CN112933096B

CN112933096B - Use of Ding Xiansai molol in preparing medicament for treating shallow phenotype, mixed or deep hemangioma

Info

Publication number: CN112933096B
Application number: CN201911268383.XA
Authority: CN
Inventors: 黄永焯; 林晓曦; 仇雅璟; 王慧媛; 陈彬; 马刚; 陈辉; 金云波; 杨希; 张世仁
Original assignee: Shanghai Institute of Materia Medica of CAS; Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Current assignee: Shanghai Institute of Materia Medica of CAS; Ninth Peoples Hospital Shanghai Jiaotong University School of Medicine
Priority date: 2019-12-11
Filing date: 2019-12-11
Publication date: 2023-06-13
Anticipated expiration: 2039-12-11
Also published as: CN112933096A

Abstract

The application relates to application of timolol to preparation of a medicine for treating shallow phenotype, mixed type or deep infant hemangioma.

Description

Use of Ding Xiansai molol in preparing medicament for treating shallow phenotype, mixed or deep hemangioma

Technical Field

The application relates to the field of pharmaceutical chemistry, in particular to application of Ding Xiansai molol in preparing medicaments for treating superficial phenotypes, mixed-type or deep hemangiomas.

Background

The angioma of infants is one of the most common benign tumors in infants, the incidence rate in caucasian race is up to 10-12%, the incidence rate in newborns in China is up to 1%, and the incidence rate in domestic ill population is up to twenty millions. About 10% of children suffer from severe complications such as ulcers, infections, pains, blindness, asphyxia, etc., and even without these complications, the fibrous adipose tissue or (and) the surface skin texture remains after the larger tumor regresses, resulting in secondary deformity, and in addition, the characteristic that it is easy to develop in the head and face, which necessitates a later plastic surgery repair, so early intervention of hemangioma treatment becomes particularly important. Traditional oral hormone, radiation therapy, laser and cryotherapy for treating infant hemangioma and the like are gradually eliminated due to side effects, along with the unexpected discovery of oral propranolol treatment for hemangioma in 2008, a series of clinical researches at home and abroad at present establish the first line position of oral propranolol in refractory infant hemangioma treatment, so that some refractory infant hemangioma is effectively treated, however, over time, the treatment of oral propranolol in infants is slowly questioned, besides systemic adverse effects of heart rate slowing, blood pressure lowering, hypoglycemia, induced bronchial asthma and the like which are known by the masses, students possibly put forward that the oral propranolol influences nervous system passages related to learning and memory through blood brain barriers of the infants, and the safety is proved by calling for longer follow-up visit. The timolol maleate preparation or the propranolol preparation for external use has the advantages of safety and no systemic absorption, has good curative effect on most of shallow-phenotype infant hemangiomas, but has poor effect on mixed or deep hemangiomas, mainly because the medicine cannot penetrate deep into the skin to reach the deep tumor body, so the hemangiomas have to be treated by oral propranolol. In order to avoid systemic adverse reactions caused by oral administration of propranolol and possible influence of long-term administration on learning and memory, a novel timolol preparation which can effectively act on mixed or deep hemangiomas through local administration and high-efficiency skin permeation and avoid adverse reactions caused by systemic administration is urgently needed at present.

For mixed or deep infant hemangio lesions, the subcutaneous component of the hemangio still progressed after initial timolol topical formulation treatment, but nonetheless, the superficial component of these hemangio still developed signs of regression during topical treatment, such as a fade in color and softening of the lesion, and the reactivity of the hemangio regression gradually increased with prolonged topical treatment time. For deep lesions, the change is not obvious, and the main reason for the phenomenon is the limitation of timolol external preparations on transdermal. Although ointments promote hydration of the skin and enhance some of the transdermal functions, if the focal area is thicker, the drug may not penetrate to a certain depth. The medicine is difficult to permeate into all parts of the tumor body, so that the special part or deep hemangioma is difficult to cure thoroughly.

The medicine which can be both oil-soluble and water-soluble (i.e. the medicine with moderate oil/water distribution coefficient value) has higher penetrability; if the drug is poorly soluble in both oil and water, transdermal absorption is difficult. The stratum corneum is just like the lipid membrane, and drugs with high lipid solubility are easy to pass through the stratum corneum. After the drug passes through the stratum corneum, it is then dispensed into the active epidermis. Therefore, the oil-water distribution coefficient is close to 1, and the transdermal permeability coefficient is better. Ionic drugs are not easily absorbed through the skin because of their strongly hydrophilic nature, which is determined by the difficulty in diffusing through the lipid cell gap.

For lipid, nonpolar skin cell membranes, more liposoluble drugs are more likely to penetrate the skin.

The inventor of the present invention found through related researches that Ding Xiansai molol with a more optimized chemical structure than timolol can promote the external medicine treatment of infant hemangioma, especially mixed or deep focus.

Disclosure of Invention

The invention aims to provide an application of timolol to preparation of a medicine for treating shallow phenotype, mixed type or deep infant hemangioma, aiming at the fact that the existing timolol cannot penetrate deeply into skin.

Preferably, in the uses described herein, the drug is administered transdermally.

More preferably, the drug is transdermally administered at a dosage concentration of 1-10mg/ml (i.e., 0.1% -1.0%). The times are 2-4 times daily, and the daily dosage of the hemangioma is 1-5 mg/square centimeter according to the surface areas of different hemangiomas of individuals.

The invention has the advantages that the Ding Xiansai molol can be used for treating all types of infant hemangiomas, in particular to mixed or deep infant hemangiomas with poor curative effect of the traditional timolol external preparation, and the skin permeation quantity is high; the drug concentration in the skin is high; has strong lipid-forming ability and promotes the regression of hemangioma.

Drawings

Figure 1 shows a graph of cumulative drug release for in vitro transdermal tests of timolol maleate, ding Xiansai molol. Wherein timolol maleate (intact skin), ding Xiansai timolol maleate (exfoliating skin), ding Xiansai timolol (exfoliating skin) are sequentially arranged from bottom left to top right.

FIG. 2 shows plasma drug concentration time profiles of timolol maleate, ding Xiansai molol

FIG. 3 shows the results of plasma drug concentration mass spectrometry detection of timolol maleate and Ding Xiansai molol

FIG. 4 shows the results of the skin drug concentration mass spectrum detection of timolol maleate and Ding Xiansai molol

Figure 5 shows the results of an apoptosis experiment of timolol maleate and Ding Xiansai molol on hemangioma endothelial cells. Wherein, the blank control is timolol maleate (100 uM), ding Xiansai timolol (100 uM) from left to right.

Figure 6 shows timolol maleate and Ding Xiansai molol oil red staining results. Wherein, HUVEC cells are induced to form lipid sequentially from top left to bottom right, 10uM timolol maleate induces HemSC,100uM timolol maleate induces HemSC, and 10uM Ding Xiansai timolol induces HemSC.

Detailed Description

Experiment raw material medicine sources:

timolol maleate is purchased from USP catalyst #1667406;

ding Xiansai molol is purchased from MCE, cat.No.: HY-102032A.

Experimental examples

Experimental example one, ding Xiansai molol in vitro transdermal test

In this experimental example, female nude mice of 8 weeks old were sacrificed and their skin was taken for in vitro transdermal test.

(1) The test mice are killed by a cervical scission method, hair on the backs of the mice is cut off by scissors, and skin on the backs of the mice is cut off;

(2) Removing subcutaneous adipose tissues by scissors, and washing the skin by distilled water and normal saline in sequence;

(3) Wiping off water stains on the surface of the skin with filter paper, sticking the filter paper on the surface of the skin with an adhesive tape, tearing off the adhesive tape after slightly pressing, and repeating for 20 times to remove the cuticle of the skin;

(4) The treated skin was placed in a double-chamber osmotic diffusion device comprising a diffusion cell, a receiving cell and a thermostatic waterbath system. The solution of the receiving pool is 75% phosphate buffer solution/ethanol, the volume of the receiving pool is 1ml, the temperature is controlled at 37 ℃, and the diffusion area is 0.785cm ² ；

(5) The test design is divided into 4 groups as shown in Table 1, sampling is carried out at regular time, fresh receiving liquid is replaced, the absorption value is measured at 294nm by an ultraviolet spectrophotometer after the sample is filtered, and the accumulated release amount of the medicine in a certain time is calculated.

TABLE 1 in vitro test design grouping

In vitro transdermal tests were performed using a double diffusion cell permeation system, and the cumulative release of drug was calculated at regular intervals (see fig. 1). From the figure we can see that the penetration of Ding Xiansai molol is much higher than timolol maleate, both for exfoliating skin and for intact skin without exfoliating, indicating a stronger transdermal performance of Ding Xiansai molol.

Experimental example III, comparison of the adipogenic and apoptotic Functions of timolol maleate and Ding Xiansai molol in hemangioma endothelial cells and Stem cells

This experimental example used infant hemangioma endothelial cells (Hemangioma derived endothelial cells, hemECs) and stem cells (Hemangioma derived stem cells, hemSCs). The separation of HemECs and HemSCs from tissue was performed by CD31 and CD133 labeled magnetic beads, respectively. The infant hemangioma specimen is from the hemangioma of the proliferation stage of the surgical excision of the ninth people hospital affiliated to Shanghai university of traffic university medical school. The specimens were placed in 50ml centrifuge tubes, washed twice with PBS, stored at 5% BSA at room temperature, and transported to the laboratory. 5ml of the culture dish containing 3mM CaCl was placed in a dish ₂ HBSS (CaCl free) ₂ MgSO (MgSO) ₄ ) Placing the solution into a specimen, removing epidermis and subcutaneous fat by using tissue scissors, and shearing the sample into 1mm ³ After the cake, the mixture was placed in a 15ml centrifuge tube, 0.2% type I collagenase was added, and the mixture was digested with shaking at 37℃for 1 hour, centrifuged at 1300rpm for 3 minutes, the supernatant was discarded, and the mixture was repeatedly washed (PBS was used for resuspension and centrifugation) 2 times to separate hemangioma endothelial cells and stem cells from CD31 and CD133 immunomagnetic beads. Finally, the selected cells were resuspended in EGM-2 containing 10% FBS and 1% diabody and seeded on type I collagen (1:100) 6cm ² In a culture dish, 37 ℃,5% CO ₂ And (5) standing and culturing in an incubator.

1. Timolol maleate and experimental result of Ding Xiansai molol apoptosis in hemangioma endothelial cells

10 ten thousand HemEC were inoculated per well in six well plates and incubated for 24 hours with each of DMSO, 100uM timolol maleate, 100uM Ding Xiansai molol 1:1000 diluted serum-depleted EGM-2 culture broth for 48 hours. 48 hours after dosing, the samples were collected by digestion with 0.25% pancreatin, treated with FITC Annexin V (BD Pharmingen Cat.559763) flow-on apoptosis kit and tested on-line.

The results of Annexin V apoptotic flow cell experiments (fig. 5) suggest that timolol maleate and Ding Xiansai molol have insignificant effects of promoting HemEC apoptosis at the same concentration (100 uM).

2. Timolol maleate and experimental result of Ding Xiansai timolol induced hemangioma stem cell adipogenic differentiation

After 10 ten thousand HemSC were inoculated per well in six well plates, incubated for 48 hours or until the plates were full, adult adipose-derived mesenchymal stem cells were subjected to oil red staining after induction culture for 14 days with DMSO, 100uM timolol maleate, 10uM Ding Xiansai molol Cheng Zhi added to adult adipose-derived mesenchymal stem cell adipogenic differentiation medium (Cyagen, product No.: HUXMD-90031). Human umbilical vein endothelial cells (HUVEC, promocell, cat. No.: C-12203) were used as negative controls and cultured and examined under the same conditions. The oil red staining experiment (fig. 6) after dosing induction of adipogenesis by infant hemangioma stem cells (HemSC) suggests that HUVEC has no adipogenesis, whereas the low concentration of Ding Xiansai molol induced the adipogenesis by HemSC comparable to the higher concentration of timolol maleate. Therefore, on the cytology level, ding Xiansai molol has stronger adipogenic capacity and promotes the regression of hemangioma compared with timolol maleate which acts on the stem cells of the infantile hemangioma.

In conclusion, ding Xiansai molol can be used as a novel topical medicine for treating the focus of infant hemangioma located in deep subcutaneous part.

Claims

1. An application of timolol to the preparation of medicaments for treating shallow phenotype, mixed type or deep infant hemangioma,

2. the use of claim 1, wherein the medicament is administered transdermally.

3. The use according to claim 1 or 2, wherein the medicament is administered transdermally at a concentration of 1-10mg/ml.

4. The use according to claim 1 or 2, wherein the medicament is used in an amount of 1-5mg per square centimeter hemangioma per person per day.