Original assignee: Horng Chi Ting
Priority date: 2020-07-07
Filing date: 2021-07-06
Publication date: 2022-01-13
Also published as: TW202202168A; CN113893335A

Abstract

Disclosed herein is an enzyme composition that includes ficin, papain, and bromelain. Also disclosed herein is use of the enzyme composition for alleviating a symptomatic vitreous opacity.

Description

ENZYME COMPOSITION AND USE THEREOF

FIELD

The present disclosure relates to an enzyme composition including ficin, papain, and bromelain. The present disclosure also relates to a method for alleviating a symptomatic vitreous opacity using the enzyme composition.

BACKGROUND

Vitreous opacities (VO) , commonly known as eye floaters, are visible deposits that form in the vitreous humor of an eye and result from degenerative changes in the gel-like vitreous of the vitreous humor. Symptomatic vitreous opacities (SVOs) are floaters that are severe enough to cause symptoms for a minimum time period of 3 months and cause significant visual impairments in a patient. SVOs may also be caused by many eye diseases/disorders, including posterior vitreous detachment (PVD) , retinal tears or retinal detachment (RD) , vitreous hemorrhage, Eales’ disease, myopia, intraocular inflammation (e.g., uveitis and vitritis) , asteroid hyalosis (AH) , non-Hodgkin’s lymphoma, amyloidosis, and trauma, or by cataract surgery.

Several treatment strategies, such as enzymatic vitreolysis with intravitreal ocriplasmin, cataract surgery combined with deep anterior vitrectomy, pars plana vitrectomy (PPV) , and neodymium-doped yttrium aluminum garnet (Nd: YAG) laser vitreolysis, have been taken to combat SVOs. However, these treatment strategies may cause undesired side effects such as cataract, glaucoma, macular degeneration, intraocular inflammation, and even blindness. Therefore, there is still a need to develop a new strategy that can be utilized in the alleviation of SVOs.

SUMMARY

Accordingly, in a first aspect, the present disclosure provides an enzyme composition for alleviating a symptomatic vitreous opacity, which can alleviate at least one of the drawbacks of the prior art, and which includes ficin, papain, and bromelain.

In a second aspect, the present disclosure provides a method for alleviating a symptomatic vitreous opacity, which can alleviate at least one of the drawbacks of the prior art, and which includes administering to a subject in need thereof the aforesaid enzyme composition.

In a third aspect, the present disclosure provides use of the aforesaid enzyme composition in the manufacture of a medicament or a food product for alleviating a symptomatic vitreous opacity in a subject. Such use can alleviate at least one of the drawbacks of the prior art.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the present disclosure will become apparent in the following detailed description of the embodiments with reference to the accompanying drawings, of which:

FIG. 1 shows the retinal images of a test subject before and after a 3-month treatment period.

DETAILED DESCRIPTION

For the purpose of this specification, it will be clearly understood that the word “comprising” means “including but not limited to” , and that the word “comprises” has a corresponding meaning.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in China or any other country.

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the present disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present disclosure. Indeed, the present disclosure is in no way limited to the methods and materials described.

The present disclosure provides an enzyme composition for alleviating a symptomatic vitreous opacity, which includes ficin, papain, and bromelain.

As used herein, the term “alleviating” or “alleviation” refers to at least partially reducing, ameliorating, relieving, controlling, treating or eliminating one or more clinical signs of a disease or disorder; and lowering, delaying, stopping or reversing the progression of severity regarding the condition or symptom being treated and preventing or decreasing the likelihood or probability thereof.

In certain embodiments, ficin, papain, and bromelain in the enzyme composition are present in a weight ratio ranging from 1: 1: 1.5 to 1: 2: 5. In an exemplary embodiment, the weight ratio of ficin, papain, and bromelain is 1: 1: 2.

Ficin, papain, and bromelain suitable for use in this disclo sure are not particularly limited, and may be obtained as commercial products, or may be prepared using techniques well-known to those skilled in the art. For instance, ficin, papain, and bromelain may be independently a natural extract isolated from a plant material or a recombinant protein made by genetic engineering technology.

According to the present disclosure, ficin may be obtained by subjecting the latex, fruit, or leaf of the plant of Ficus genus to an extraction treatment using water.

According to the present disclosure, papain (also known as papaya proteinase I) may be obtained by subjecting the latex from the fruit of Carica papaya or Vasconcellea pubescens to an extraction treatment using water.

According to the present disclosure, bromelain may be obtained by subjecting the fruit and/or stem of the plant of Bromeliaceae family to an extraction treatment using water. Accordingly, bromelain may be selected from the group consisting of fruit bromelain, stem bromelain, or a combination thereof.

According to the present disclosure, the procedures and conditions of the water extraction treatment are within the expertise and routine skills of those skilled in the art.

In certain embodiments, the water extraction treatment may be performed by a process including the steps of:

a) subjecting a fruit of a plant to an extraction treatment using water at a temperature ranging from 40℃ to 50℃, followed by partitioning with dichloromethane; and

b) collecting the organic layer obtained in step (a) , followed by drying and recrystallization.

In certain embodiments, the enzyme composition of the present disclosure may be formulated as a food product using a standard technique well known to one of ordinary skill in the art. For instance, the aforesaid enzyme composition may be directly added to the edible material, or may be utilized for preparing an intermediate composition (e.g., a food additive or a premix) suitable to be subsequently added to the edible material.

According to the present disclosure, the food product may be in the form of fermented foods, processed foods, health foods, or dietary supplements.

In certain embodiments, the enzyme composition of the present disclosure may be prepared in the form of a pharmaceutical composition. The pharmaceutical composition may be formulated into a suitable dosage form for oral, parenteral or topical administration using technology well known to those skilled in the art.

For parenteral administration, the pharmaceutical composition according to the present disclosure may be formulated into an injection, e.g., a sterile aqueous solution or a dispersion.

The pharmaceutical composition according to the present disclosure may be administered via one of the following parenteral routes: intraperitoneal injection, intrapleural injection, intramuscular injection, intravenous injection, intraarticular injection, intrasynovial injection, intrathecal injection, intracranial injection, intraepidermal injection, subcutaneous injection, intradermal injection, intralesional injection, intravitreal injection, and sublingual administration. In an exemplary embodiment, the pharmaceutical composition is formulated into a dosage form for intravitreal injection.

According to the present disclosure, the suitable dosage form for oral administration includes, but is not limited to, sterile powders, tablets, troches, lozenges, pellets, capsules, dispersible powders or granules, solutions, suspensions, emulsions, syrup, elixir, slurry, and the like.

According to the present disclosure, the suitable dosage form for topical administration includes, but is not limited to, emulsions, gels, ointments, creams, patches, liniments, powders, aerosols, sprays, lotions, serums, pastes, foams, drops, suspensions, salves, and bandages.

According to the present disclosure, the pharmaceutical composition may further include a pharmaceutically acceptable carrier widely employed in the art of drug-manufacturing. For instance, the pharmaceutically acceptable carrier may include one or more of the following agents: solvents, buffers, emulsifiers, suspending agents, decomposers, disintegrating agents, dispersing agents, binding agents, excipients, stabilizing agents, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delaying agents, liposomes, and the like. The choice and amount of the aforesaid agents are within the expertise and routine skills of those skilled in the art.

The present disclosure provides a method for alleviating a symptomatic vitreous opacity, which includes administering to a subject in need thereof the aforesaid enzyme composition.

The present disclosure also provides use of the aforesaid enzyme composition in the manufacture of a medicament or a food product for alleviating a symptomatic vitreous opacity in a subject. In certain embodiments, the medicament is in a dosage form for oral administration.

The dose and frequency of administration of the pharmaceutical composition may vary depending on the following factors: the severity of the illness or disorder to be treated, routes of administration, and age, physical condition and response of the subject to be treated. In general, the pharmaceutical composition may be administered in a single dose or in several doses.

The disclosure will be further described by way of the following examples. However, it should be understood that the following examples are solely intended for the purpose of illustration and should not be construed as limiting the disclosure in practice.

EXAMPLES

General Procedures:

1. Non-mydriatic fundus photography (NMFP)

In the following examples, the respective test subject was subjected to ocular fundus image analysis and the ocular fundus photograph was obtained using a non-mydriatic retinal camera (KOWA, nonmyd 8) , so as to observe whether there was a large blood clot or there were obvious symptomatic vitreous opacities (SVOs) in the vitreous humor of the respective test subject.

2. B-scan ultrasonography

In the following examples, the respective test subject was subjected to B-scan ultrasonography using

imaging system (Alcon Laboratories Co., Ltd. ) , so as to determine the number of SVO and observe whether there were any symptoms of retinopathy.

3. Optical coherence tomography (OCT)

In the following examples, the respective test subject was subjected to OCT using CIRRUS ^TM HD-OCT 500 (ZEISS) , so as to determine the number of SVO and observe whether there were any symptoms of retinopathy.

Test subjects:

280 test subjects participating in the following tests were enrolled from Kaohsiung Armed Forces General Hospital, Taiwan, China. The tests were approved by the Institutional Review Board of Kaohsiung Armed Forces General Hospital and were conducted according to the principles of the Declaration of Helsinki. In addition, written informed consent was obtained from each of the test subjects.

These test subjects (at the age between 20 and 60 years and having an average age of 48 years ± 3 years) were diagnosed with a SVO (s) between April and December in 2017, and were selected and enrolled according to the inclusion and exclusion criteria as outlined in Table 1.

Table 1

Example 1. Preparation of enzyme composition of present disclosure

190 mg of fruit bromelain (Sgonek Biologic Technology Co., Ltd., Xi’ an City, China) , 95 mg of papain (Hongda Phytochemistry Co., Ltd., Shaanxi City, China) , and 95 mg of ficin (Hongkong Gotopharm Co., Ltd., Taiwan, China) were evenly mixed, followed by encapsulating the resultant mixture with a capsule shell consisting of hydroxypropyl methylcellulose (HPMC) and pectin (Yung Shin Pharmaceutical Industrial Co., Ltd., Taiwan, China) , so as to obtain a capsule containing an enzyme composition of the present disclosure.

Example 2. Evaluation for the effect of enzyme composition according to this disclosure on alleviating SVOs

Methods:

Prior to the start of the test (i.e., on Month 0) , 120 test subjects were subjected to NMFP examination to investigate whether there was a large and obvious SVO in the vitreous humor of the respective test subject, and the number of SVO was determined by OCT examination.

Based on the aforementioned OCT examination results, the 120 test subjects were divided into two experimental groups, including experimental group 1 (in which each of the test subjects had a single SVO in the vitreous humor) (n=80) , and experimental group 2 (in which each of the test subjects had multiple SVOs in the vitreous humor) (n=40) . Each test subject was orally administered with two capsules obtained in Example 1 per day for a 3-month treatment period. At the ends of Months 1, 2, and 3, each test subject was subjected to best-corrected visual acuity (BCVA) measurement, and to NMFP examination, B-scan ultrasonography examination, and OCT examination according to the General Procedures, and the number of SVO was counted, so as to evaluate the therapeutic effect on SVO and the presence or absence of side effects.

In the experimental group 1, if the number of SVO of the test subject was reduced to 0, it is considered that the single SVO has been alleviated. In the experimental group 2, if the number of SVO of the test subject was reduced to 1 or 0, it is considered that the multiple SVOs have been alleviated.

The number of test subjects with a non-alleviatedSVO (s) at Month 0 (i.e., prior to the oral administration of the capsule of the present disclosure) and that at the ends of Months 1, 2, and 3 after starting oral administration of the capsule of the present disclosure were counted.

The alleviation rate (%) was calculated using the following Equation (I) :

A= [ (B-C) /B] ×100 (I)

where A=alleviation rate (%)

B = the number of test subjects with a non-alleviated SVO (s) in the respective experimental group at Month 0

C = the number of test subjects with a non-alleviated SVO (s) in the respective experimental group at Month 3

The data thus obtained were statistically analyzed using SAS 9.0 statistics software (SAS Institute Inc., Cary, NC, USA) and paired Student’s t-test, so as to assess the difference between the groups. Statistical significance is indicated by p＜0.05.

Results:

A. Evaluation of therapeutic effectiveness

As shown in Table 2 below, the number of test subjectswith a non-alleviated SVO in the experimental group 1 at the end of Month 3 was lower than that at Month 0, and a similar satisfactory result was observed with respect to the experimental group 2. In addition, after the 3-month treatment period, the alleviation rates of the experimental groups 1 and 2 reached 70%and 72.5%, respectively.

Table 2

*: When the experimental data obtained at the end of Month 3 was compared to that obtained at Month 0, p<0.05.

FIG. 1 shows the retinal images of a test subject in the experimental group 2 be fore and after the 3-month treatment period. It can be seen from FIG. 1 that the SVOs in the vitreous humor of the test subject disappeared after the 3-month treatment period (see the images obtained from OCT and B-scan ultrasonography) , and large numbers of dark floaters or cobwebs in the vitreous humor of the test subject disappeared after the 3-month treatment period (see the image obtained from NMFP) .

Besides, the visual acuity of each test subject in the experimental group 1 and the experimental group 2 was improved (data not shown) .

These results indicate that the enzyme composition of the present disclosure can effectively alleviate SVOs.

B. Evaluation of side effects

During the 3-month treatment period, all the test subjects were found normal in angle of anterior chamber, lens status, cornea, and zonular fibers, and no retinal damage, ocular inflammation, or other side effects were observed on all the test subjects. In addition, all the test subjects had normal intraocular pressure, blood sugar, and blood pressure.

These results indicate that the enzyme composition of the present disclosure does not cause undesired side effects in the human body (especially the normal collagen fibrils in the ocular tissues) .

Example 3. Evaluation for the effect of enzyme composition according to this disclosure at different dosages on alleviating SVOs

Methods:

Prior to the start of the test (i.e., at Month 0) , 160 test subjects were subjected to NMFP examination to investigate whether there was a large and obvious SVO in the vitreous humor of the respective test subject, and the number of SVO was determined by OCT examination. The 160 test subjects were randomly divided into 4 groups, including one control group and three experimental groups (i.e., experimental groups 1 to 3) (n=40 per each group) .

The test subjects of the experimental group 1, the experimental group 2, and the experimental group 3 were respectively orally administered with one capsule, two capsules, and three capsules obtained in Example 1 per day for a 3-month treatment period. The test subjects of the control group received no treatment.

During the 3-month treatment period, all the test subjects were subjected to ophthalmic examination according to the procedures described in Example 2, and the alleviation rate of each group was determined using the abovementioned Equation (I) .

The data thus obtained were statistically analyzed using SAS 9.0 statistics software (SAS Institute Inc., Cary, NC, USA) and Scheffé's test, and Williams’ test was used to assess the difference between the groups. Statistical significance is indicated by p＜0.05.

Results:

As shown in Table 3 below, the number of test subjects with a non-alleviated SVO (s) in the experimental group 1 at the end of Month 3 was lower than that at Month 0, and similar satisfactory results were observed with respect to the experimental groups 2 and 3. In addition, after the 3-month treatment period, the alleviation rates of the experimental groups 1, 2, and 3 reached 65%, 70%, and 75%, respectively. A significant dose-dependent effect of the enzyme composition of the present disclosure on the alleviation rate was observed.

Table 3

^#: When compared to the control group, p<0.05.

Summarizing the above test results, it is clear that the enzyme composition of the present disclosure is effective in alleviating SVOs.

While the disclosure has been described in connection with what are considered the exemplary embodiments, it is understood that this disclosure is not limited to the disclosed embodiments but is intended to cover various arrangements included within the spirit and scope of the broadest interpretation so as to encompass all such modifications and equivalent arrangements.

Claims

An enzyme composition for alleviating a symptomatic vitreous opacity, comprising ficin, papain, and bromelain.
The enzyme composition as claimed in Claim 1, wherein ficin, papain, and bromelain are present in a weight ratio ranging from 1: 1: 1.5 to 1: 2: 5.
The enzyme composition as claimed in Claim 2, wherein ficin, papain, and bromelain are present in a weight ratio of 1: 1: 2.
The enzyme composition as claimed in Claim 1, which is formulated as a food product.
The enzyme composition as claimed in Claim 1, which is formulated as a pharmaceutical composition.
The enzyme composition as claimed in Claim 5, wherein the pharmaceutical composition is in a dosage form for oral administration.
The enzyme composition as claimed in Claim 1, wherein bromelain is selected from the group consisting of fruit bromelain, stem bromelain, or a combination thereof.
A method for alleviating a symptomatic vitreous opacity, comprising administering to a subject in need thereof an enzyme composition as claimed in Claim 1.
The method as claimed in Claim 8, wherein ficin, papain, and bromelain are present in a weight ratio ranging from 1: 1: 1.5 to 1: 2: 5.
The method as claimed in Claim 9, wherein ficin, papain, and bromelain are present in a weight ratio of 1: 1: 2.
The method as claimed in Claim 8, wherein the enzyme composition is a pharmaceutical composition in a dosage form for oral administration.
The method as claimed in Claim 8, wherein bromelain is selected from the group consisting of fruit bromelain, stem bromelain, or a combination thereof.
Use of an enzyme composition in the manufacture of a medicament or a food product for alleviating a symptomatic vitreous opacity in a subject, wherein the enzyme composition is as claimed in Claim 1.
The use as claimed in Claim 13, wherein ficin, papain, and bromelain are present in a weight ratio ranging from 1: 1: 1.5 to 1: 2: 5.
The use as claimed in Claim 14, wherein ficin, papain, and bromelain are present in a weight ratio of 1: 1: 2.
The use as claimed in Claim 13, wherein the medicament is in a dosage form for oral administration.
The use as claimed in Claim 13, wherein bromelain is selected from the group consisting of fruit bromelain, stem bromelain, or a combination thereof.