Patent Application
20160235757
The present invention relates to topical skin pharmaceutical compositions containing Icotinib or its pharmaceutically acceptable salts and uses thereof.
Dermatologic diseases relate to skin disorders. Some of the common and frequently-occurring diseases seriously affecting people's health are leprosy, scabies, fungal disease, psoriasis, and skin bacterial infections. In dermatologic diseases, the morphologies, structures and functions of skin (including hairs and nails) change after they are affected by internal and external factors, resulting in the pathological process, and accordingly generating a variety of clinical manifestations. The incidence of dermatologic diseases is very high. While the majority of them are mild and often are not life-threatening, (but only a few are severer and life threatening can even threaten life), they can seriously affect the appearance of patients, causing a severe psychological burden, thereby affecting the patients' daily works and lives.
Among them, psoriasis is a common chronic inflammatory skin disease, the incidence rate of which is about 2% in Western countries and about 0.3% in Asia. The incidence rate, however, has increased rapidly in recent years. The typical skin manifestations of psoriasis include demarcated red patches with silvery white scales, which can seriously affect the patient's appearance. Thus, although psoriasis is not life-threatening, it causes a heavy psychological burden, thereby affecting the patient's daily works and lives. Improper treatment can make it worse and increase the psychological and economic burdens of the patients. For example, erythrodermic and pustular psoriasis can cause metabolic disorders of the whole body, multiple organ (such as cardiovascular and lung) complications, and infections, which can be life-threatening. Although some of them are caused by unknown etiology, a considerable amount of erythrodermic and pustular psoriasis, including onset and exacerbation, are caused by improper treatment.
Early studies suggest that the pathogenesis of psoriasis is mainly caused by the abnormal differentiation of epidermal hyperplasia and the activation of the immune system. Tazarotene (a vitamin A derivative), lente and potent glucocorticoid, and calcipotriol (a Vitamin D3 derivative) are recommended as topical treatments according to China's psoriasis treatment guidelines of 2008. However, the long-term use of corticosteroids can cause many side effects, including skin atrophy, telangiectasia, folliculitis, pigmentation and hypopigmentation. Long-term use of potent glucocorticoid preparations may cause systemic reactions, and even induced pustular or erythrodermic psoriasis after withdrawal. The vitamin A derivatives can irritate a mucocutaneous zone, especially for patients prone to allergies. Common side effects of calcipotriol include pruritus, skin irritation, burning sensation, tingling, dry skin, erythema and rash, and calcium metabolism to a certain extent. Therefore, only a limited number of products, with significant effect and small side effects, are suitable for psoriasis patients.
Recent studies find that in normal human epidermal layers, the expression of epidermal growth factor receptor (EGFR) is inconsistent, and a large amount of EGFR is expressed in the actively dividing basal layers and part of the prickle cell layer near the basal layer. However, in the skin of someone with psoriasis vulgaris, EGFR is expressed in all the layers, and in the layers above the prickle cell layer. The expression level is eight times higher than the normal control. The overexpression of EGFR in the skin lesions of psoriatic vulgaris suggests that psoriasis may be associated with excessive proliferation of epidermal cells and abnormal differentiation. Controlling the abnormal expression of EGFR may open up new ways and provide new drugs for the treatment of psoriasis.
Tyrosine kinase receptors are transmembrane proteins involved in signal transduction, in which the receptors transduce the growth factor signals from the cell surface to intracellular molecules that control critical cellular functions, for example, cell growth, mutation, angiogenesis and apoptosis inhibition. Epidermal growth factor receptor (EGFR) tyrosine kinase is one type of \ such receptors. There has not been an effective tyrosine kinase inhibitor in the market for treating psoriasis nowadays.
In light of the limitations of existing technology, the present invention provides topical pharmaceutical compositions inhibiting tyrosine kinase and the preparation methods thereof.
First, the present invention provides a topical skin pharmaceutical composition, which comprises the active ingredient inhibiting tyrosine kinase and excipients of the topical preparation, wherein the active ingredient is Icotinib or a pharmaceutically acceptable salt thereof, and the excipients comprise dispersion mediums, emulsifiers and/or one or more other pharmaceutically acceptable excipients of the topical preparation.
The present invention also provides preferred technology for the above technical solutions:
Preferably, the active ingredient can be Icotinib freebase, Icotinib hydrochloride, Icotinib maleate or Icotinib phosphate;
Preferably, the concentration of the active ingredient is 0.1-11 wt %, more preferably 0.3-5 wt %, further preferably 0.9-4.3 wt %, and especially preferably 1-1.5 wt %;
Preferably, the dispersion mediums include water soluble matrices and/or oily matrices;
Preferably, the water soluble matrices comprise water, glycerin, gelatin, ethanol, polyethylene glycol, propylene glycol, DMSO and/or cellulose derivatives;
Preferably, the concentration of the water soluble matrices is 40-100 wt %, more preferably 65-85 wt %;
Preferably, the oily matrices comprise hydrocarbon matrices, oil matrices, lipid matrices and/or organosilicon oxide polymers;
Preferably, the hydrocarbon matrices comprise hexadecanol, octadecanol and/or liquid paraffin; the oily matrices comprise soybean oil, castor oil, glycerin mono-, di-stearate and/or Vaseline; the lipid matrices comprise lanolin and/or beeswax; the organosilicon oxide polymers are dimethylsiloxane polymer;
The invention also provides a use for the preparation of ointments, preferably cream, from the topical pharmaceutical compositions.
The invention also provides a use for the preparation of gels, preferably transparent, from the topical pharmaceutical compositions.
The invention also provides a use for the preparation of medicines from the topical pharmaceutical compositions for the treatment of non-malignant excessive hyperplasia disorders or tumor and complications thereof.
The invention also provides preferred technology for the above the technical solutions preferably if:
The invention also provides a method for treating mammalian tissue excessive hyperplasia disease, comprising administering a therapeutically effective amount of the pharmaceutical compositions to a patient suffering from tissue excessive hyperplasia disease.
The invention also provides preferred technologies for the above technical solutions preferably if:
In particular, the active ingredient of the skin topical pharmaceutical compositions provided by the invention is Icotinib freebase, which is prepared by the following method: Icotinib hydrochloride was dissolved in a mixture of ethanol and water. The sodium hydroxide solution was added dropwise into the solution of Icotinib hydrochloride at a certain temperature until the pH value of the mixture is greater than or equal to 13. The reaction solution was stirred, cooled to room temperature, and precipitated. The precipitation was filtered, washed by pure water, and dried under vacuum below a certain temperature to obtain the Icotinib freebase. The Icotinib freebase of the present invention includes, but is not limited to, the molecules prepared from the above preparation method. The structure of Icotinib freebase is as follows:
In particular, the active ingredient of the skin topical pharmaceutical compositions provided by the invention is Icotinib hydrochloride, preferably with the crystal form I of Icotinib hydrochloride, which includes, but is not limited to, the molecules prepared according to the preparation method disclosed in the international application of WO2010/003313A1. The structure of Icotinib hydrochloride is as follows:
In particular, the active ingredient of the skin topical pharmaceutical compositions provided by the invention is Icotinib maleate, which can be prepared by the following method. First, Icotinib freebase was dissolved in acetone achieving the Icotinib solution. In addition, maleic acid was dissolved in acetone achieving the maleic acid solution. The maleic acid solution was added to the Icotinib solution, the reaction mixture stirred and reacted, and then Icotinib maleate was isolated. The Icotinib maleate includes, but is not limited to, the molecules prepared from the above preparation method. The structure of Icotinib maleate is as follows:
In particular, the active ingredient of the skin topical pharmaceutical compositions provided by the invention is Icotinib phosphate, which can be prepared by the following method. First, cotinib freebase was dissolved in isopropanol, achieving the Icotinib solution. In addition, the phosphoric acid solution was added to isopropanol achieving the phosphoric acid solution. The phosphoric acid solution was added to the Icotinib solution, and the reaction mixture was stirred and isolated to obtain Icotinib phosphate. The Icotinib phosphate includes, but is not limited to, the molecules prepared from the above preparation method. The structure of Icotinib phosphate is as follows:
In particular, the Icotinib or its pharmaceutically acceptable salts thereof in the present invention refer to any pharmaceutically acceptable materials containing the structure of Icotinib, including, but not limited to: Icotinib freebase, Icotinib hydrochloride, Icotinib maleate, Icotinib phosphate, Icotinib solvate, Icotinib chelate, Icotinib hydrate, and all crystal forms of the above materials.
The Icotinib or pharmaceutically acceptable salts thereof in the invention can contain an asymmetric center, chiral axis and chiral surface (see pages 1119-1190 on “Stereochemistry of Carbon Compounds” (edited by E. X. Eliel and S. H. Wilen) published by John Wiley & Sons, Inc. in 1994), and can include racemate, racemic mixture, individual diastereomers, all possible isomers and their mixtures, including optical isomers and all stereo isomers included in the invention. In addition, tautomers can include the Icotinib or pharmaceutically acceptable salts thereof in the invention. And all the tautomers are also included in the scope of the present invention.
The Icotinib or pharmaceutically acceptable salts thereof in the invention can exist in a large number of different crystalline forms.
Any kind of drug for clinical use should be made in forms suitable for different medical and preventive applications. These forms are called dosage forms, while the dosage forms are known as pharmaceutical preparations. To develop and produce pharmaceutical dosage forms and preparations that are of high curative effect, of minimal side effect, easy to take, convenient for transportation and storage, and of stable quality, the production mainly depends on the pharmaceutical excipients, although it also relates to production technologies, production facilities, preparation processes, quality controls and so on. For any kind of preparation, in addition to the active ingredients (main drug), the rest are pharmaceutical excipients. Therefore, the quality of pharmaceutical excipients, and the scientificity and rationality of the excipient formulas, directly affect the quality of the preparations.
The topical pharmaceutical compositions of Icotinib or the pharmaceutically acceptable salts thereof should be made to allow the drug to be released through the epidermis to apply its therapeutic effect in the skin. The skin, by covering the whole body, prevents the body from losing water, electrolytes and other substances, and also is a barrier against the invasion of foreign substances, thereby playing an important role in protecting the body. The barrier function of the skin is mainly undertaken by the corneum, which is a thin film layer with a certain mechanical strength, and a major barrier for the transdermal absorption of the drug. It is generally believed that the corneum can hold a proper concentration of water-soluble and fat-soluble substances, and drugs with small molecular weight can diffuse into the endothecium through the intercellular space. Hair follicles and cryptae can penetrate the corneum, thereby providing another passway for the drug absorption.
There are many factors that affect the transdermal absorption of the drug, such as physiological factors, the physicochemical properties of drugs, and the type and composition of matrices. Although the inherent activity of the drug is the most important factor that determines its therapeutic use, the release and transdermal absorption of the drug are influenced by the matrices to a great extent.
Icotinib (with a chemical name of 4-[(3-ethynyl phenyl) amino] quinazoline [6,7-b]-12-crown-4) or a pharmaceutically acceptable salt thereof is the active ingredient of the preparation.
Transcutol P has a chemical name of diethylene glycol monoethyl ether. It can be used as the solvent and transdermal absorption enhancer for the active drug in pharmaceutical manufacturing.
Labrasol, with a Chinese chemical name of caprylic capric acid macrogol glycerides and an English name of Caprylocaproyl macrogol-8 glycerides, is a mixture of mono-, di-, tri-glycerides and mono-, di-fatty acid polyethylene glycol ester with a certain ratio, which is commonly used as an emulsifier and a transdermal absorption enhancer in pharmaceutical manufacturing.
Carbopol, with a Chinese name of carbomer, is a white, “fluffy”, acidic powder that can absorb water and has a slight special smell. It is a polymer of acrylic bonded allyl sucrose or pentaerythritol allyl ether, and is commonly used as a gel matrix.
Tefose 63®, with a chemical name of polyethylene glycol-7 stearate, is a mixture of polyethylene glycol-6 stearate (PEG-6 stearate), ethylene glycol palmitostearate, and polyethylene glycol-32 stearate (PEG-32 stearate).
Labrafil M 1944®, with a chemical name of oleic acid polyethylene glycol glyceride (EP), is a mixture composed of mono-, di-, tri-glycerides and mono-, di-fatty acid polyethylene glycol ester with a certain ratio.
DMSO, with a chemical name of dimethyl sulfoxide, is a colorless liquid. It can solve with many organic solvents and water.
To prepare the preparation provided by the invention, one needs to pay attention to the insolubility between the excipients, for example, carbomer being discolored when encountering m-dihydroxybenzene, and being insoluble with phenol, cationic polymers, strong acid, and electrolyte with high concentration. Trace amounts of iron or other transition metals are capable of catalyzing and degrading carbopol dispersions. Large amounts of heat can be produced when carbomer comes in contact with strong alkaline substances, such as ammonia, potassium hydroxide, sodium hydroxide, or strongly alkaline organic amine. Some amino-containing drugs can form soluble complexes with carbomer, and, generally speaking, the occurrence of such cases can be prevented by adjusting the solubility parameter of the liquid with appropriate alcohol or polyatomic acid.
The excipients and the insolubility between them in the preparations are common knowledge in the field, which can be found in “The Handbook of Pharmaceutical Excipients” (edited by [EN] R. C. Rowe, [US] P. J. Sheskey, and [EN] P. J. Weller, mainly translated by Junmin Zheng, and published by Chemistry Industry Press), “The Handbook of Commonly Used Pharmaceutical Excipients” (mainly edited by Jiewei Li, Jixiang Liu, and published by The Second Military Medical University Press) and “Pharmaceutical Excipients Daquan” (mainly edited by Mingsheng Luo, and Tianhui Gao, and published by Sichuan Science & Technology Press), none of which are explained in detail here.
The present invention provides topical preparations containing Icotinib or pharmaceutically acceptable salts thereof and the preparation methods. The topical preparations provided in the invention are with minimal skin irritation, or no adverse effects such as pruritus, burning sensations, tingling, dry skin, erythema, and rashes. The preparation will neither incur parahormone-related side effects such as skin atrophy, pigmentation or hypopigmentation after long-term use, nor cause any related dermatological symptoms after drug withdrawal. The preparation methods are easily understood, operable, and controllable, and suitable for industrialization.
The following examples shall be only used to illustrate specific embodiments of the present invention so that those skilled in the art can implement the present invention, but not used to limit the scope of the present invention. In the embodiments of the present invention, the techniques or methods, with no special instructions, are conventional techniques or methods in the field.
The embodiments of the topical preparations provided in the invention are as follows. The values in the embodiments mean “wt %” with no special instructions.
(1) The prescribed amount of carbomer was weighed, and fully swollen in propylene glycol;
(2) The prescribed amounts of Icotinib hydrochloride, Transcutol P and/or Labrasol were weighed individually and mixed evenly;
(3) The mixture obtained in step (2) was added to the swollen carbomer solution obtained in step (1);
(4) The mixture obtained in step (3) was stirred until it turned transparent at room temperature.
(1) The prescribed amounts of Icotinib hydrochloride, Transcutol P, and Labrasol were weighed individually and mixed evenly;
(2) The mixture obtained in step (1) was added to propylene glycol solution;
(3) The mixture obtained in step (2) was stirred until it turned transparent at room temperature.
(1) The prescribed amounts of carbomer and ethylparaben were weighed individually, and fully swollen in propylene glycol;
(2) The prescribed amounts of Icotinib hydrochloride, Transcutol P, and Labrasol were weighed and mixed evenly;
(3) The mixture obtained in step (2) was added to the swollen carbomer solution obtained in step (1);
(4) The mixture obtained in step (3) was stirred to until it turned transparent at room temperature.
(1) The prescribed amount of carbomer was weighed, and fully swollen in propylene glycol;
(2) The prescribed amounts of Icotinib hydrochloride, Transcutol P, and Labrasol were weighed individually and mixed evenly;
(3) The mixture obtained in step (2) was added to the swollen carbomer solution obtained in step (1);
(4) The prescribed amount of glycerol or ethanol was added to the mixture obtained in step (3), and stirred until it turned transparent at room temperature.
(1) The prescribed amounts of PVP-K30, carbomer, CMC-Na and/or HPMC were weighed individually, and fully swollen in propylene glycol;
(2) The prescribed amounts of Icotinib hydrochloride, Transcutol P, and Labrasol were weighed individually and mixed evenly;
(3) The mixture obtained in step (2) was added to the swollen carbomer solution obtained in step (1), and stirred until it turned transparent at room temperature.
A, Preparation Methods of Examples 30-32
(1) The prescribed amounts of Tefose 63, Labrafil M 1944, Vaseline, octadecanol, hexadecanol, ethylparaben and/or liquid paraffin were weighed individually, mixed, stirred, and heated to 75-80° C., until diatexis;
(2) In addition, the prescribed amounts of Icotinib hydrochloride, water and/or glycerol were weighed individually, mixed, heated to 60-70° C., and stirred evenly;
(3) The mixture obtained in step (2) was added to the mixture obtained in step (1), emulsified for 20 minutes, and cooled to room temperature in a water bath.
B, Preparation Methods of Examples 33-36
(1) The prescribed amounts of Vaseline, octadecanol, liquid paraffin and ethylparaben were weighed individually, mixed, stirred, and heated to 75-80° C., until diatexis;
(2) In addition, the prescribed amounts of glycerol, sodium lauryl sulfate, water, DMSO and/or propylene glycol, mixed, heated to 60-70° C., and stirred evenly;
(3) The mixture obtained in step (2) was added to the mixture obtained in step (1), and emulsified for 10-30 minutes;
(4) The prescribed amount of Icotinib hydrochloride was added to the mixture obtained in step (3), emulsified sequentially for 10 minutes, and cooled to room temperature.
(1) The prescribed amount of carbomer was weighed, dissolved in water, added to triethanolamine, and stirred until fully swollen;
(2) In addition, the prescribed amount of Icotinib hydrochloride was weighed, added with water, stirred at 40° C., added with the prescribed amount of sodium hydroxide, and stirred evenly;
(3) In addition, the prescribed amounts of citric acid and/or the rest of the triethanolamine were weighed, dissolved in water, added to the solution obtained in step (2) dropwise, added with the carbomer solution obtained in step (1), emulsified for 5 minutes, and then heated to 60-70° C.;
(4) In addition, the prescribed amounts of Tefose 63, Labrafil M 1944, sodium lauryl sulfate, octadecanol, hexadecanol, liquid paraffin, ethylparaben and/or glycerol were weighed individually, mixed, stirred, heated to 75-80° C. until diatexis, added to the solution obtained in step (3), emulsified for 15 minutes, stirred in a water bath, and cooled to room temperature.
(1) The prescribed amounts of Tefose 63, Labrafil M 1944, Vaseline, octadecanol, hexadecanol, soybean oil, stearic acid, ethylparaben and/or liquid paraffin were weighed individually, mixed, stirred, and heated to 75-80° C. until diatexis;
(2) In addition, the prescribed amounts of Icotinib freebase, triethanolamine, citric acid, sodium citrate, water and/or carbomer were weigh individually, mixed, stirred, and heated to 60-70° C.;
(3) The mixture obtained in step (2) was added to the mixture obtained in step (1), emulsified for 20 minutes, and cooled to room temperature in a water bath.
B, Preparation Methods of Examples 48-50
(1) The prescribed amounts of Vaseline, octadecanol, liquid paraffin and ethylparaben were weighed individually, mixed, stirred, and heated to 75-80° C. until diatexis;
(2) In addition, the prescribed amounts of citric acid, carbomer, water and/or triethanolamine were weighed individually, mixed, stirred, and heated to 60-70° C.;
(3) The mixture obtained in step (2) was added to the mixture obtained in step (1), and emulsified for 10 minutes;
(4) The prescribed amount of Icotinib freebase was added to the mixture obtained in step (3), sequentially emulsified for 10 minutes, and cooled to room temperature.
(1) The prescribed amounts of Tefose 63, Labrafil M 1944, beeswax, simethicone, octadecanol, liquid paraffin, ethylparaben, benzoic acid, chlorobutanol and/or chlorcresol were weighed individually, mixed, stirred, and heated to 75-80° C. until diatexis;
(2) In addition, the prescribed amounts of Icotinib hydrochloride, Icotinib maleate, Icotinib phosphate, triethanolamine, citric acid, sodium citrate, water and/or sodium lauryl sulfate were weighed individually, mixed, stirred, and heated to 60-70° C.;
(3) The mixture obtained in step (2) was added to the mixture obtained in step (1), emulsified for 20 minutes, and cooled to room temperature.
100 g Icotinib hydrochloride was dissolved in the mixture of 300 ml ethanol and 200 ml water. A solution of 11.2 g sodium hydroxide in 100 ml water was added dropwise at 60° C. to the Icotinib hydrochloride solution until the pH value of the reaction solution reached 13. The reaction solution was then stirred for an hour and then cooled to room temperature. The precipitate was filtered and washed with purified water and dried for 8 hours under vacuum below 60° C. to obtain 90 g Icotinib freebase.
10 mg Icotinib freebase was dissolved in 1 ml acetone to obtain Icotinib solution. In addition, 34.82 mg maleic acid was dissolved in 3 ml acetone to obtain a 0.1 mol/L maleic acid solution. 0.26 ml of the 0.1 mol/L maleic acid solution was added to the Icotinib solution and the reaction mixture was stirred for 24 hours to obtain Icotinib maleate.
10 mg Icotinib freebase was dissolved in 1 ml isopropanol to obtain Icotinib solution. In addition, 18.9 μL phosphoric acid was dissolved in 3 ml isopropanol to obtain a 0.1 mol/L phosphoric acid solution. 0.26 ml of the 0.1 mol/L phosphoric acid solution was added to the Icotinib solution and the reaction mixture was stirred for 24 hours, and then the Icotinib phosphate was isolated.
Test Materials:
Test drugs: Icotinib hydrochloride cream preparations, with the specification of 1 g/100 ml (1%);
Negative control cream matrix group: it does not contain active ingredients (Icotinib hydrochloride), but contains the other ingredients are the same as the test drug;
Positive control drugs: Halometasone Cream, with the specification of 15 g: 7.5 mg, Hongkong Aomei pharmaceutical factory, with the batch number of: 0911501;
Test animals: ICR mice, male, 30;
Test instrument: OLYMPUS biological microscope.
Test Method:
30 ICR male mice were randomly divided into 3 groups, with 10 mice in each group: the negative control cream matrix group, the positive control drug group of Halometasone Cream, and the 1% Icotinib hydrochloride cream group. The creams were applied to the tail skin of the mice. Before each administration, the mouse tails were gently wiped with a cotton swab and water, and the mice tails in different groups were applied with a thin layer of different drugs, 2 times a day, for 14 consecutive days. After treatment the mice were killed, and the skin with a size of 1.5 cm×0.2 cm was taken at 1-2 cm from the distal end of the mouse tail, fixed with 4% formalin, embedded with paraffin, sliced, and stained with Hematoxylin and Eosin (“H&E”).
The changes of skin keratosis layer, the granular layer, the prickle cell layer, the basal cell layer, the dermis, the follicles and other changes of the mouse tail were observed under a light microscope. The continuous granular layer cells that exist between any two adjacent follicular epidermises (i.e., scale epidermis) are called scales with granular layer. 50 scales were observed for each animal, and the scales with the formation of granular layer (SG) were calculated. The data are shown in table A.
As shown from table A: Icotinib hydrochloride cream preparation could significantly promote the formation of scale granular layers on the mouse tail compared with the negative control matrix group, and the effect is stronger than the positive control group of Halometasone Cream.
Test Materials:
Test drugs: Icotinib hydrochloride gel preparation: with the specification of 1 g/100 ml (1%); and the specification of 1.5 g/100 ml (1.5%);
Negative control gel matrix group: does not contain active ingredients (Icotinib hydrochloride), but the other ingredients are the same as the test drug;
Positive control drugs: Clobetasonl Propionate Cream, with the specification of 10 g: 2 mg (0.02%), Guangdong Shunfeng Pharmaceutical Co. Ltd., with the batch number of: 20110304;
Test animals: ICR mice, male, 40;
Test instrument: biological microscope.
Test Method:
40 ICR male mice were randomly divided into 4 groups, with 10 mice in each group: the negative control gel matrix group, the 1% Icotinib hydrochloride gel group, the 1.5% Icotinib hydrochloride gel group, and the positive drug Clobetasonl Propionate Cream group. The creams were applied to the tail skin of the mice. Before each administration, the mouse tails were gently wiped with a cotton swab and water, and the mice tails in different groups were applied with a thin layer of different drugs, 2 times a day, for 14 consecutive days. After treatment the mice were killed, and the skin with a size of 1.5 cm×0.2 cm was taken at 1-2 cm from the distal end of the mouse tails, fixed with 4% neutral formalin, embedded with paraffin, sliced, and stained with H&E.
The changes of the skin keratosis layer, the granular layer, the prickle cell layer, the basal cell layer, the dermis, the follicles and other changes of the mouse tail were observed under a light microscope. The continuous granular layer cells exist between any two adjacent follicular epidermises (i.e., scale epidermis) are called scales with granular layer. Each animal was observed, and the ratios of the number of the scales with the formation of granular layer to the number of the total scales were calculated. The data are shown in table B.
As shown from table B: the 1% Icotinib hydrochloride gel group and 1.5% Icotinib hydrochloride gel group all have the effect of increasing the number of the scales with granular layer on mouse tails.
Test Materials:
Test drugs: Icotinib hydrochloride cream preparation: with the specification of 1 g/100 ml (1%); with the specification of 2 g/100 ml (2%); and with the specification of 4 g/100 ml (4%);
Negative control cream matrix group: does not contain active ingredients (Icotinib hydrochloride), but the other ingredients are the same as the test drug;
Positive control drugs: Calcipotriol Ointment, with the batch number of EH4129, LEO Laboratories Ltd.;
Test animals: cavies, 250-300 g, male and female each half, 70;
Test instrument: biological microscope.
Test Method:
5% propranolol liniment: 5 g propranolol hydrochloride was dissolved in an appropriate amount of 50% ethanol, added with 5 ml laurocapram (Azone-propylene glycol) as composite accelerator, added with 5 g polyvinylpyrrolidone (PVP-K30) as film-forming material, added with 50% ethanol to reach 100 ml in the final mixture, and mixed.
For the 70 healthy adult cavies, 10 cavies were randomly selected (male and female each half) as normal control group. For the rest of the cavies, the dorsal skins on both sides of auriculas were applied with 5% propranolol hydrochloride emulsion liniment (80 μl/ear), once every morning and once every evening, for 2 consecutive weeks. After the completion of the modeling, the cavies were randomly divided into 6 groups, namely the model group, the negative control matrix group, the 1% Icotinib hydrochloride cream group, the 2% Icotinib hydrochloride cream group, the 4% Icotinib hydrochloride cream group, and the Calcipotriol Ointment group. Left and right ears of the animals in the model group were not applied with drugs. The left and right ears of the animals in the negative control matrix group were applied with the negative control matrix. The left and right ears of the animals in the administration group were smeared drugs. After treatment for 2 weeks, the animals were killed with CO2 anesthesia, and the skin with a size of 10 mm×5 mm was taken at the middle from both sides of auriculas of the animals in each group, fixed with 4% neutral formalin, embedded with paraffin, and stained with H&E.
The changes of the skin keratosis layer, the granular layer, the prickle cell layer, the basal cell layer, the dermis, and other changes of the cavies auriculas were observed under a light microscope. The thickness of the epidermal layer was measured under the light microscope at 100× magnification. The grid micrometer with 25×25 grids number was counted. The thickness (mm)=grids number×0.0212.
The changes of skin keratosis layer, the granular layer, the prickle cell layer, the basal cell layer, the dermis, and other changes of the cavies auriculas were observed under light microscope, and the thickness of the epidermal layer was measured with 100 times light microscope, the grids number of 25×25 grid micrometer being counted, grids number×0.0212=thickness (mm), the data being shown in Table C. (Note: an animal died in each group of the normal control group and 2% Icotinib hydrochloride cream group, and a slice of an animal from each of the model group and the calcipotriol group was not processed properly for observation.)
ΔΔP < 0.01, vs. negative control matrix group, all data (Mean ± SD) were carried on one-way analysis of variance with SPSS17.0.
One week after modeling, the cavies lost hair on the ear, and the local skin was red and swollen, and covered with a small amount of fine silver white scales. During the first and second weeks after treatment, on the cavies' auriculas appeared erosion, crusting, and peeling, while both ears of the animals of the control group were normal. After treatment for 2 weeks, on both ears of the animals appeared skin erosion and peeling in the model group, while the conditions for animals in the treatment group were improved. Particularly, the new hair was observed on the ear surface for the animals in the Icotinib hydrochloride cream treatment group, while no new hair was observed in the model group.
Under the light microscope, in the normal control group, the epidermal horny layers of the animals were completely homogeneous, the granular layer appeared single linear, the basal layer was composed of single columnar cells, the dermo-epidermis junction appeared wavy, the capillary vessels were in absence of congestion, and the structure appeared normal. In comparison, in the model group, the horny layer of the animals appeared akeratosis or hyperkeratosis, the prickle cell layer thickened, the trochanterellus extended, appearing club-shaped, the dermal papillae extended upwards, appearing rod-shaped, and the thickness of the ear epidermis significantly increased compared with the normal control group (P<0.01).
After administration of 1%, 2%, and 4% Icotinib hydrochloride cream, hyperkeratosis or akeratosis was lessened, the prickle cell layer appeared thinner, the trochanterellus extension and the dermal papillae upward extension were significantly reduced, and the thickness of the epidermis was significantly decreased compared with the model group (P<0.01).
Comparing the 2% cream group with the 1% cream group, there was no significant difference in the reduction of the epidermis thickness (P=0.250). Comparing the 4% cream group with the 1% cream group, the thickness of epidermis was significantly reduced (P<0.01).
The above-described examples are only for a full illustration of the embodiments of the present invention. The scope of the present invention is defined by the appended claims, but not limited to the embodiments. It is to be noted that various changes and modifications may be well known to those skilled in the art, and such changes and modifications are understood to be included within the scope of the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| PCT/CN2013/085042 | Oct 2013 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2014/088344 | 10/11/2014 | WO | 00 |
