JP7567109B2 - Wound healing agent - Google Patents

Description

The present invention relates to a wound healing agent.

As described in Patent Document 1, it is known that a complex of hydroxyapatite and agarose gel as a carrier is used as a material for forming artificial bones and artificial dental roots.
As described in Patent Documents 2 and 3, crystals of salts such as calcium polyphosphate are also known to be used as hemostatic agents.
The materials for forming artificial bones and artificial dental roots and the hemostatic agents described in these documents may, in clinical practice, impair healing in some cases or cause a foreign body reaction between the tissue and the living body.
In addition, Fiblast Spray is used as a drug for such healing purposes, but Fiblast Spray is not approved for use on cancer patients or those who have had skin cancer.

International Publication No. 2007/061001 Special Publication No. 2016-535041 Special Publication No. 2008-531692

When hydroxyapatite and agarose composite materials were used in clinical practice, they sometimes led to poor healing or foreign body reactions between the wound and the body. In addition, Fiblast Spray is expensive to manufacture, and when used for wound healing, it needs to be administered frequently and causes a strong inflammatory reaction, so the treatment is laborious. In addition, because it is a protein preparation, it is prone to side effects such as irritation, pain, and redness after administration when there are many inflammatory cells at the wound site.
To provide a wound healing agent which can more reliably stop bleeding and more reliably heal wounds in clinical practice, and which can be used by cancer patients and those who have had skin cancer, and which can be administered at lower cost and with reduced frequency, thereby easing the labor of treatment.

The present invention is an invention for solving the above problems, and for that purpose, the following configurations are adopted.
A wound healing agent containing 0.50 to 3.00 parts by weight of mono-needle and/or spherical crystals of Ca(HPO4)(H2O)2 per 1.0 part by weight of agarose gel.

The wound healing agent contains monoacicular and/or spherical crystals of Ca(HPO4)(H2O)2 and agarose gel, and the hemostatic composition is applied or coated on a wound in a living body, thereby enabling wound healing to occur quickly and reliably.

Treatment rate versus number of days of treatment Figure showing the total treatment rates for groups A to D A graph showing the healing promotion rate for each of groups B to D. Scanning electron micrograph of calcium phosphate agarose gel 2 (containing monocrystalline Ca(HPO4)(H2O)2)

The wound-healing agent of the present invention will be described below.
The wound healing agent of the present invention is a composition for applying to a bleeding part of a wound to effect wound healing. It contains 0.50 to 3.00 parts by weight of mono-needle crystal Ca(HPO4)(H2O)2 and 1.0 part by weight of agarose gel.

<Single-needle and/or spherical crystals of Ca(HPO4)(H2O)2>
In the present invention, Ca(HPO4)(H2O)2 is used as calcium phosphate.
The means for obtaining such Ca(HPO4)(H2O)2 is not particularly limited, and among Ca(HPO4)(H2O)2, mono-needle crystals and/or spherical crystals are used. Ca(HPO4)(H2O)2 having both mono-needle crystal parts and spherical crystal parts may be used.
The monoacicular Ca(HPO4)(H2O)2 of the present invention can be obtained by, for example, preparing a 100-1000 mM calcium chloride solution having a pH of 7.4 at 20° C. and a 30-500 mM sodium hydrogen phosphate (such as disodium hydrogen phosphate dodecahydrate) solution having a pH of 7.4 at 20° C. Then, one cycle of immersion in the 100-1000 mM calcium chloride solution having a pH of 7.4 at 20° C. for 2 hours, washing with pure water, and then immersion in a 30-500 mM sodium hydrogen phosphate solution having a pH of 7.4 at 20° C. for 2 hours and washing with pure water is performed, and this cycle is repeated for a total of 5-20 times.
The calcium phosphate contained in the wound healing agent of the present invention preferably contains 100% by mass of mono-needle and spherical crystals of Ca(HPO4)(H2O)2, but other calcium phosphates may also be contained within a range that does not impair the effects of the present invention. As a result, the content ratio of mono-needle crystals of Ca(HPO4)(H2O)2 in the calcium phosphate may be 95% by mass or more, or may be 90% by mass or more. The higher this ratio, the better the wound healing effect.
Other examples of calcium phosphate include amorphous, monoclinic, hexagonal and orthorhombic calcium phosphate, amorphous calcium polyphosphate, calcium polyphosphate other than monoacicular, hydroxyapatite (Ca10(PO4)6(OH)2), and the like.

The average particle size of each particle of the monoacicular and spherical crystals of Ca(HPO4)(H2O)2 of the present invention is preferably 1.00 μm or more, more preferably 2.00 μm or more, and even more preferably 3.00 μm or more. Also, it is preferably 200 μm or less, more preferably 100 μm or less, even more preferably 30.0 μm or less, and most preferably 10.0 μm or less.
The content ratio of the mono-acicular and spherical crystals of Ca(HPO4)(H2O)2 in the total weight of the wound healing agent in a dry state is 0.50 to 3.00 parts by weight when the weight of the agarose gel is 1 part by weight, in terms of dry weight. The content is preferably 0.70 parts by weight or more, more preferably 0.80 parts by weight or more, even more preferably 0.90 parts by weight or more, and most preferably 1.00 parts by weight or more. The content is preferably 2.50 parts by weight or less, more preferably 2.00 parts by weight or less, even more preferably 1.70 parts by weight or less, and most preferably 1.50 parts by weight or less. The mass measurement was performed by measuring the total mass, measuring the mass of calcium by ICP mass spectrometry, measuring the mass of calcium phosphate based on the chemical formula, and subtracting the mass from the total mass to obtain the mass of agarose, and then calculating the weight ratio. The term "single-needle crystals" used here refers to calcium phosphate particles that have been subjected to X-ray analysis (using a fully automated multipurpose X-ray analyzer manufactured by Rigaku) and the results show that they are crystals, based on the distribution of peaks indicating single-needle crystals. Spherical crystals can also be identified in the same way.
The average particle size is an arithmetic mean diameter based on the number of particles determined by a particle size distribution measuring device.

<Agarose gel>
The agarose gel in the present invention is a known material obtained by hydrogelling agarose, which is a type of polysaccharide.
Of these, agarose gels that are partially crosslinked are preferred, and known products such as agarose gel granules (Pharmacia Biotech), Biogel (trademark) A (Bio Rad Laboratories), and Lonza can be used.

<Other hydrogel-forming polymers>
A hydrogel-forming polymer may be used together with the agarose gel as long as the effects of the present invention are not impaired. The hydrogel-forming polymer may be partially physically or chemically crosslinked, and is 1) a polysaccharide selected from the group consisting of alginic acid, pullulan, pectin, starch, pregelatinized starch, hyaluronic acid, cellulose, methylcellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylcellulose, mannan, chitosan, chitin, chitosan-chitin copolymer, dextran, and dextran sulfate, or 2) a polypeptide selected from the group consisting of polyaspartate, collagen, gelatin, poly-γ-glutamic acid, polylysine, and casein, or 3) another biodegradable polymer selected from the group consisting of polylactic acid, polyglycolic acid, and polylactic acid-glycolic acid copolymer, or 4) a biocompatible polymer selected from the group consisting of lignin, polyornithine, polyrotaxane, polyethylene succinate, polyvinyl alcohol, polyethylene glycol, polyacrylamide, polyvinylpyrrolidone, and poly(hydroxyethyl)(meth)acrylate.

<Form of wound healing agent>
The wound healing agent of the present invention may contain Ca8(H2PO4)6.5(H2O) or the like within a range that does not impair the effect of the present invention.
The wound healing agent may be in a wet state containing moisture or in a dry state. In each state, any form such as a sheet, particle, flake, powder, etc. may be adopted.
In order to improve adhesion to the wound, it is necessary to adhere and fix the wound in any form, such as an agent in which the wound-healing agent is soaked in a known solvent, a cream or ointment in which the wound-healing agent is dispersed in a known medium, or a patch sheet in which the wound-healing agent is integrated with a known sheet.

<Inclusion forms of single needle and spherical crystals of Ca(HPO4)(H2O)2 and agarose gel>
In addition to the above, the mono-needle and/or spherical crystals of Ca(HPO4)(H2O)2 prepared separately can be simply mixed with agarose gel, either in a solvent or without a solvent, and optionally further molded, and the resulting mixture or molded product can be used as a wound healing agent.
Figure 4 shows a scanning electron microscope photograph of calcium phosphate agarose gel 2 (containing mono-needle crystals of Ca(HPO4)(H2O)2). The photograph in the upper right corner of Figure 4 is an enlarged view of a portion of the center of the photograph in the upper left, and the photograph below that is an even larger view of the center of the upper right corner, with the highest magnification. The white portion in the photograph with the highest magnification is Ca(HPO4)(H2O)2.

As another incorporation form, a first aqueous solution containing calcium (a 400 mM calcium chloride solution at pH 7.4) and a second aqueous solution containing no calcium phosphate ions (a 120 mM sodium hydrogen phosphate solution (disodium hydrogen phosphate dodecahydrate) at pH 7.4) are prepared, and a sheet-like or rod-like hydrogel-like agarose (agarose gel) such as a disk is alternately immersed in these two aqueous solutions to form and support mono-needle Ca(HPO4)(H2O)2 on the surface and/or inside of the agarose gel. By carrying out this alternate immersion process several to several tens of times (alternate immersion method) according to the purpose, mono-needle Ca(HPO4)(H2O)2 particles of the desired particle size can be supported in the agarose gel.
In the step of alternately immersing the agarose gel, it is preferable not to use a metal support member such as stainless steel that supports the agarose gel and is immersed together with the agarose gel, in order to form mono-needle crystals of Ca(HPO4)(H2O)2.

Therefore, to make the agarose gel support the monoacicular and/or spherical crystals of Ca(HPO4)(H2O)2, it is preferable to perform the treatment by the above-mentioned alternate immersion method, for example, by directly holding only the end of the agarose gel and immersing only the agarose gel without immersing the holding member in the above-mentioned aqueous solution. If a metal or resin supporting member is also immersed in the first aqueous solution and the second aqueous solution together with the agarose gel, there is a possibility that the calcium phosphate particles generated are not monoacicular and/or spherical crystals of Ca(HPO4)(H2O)2.
Instead of directly holding the agarose gel, glass, quartz, ceramics, drug-resistant thermoplastic polyurethane, etc. can be used as the support member. In this case, the agarose gel sheet is placed on a plate, dish-shaped, or container-shaped support member made of glass, quartz, ceramics, or drug-resistant thermoplastic polyurethane, and the support member is alternately immersed in the first aqueous solution and the second aqueous solution. This allows calcium phosphate to permeate the agarose gel sheet from the upper surface, and Ca(HPO4)(H2O)2 to be crystallized as single needle crystals within the agarose gel sheet.

The wound healing agent of the present invention can contain agents such as growth factors of various cells (e.g., epidermal growth factor, fibroblast growth factor, etc.), blood coagulation factors (e.g., thrombin, factor VIII, etc.), or antibacterial agents (antibiotics) depending on the purpose of application or implantation (e.g., to promote bone regeneration or tissue regeneration or promote hemostatic action at the same time as hemostasis) or the site. These other agents can be supported in the agarose gel matrix or on Ca(HPO4)(H2O)2 by being present in any of the aqueous solutions used in the immersion step in the above-mentioned alternating immersion method, or by being mixed and encapsulated in the agarose gel matrix during or after the formation of the agarose gel.

The wound healing agent of the present invention thus obtained can be quickly applied to or embedded in an appropriate amount (usually so as to fill the entire cavity) at a bleeding site in an open wound, such as an open wound such as an abrasion, a tooth extraction socket, a periodontal pocket, or a closed wound, such as a cyst or bone defect, in a mammal, particularly a human, to rapidly stop bleeding or significantly shorten the time required for hemostasis or blood clotting.Furthermore, tissue healing can be promoted through blood clot retention.
In particular, in the field of dentistry, it can be used for the treatment of periodontal disease, implant surgery, hemostasis after tooth extraction, and osteomyelitis treatment, and in the field of orthopedics, it can be used for hemostasis and wound protection in the event of fractures or trauma, and for tissue healing of abrasions, bedsores, etc.
The composite material of the present invention can regenerate lost bone and maintain the bone morphology by densely filling a bone defect. The composite material of the present invention may be used in a state supported on a suitable support, such as a sheet member or a bandage. It can also be made into an ointment by mixing with a known suitable base material.

The present invention will be described in detail below by way of specific examples, but it is not intended that the present invention be limited to these examples.
<Preparation of abrasion model>
The state of hair growth on the back skin of the hairless rats was checked, and hair was removed using electric clippers and an electric shaver if necessary.
Under isoflurane inhalation anesthesia, a clinical skin defect creation knife was used to create an abrasion wound approximately 20 × 25 mm in size and 400 to 500 μm deep, extending into the dermis, on the back.

<Measurement of wound surface diameter, selection of animals and grouping>
Grouping was performed on the day the abrasion model was made. The wound surface diameter (long diameter and short diameter) was measured using a caliper, and the calculated wound area (long diameter x short diameter (mm2)) was used as an index to perform grouping according to Table 1 using a stratified sequential randomization method. No treatment was given to the six animals in group A, and either calcium phosphate agarose gel 1 or 2 or Fiblast Spray 500 (Kaken Pharmaceutical Co., Ltd.) was administered to the six animals in groups B, C, and D, as shown in Table 1. At the time of grouping,
Animals with poor wound conditions were excluded in advance. Six hairless rats in each of groups A, B, C, and D were assigned animal numbers.

<Preparation of wound healing agent>
The test substances, calcium phosphate agarose gels 1 and 2 (powders), were obtained by the following method.
Another test substance, Fiblast Spray 500, was prepared by dissolving the freeze-dried powder in 5 mL of the attached dissolving solution and storing it in a refrigerator (expiry date was 2 weeks), and the required amount was dispensed on the day of administration.

<Measurement of wound surface diameter>
The tapes were removed under isoflurane inhalation anesthesia. The gauze attached to the wound surface was thoroughly moistened with saline and slowly peeled off to remove it. The wound surface was lightly washed with saline and pressed with absorbent cotton to remove moisture, and the wound diameter (long diameter and short diameter) was measured with a vernier caliper. The measurement period was from the day after group division (day 1 of administration) to day 14 of administration for groups A and D, and from days 3 and 6 to 14 of administration for groups B and C, and the measurement frequency was once a day.

Administration of test substance
The test substances were administered to groups B and C once a day on days 0 and 3, and from day 6 onwards if there was bleeding or wetness, while group D was administered once a day from day 0 onwards. The administration period for all groups was 14 days, and after measuring the wound diameter, the administration was performed under isoflurane inhalation anesthesia.
For groups B and C, powdered test substance was spread over the entire wound, two layers of 3x3cm gauze were placed over the wound, lightly pressed with a wooden spatula, and a 4x4cm waterproof film was applied on top of the gauze. A 5x5cm adhesive elastic bandage was then applied over the gauze, which was then covered with a 7.5x7.5cm Bemcot, and the adhesive elastic bandage was then wrapped around the torso and fixed (fixed on days 0 and 3 of administration, and changed daily from day 6 onwards).
In group D, 100 μL of the liquid was applied to the entire wound surface, and the wound was secured in the same manner as in groups B and C using gauze, waterproof film, adhesive elastic bandage, and Bemcot (changed daily).
Group A was treated in the same way as the test substance group, except that the test substance was not administered (changed daily).

<Skin collection>
All animals in all groups were sacrificed by exsanguination after total blood collection from the posterior vena cava on the 14th day after administration, and the wounded skin including the normal area was excised and fixed in 10% neutral buffered formalin.

<Cure rate>
The wound area (long diameter x short diameter) in mm2 was calculated from the wound surface diameter, and the healing rate (%) and total healing rate (AUC0-14, %/day) were calculated using the following formula.
Cure rate (%) =
(Wound area on the day administration started - Wound area at time of measurement) ÷ (Wound area on the day administration started) × 100
(Note that the "start date of administration" here refers to the date of the first administration.)
<Total cure rate (AUC0-14, %/day, Groups A and D)>
Total cure rate =
(Cure rate on day 0 of administration + cure rate on day 1 of administration) x 1/2
+ (cure rate on the first day of administration + cure rate on the second day of administration) x 1/2
+ (cure rate on the second day of administration + cure rate on the third day of administration) x 1/2
+ (cure rate on the 3rd day of administration + cure rate on the 4th day of administration) x 1/2
+ (cure rate on the 4th day of administration + cure rate on the 5th day of administration) x 1/2
+ (cure rate on the 5th day of administration + cure rate on the 6th day of administration) x 1/2
+ (cure rate on the 6th day of administration + cure rate on the 7th day of administration) x 1/2
+ (cure rate on the 7th day of administration + cure rate on the 8th day of administration) x 1/2
+ (cure rate on the 8th day of administration + cure rate on the 9th day of administration) x 1/2
+ (cure rate on the 9th day of administration + cure rate on the 10th day of administration) x 1/2
+ (cure rate on the 10th day of administration + cure rate on the 11th day of administration) x 1/2
+ (cure rate on the 11th day of administration + cure rate on the 12th day of administration) × 1/2
+ (cure rate on the 12th day of administration + cure rate on the 13th day of administration) x 1/2
+ (cure rate on the 13th day of administration + cure rate on the 14th day of administration) × 1/2

<Total cure rate (AUC0-14, %/day, Groups B and C)>
Total cure rate =
(Cure rate on day 0 of administration + cure rate on day 3 of administration) x 3/2
+ (cure rate on the 3rd day of administration + cure rate on the 6th day of administration) x 3/2
+ (cure rate on the 6th day of administration + cure rate on the 7th day of administration) x 1/2
+ (cure rate on the 7th day of administration + cure rate on the 8th day of administration) x 1/2
+ (cure rate on the 8th day of administration + cure rate on the 9th day of administration) x 1/2
+ (cure rate on the 9th day of administration + cure rate on the 10th day of administration) x 1/2
+ (cure rate on the 10th day of administration + cure rate on the 11th day of administration) x 1/2
+ (cure rate on the 11th day of administration + cure rate on the 12th day of administration) × 1/2
+ (cure rate on the 12th day of administration + cure rate on the 13th day of administration) x 1/2
+ (cure rate on the 13th day of administration + cure rate on the 14th day of administration) × 1/2

<Healing promotion rate>
Healing promotion rate (%) = (sum of healing rates of each individual - average of sum of healing rates of controls) ÷ average of sum of healing rates of controls × 100

<Statistical processing>
The obtained values (body weight, cure rate, total cure rate), their average value and standard error were calculated for each group. The significant difference between each group was tested for homogeneity of variance by the Bartlett method, and since all values were homogeneous, one-way analysis of variance was performed. The significance level for the Bartlett method and one-way analysis of variance was set at a risk level of 5%, and for the Tukey method, the significance levels were set at a risk level of 5% and 1%. All statistical analyses were performed using SPSS14.0J (IBM Japan, Ltd.), Microsoft Excel2013 (Microsoft Corp.), and OS (Windows 10).

(a) Calcium phosphate agarose gel 1 (Comparative example)
0.45 g of agarose (Lonza) was dissolved in hot water (15 ml) and then cooled to gel, to prepare a sheet-like agarose gel (thickness: about 1 mm). The prepared sheet-like agarose gel was punched out to a diameter of 10 mm and formed into a disk shape, which was used in the following alternate immersion method.
To obtain a composite of Ca(ClO)2.Ca3(PO4) and agarose gel, a 200 mM calcium chloride solution at 4°C and a 120 mM sodium hydrogen phosphate solution (disodium hydrogen phosphate dodecahydrate) solution at pH 7.4 at 4°C were prepared. The above-mentioned sheet-shaped agarose gel with a diameter of 10 mm was immersed in a 200 mM calcium chloride solution at 4°C for 2 hours, washed with pure water, and then immersed in a 120 mM sodium hydrogen phosphate solution (disodium hydrogen phosphate dodecahydrate) solution at pH 7.4 at 4°C for 2 hours and washed with pure water, for a total of 2 immersions and washings as one cycle, for a total of 12 cycles. During the immersion, no support member made of metal such as stainless steel or resin, which is immersed together with the agarose gel to support the agarose gel, was used. The edge of the disk-shaped agarose gel was held, and the disk was treated by an alternate immersion method in which only the agarose gel portion was immersed, while the held portion was not immersed.
The particle size of the obtained Ca(ClO)2·Ca3(PO4) was 6.2 μm±1.8 μm, and the content of Ca(ClO)2·Ca3(PO4) was 0.67 parts by weight per part by weight of agarose.

(b) Calcium phosphate agarose gel 2 (Example)
0.45 g of agarose (Lonza) was dissolved in hot water (15 ml) and then cooled to gel, to prepare a sheet-like agarose gel (thickness: about 1 mm). The prepared sheet-like agarose gel was punched out to a diameter of 10 mm and formed into a disk shape, which was used in the following alternate immersion method.
To obtain a composite of monoacicular Ca(HPO4)(H2O)2 and agarose gel, a 400 mM calcium chloride solution at pH 7.4 at 20° C. and a 120 mM sodium hydrogen phosphate solution (disodium hydrogen phosphate dodecahydrate) at pH 7.4 at 20° C. were prepared. The above-mentioned sheet-shaped agarose gel with a diameter of 10 mm was immersed in a 400 mM calcium chloride solution at pH 7.4 at 20° C. for 2 hours, washed with pure water, and then immersed in a 120 mM sodium hydrogen phosphate solution (disodium hydrogen phosphate dodecahydrate) at pH 7.4 at 20° C. for 2 hours and washed with pure water, for a total of 12 cycles of immersion and washing.
During the immersion, no support member made of metal such as stainless steel or resin was used to support the agarose gel and to be immersed together with the agarose gel. The end of the disk-shaped agarose gel was held, and the treatment was performed by the alternate immersion method in which only the agarose gel portion was immersed, while the held portion was not immersed.
The particle size of the obtained Ca(HPO4)(H2O)2 was 4.05 μm±1.36 μm. In terms of dry weight, the content of Ca(HPO4)(H2O)2 was 1.13 parts by weight per part by weight of agarose.

<Test Method>
The above calcium phosphate agarose gel 1, calcium phosphate agarose gel 2, and Fiblast Spray 500 were used to treat the abrasion wounds of rats, and the progress was observed for 14 days. Even after 14 days, when the wounds were treated with Fiblast Spray 500, bleeding could not be stopped and an inflammatory reaction occurred, so that abundant infiltration of inflammatory cells was observed. In contrast, when the wounds were treated with Gel 2, no inflammatory cells were observed.

(Dosage)
The dosages of Group B (calcium phosphate agarose gel 1) and Group C (calcium phosphate agarose gel 2) were calculated by dividing the daily dosage by the number of animals used, and the results are shown in the following table. The dosages were calculated based on the weight of calcium phosphate agarose gel.

The additional dose in Table 2 is the amount additionally administered to each animal due to bleeding or wetting after the initial administration.
The total dose is the total dose administered per animal during the administration period (14 days) including the first administration.
In both groups B and C, the total dose was approximately 1.4 times the initial dose, and the number of additional doses during the administration period was one or two.
In addition, even when spherical crystals of Ca(HPO4)(H2O)2 are used instead of the mono-needle crystals of Ca(HPO4)(H2O)2, the same effects as those in the case of the mono-needle crystals are exhibited.

Group D required daily administration of Fiblast Spray 500 for 14 days. The dosage of its main ingredient, trafermin, was 10 μg/day per mouse in a volume of 100 μL/day, and the total dosage during the administration period (14 days) was 140 μg/mouse, 14 times that amount.
Group A (control) received no treatment.

<Cure rate and total cure rate>
The cure rates are shown in Figure 1, and the total cure rates are shown in Figure 2.
Group A: No medication was administered to the control group A. The cure rate gradually increased over time, and on the 14th day of administration, the cure rate was 62.3±2.5%, and the total cure rate was 446.3±26.5%/day.
Group B: The cure rate in group B, which was administered (calcium phosphate agarose gel 1 (Ca(ClO)2·Ca3(PO4))), gradually increased with the number of days of administration, and on the 14th day of administration the cure rate was 68.9±1.4%, and the total cure rate was 490.9±13.8%/day.
Group C: The cure rate of group C, which was administered calcium phosphate agarose gel 2 (mononeedle crystals of Ca(HPO4)(H2O)2), gradually increased with the number of days of administration. The cure rate on the 14th day of administration was 75.8±2.5%, and the total cure rate was 560.7±18.2%-days.
Group D: The cure rate of group D, which was administered (Fiblast Spray 500), gradually increased with the number of days of administration, and the cure rate on the 14th day of administration was 70.6±1.9%, and the total cure rate was 552.0±17.6%-days.
The cure rates for group B from the 6th day after administration, group C from the 3rd day after administration, and group D from the 1st day after administration were A.
In addition, there was a significant difference between the cure rate and the sum of cure rates from 7 to 14 days after administration in group C and the cure rate and the sum of cure rates from 6 to 11 days after administration in group D.

The cure rate and the sum of cure rates from 7 to 14 days after administration in group C, and the cure rate and the sum of cure rates from 6 to 11 days after administration in group D were significantly higher than those in groups A and B. In addition, the cure promotion rate calculated by setting the sum of cure rates in group A as 100% was 10.0% in group B, 25.6% in group C, and 10.0% in group D.
A 23.7% increase in healing was observed in group D.
From the above, under the conditions of this test, it was confirmed that the test substance was administered percutaneously to the rat abrasion model in groups C and D, and it was suggested that group C had the same effect as group D, which is already on the market. However, group D required daily administration, while group C only required several administrations due to bleeding and wetness. Considering this history, it can be understood that although groups C and D seemingly have the same effect, the wound healing agent of the present invention in group C is more usable due to the difference in the number of administrations. Furthermore, in group D, strong inflammation occurred at the wound site during the administration period, but in groups B and C, only weak inflammation occurred to the same extent as group A, which was not administered.
Group B also showed a tendency to promote healing, although this tendency was weaker than that of group D. However, healing in group C was clearly more advanced than that in group B.
According to these results, the wound healing agent of the present invention is not a protein preparation, and therefore can be produced relatively inexpensively and can be administered less frequently, thereby reducing the burden of treatment compared to Fiblast Spray, which requires daily administration.

Claims (1)

A wound healing agent containing 0.50 to 3.00 parts by weight of mono-needle and/or spherical crystals of Ca(HPO4)(H2O)2 per 1.0 part by weight of agarose gel.