CN1452956A - Application of hypocrellin photosensitizer in pharmacy - Google Patents
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Abstract
The present invention is the application of hypocrellin photosensitizer in pharmacy. Hypocrellin photosensitizer is used in preparing photodynamic medicine for treating nevus flammeus, senile macular degeneration and other blood capillary diseases.The said hypocrellin includes hypocrellin A, hypocrellin B and medicinal carrier. The present invention utilizes the photophysical and chemical features of hypocrellin photosensitizer, especially its maximum absorption spectrum value at 450-580 nm wavelength range, the light of which has effective penetrating depth of 1-2 mm to provide new clinical indications for the hypocrellin.
Description
Technical field
The present invention relates to the purposes of hypocrellin class photosensitizer, relate in particular to hypocrellin (HA) or the application of HB Hypocrellin B (HB) in pharmaceutical field.
Background technology
Photodynamic therapy (photodynamic therapy, abbreviation PDT) shows the human body photosensitizer administration, when medicine quick dose of illumination exciting light when focus has maximum distribution, in the presence of oxygen, produce active oxygen and other reactive intermediate, reach the purpose of treatment disease by selective killing or inhibition target cell.Photodynamic therapy begins to be used for the clinical diagnosis and the treatment of malignant tumor in later 1970s, the nineties begins to be used for the treatment of benign diseases such as nevus flammeus, degeneration of macula, atherosclerosis and psoriasis, being the first-selected Therapeutic Method of nevus flammeus at present, is unique effective therapy of degeneration of macula.
At present approved is used for clinical and is being optical dynamic therapy at tumor mostly at the photosensitizer of clinical experiment just, because tumor phototherapy window is 600nm-900nm, therefore requires the effective light absorption wavelength of photosensitizer at least more than 600nm.Because light is directly proportional to the penetration depth and the optical wavelength of tissue, effective treatment degree of depth of these photosensitizer is all more than 5 millimeters.Yet nevus flammeus is that a kind of congenital dilatation deformity sexually transmitted disease (STD) of high dermis blood capillary becomes; Degeneration of macula is a kind of Senile disease that new capillary vessel appears in optical fundus macula lutea choroid, and the pathological changes degree of depth of the two all is no more than 1mm.If use photosensitizer treatment nevus flammeus and the degeneration of macula of these light absorption wavelength more than 600nm, if adopt the LASER Light Source that is complementary with it to excite, then will cause the damage of deep layer normal structure, cause severe complications; If adopt the LASER Light Source about wavelength 530nm to shine to these photosensitizer, then cause the light dynamic efficiency low, thereby affect the treatment because of efficiency of light absorption is low.
Hypocrellin is the non-porphyrin class photoactive substance of a group of special product of China, and its structure, photosensitization character, structural modification and corresponding light power function have been had a large amount of reports (Jiang Lijin, He Yuying, Science Bulletin (summary), 45 (2000), 2019-2032; J.H.Ma, J.Q.Zhao, L.J.Jiang, Photochem.Photobiol., 2001,74,143-148 (inviting summary)).They have that easy purification, cost are low, photosensitizer triplet productive rate and creating singlet oxygen by using quantum yield height, phototoxicity height, dark toxicity are low, from advantages such as the removing speed of normal structure are fast.Up to now, mainly be optical dynamic therapy about the research of hypocrellin clinical indication at tumor, some antiviral experiments reports and treatment psoriasis, leukodermic application report are also arranged.Because the characteristics of hypocrellin absorption spectrum, effectively the treatment degree of depth can not finely satisfy the needs of entity tumor optical dynamic therapy, with novel porphyrin, that the phthalocyanines photosensitizer is compared advantage is not obvious, it is developed to useful clinically smooth power antitumor class medicine certain difficulty.Therefore the antitumor light power effectiveness research of hypocrellin still rests on the basic research stage at present.And hypocrellin as phototherapy medicament treat psoriasis, leukodermic clinical efficacy does not gain public acceptance as yet.
Summary of the invention
The object of the invention is to provide the new purposes of hypocrellin class photosensitizer, i.e. new application in pharmacy.
Relate to Hypocrella bambusae (Bet Br). Sace class photosensitizer as the application in the medicine of preparation optical dynamic therapy shallow-layer microvascular disease.
Further, be specifically related to Hypocrella bambusae (Bet Br). Sace class photosensitizer as the application in the medicine of preparation optical dynamic therapy nevus flammeus disease.Relate to Hypocrella bambusae (Bet Br). Sace class photosensitizer as the application in the medicine of preparation optical dynamic therapy agedness yellow spot degenerative disease.Also relate to the application in the medicine of newborn venule disease of esophagus that Hypocrella bambusae (Bet Br). Sace class photosensitizer brings out as preparation optical dynamic therapy portal hypertension, gastric mucosa.
The present invention makes full use of the optical physics and the photochemical properties of hypocrellin class photosensitizer, promptly utilize the characteristics of its absorption spectrum maximum at 450nm-580nm, and the Effective depth penetration of this wave-length coverage light is about the characteristic of 1-2 millimeter, maximize favourable factors and minimize unfavourable ones, for hypocrellin class photosensitizer provides new clinical indication.
In order to understand essence of the present invention better, to give detailed description to the concrete using method and the mechanism of action used in the medicine of Hypocrella bambusae (Bet Br). Sace class photosensitizer as preparation treatment shallow-layer microvascular disease below, and with the pharmacological testing of hypocrellin class photosensitizer and its new function in pharmaceutical field of presentation of results and new purposes.
Concrete using method:
1, photosensitizer medication: should select thicker vein, prevent that medicinal liquid from leaking into outside the blood vessel., push away Bi Houying and push several ml physiological salines again keeping away direct intravenous injection under the high light condition with photosensitizer stock solution (or through suitably dilution), make injection place intravascular drug lowering of concentration, prevent that this place from photosensitivity reaction taking place.Photosensitizer all need be avoided exposing under light or daylight of a specified duration in preparation, preservation and injection process, in order to avoid reduce drug effect.
2, photosensitizer: dosage is 0.1～1mg/kg, and optimal dose is 0.2～0.6mg/kg.
3, laser instrument: can use following several laser: argon laser, as the AURORA/M type argon ion pump dye laser that U.S. Coorper company produces, output wavelength is 488.0nm and 514.5nm, continuously output.KTP/532 laser, as the JY-B type frequency multiplication YAG laser instrument that China's Ministry of Information Industry's electronics the 11 institute is produced, output wavelength 532nm, pulse output, pulse frequency 1～10KHz, pulsewidth 200～300ns.Copper vapor laser, as the IECu-10 type copper vapor laser that IEAS produces, output wavelength 510.6nm～578.2nm, pulse output, pulse frequency 6KHz, pulsewidth 20～40ns.
4, laser irradiating method: give laser irradiation simultaneously in the photosensitizer intravenous injection.Laser is exported vertical irradiation during the treatment nevus flammeus by optical fiber; To note adjusting irradiating angle in the irradiation process, make every effort to whole treatment region and obtain uniform irradiation; The big person of pathological changes area can once successively treat two focal zones, i.e. two hot spot treatments.Need to focus on the direct irradiation macular area through slit lamp during the treatment age-related macular degeneration.
5, exposure dose: irradiation power density is 30～150mW/cm
2, optimum power intensity is 80～100mW/cm
2Energy density is 80～300J/cm
2, optimum capacity density is 100～200J/cm
2The variation of required horsepower meter monitoring output in irradiation front and back and the irradiation process.
6, lucifuge: promptly should avoid daylight or accent light irradiation whole skin after in the photosensitizer input body, generally need lucifuge 2～3 days, after this can increase gradually and be subjected to light intensity, as not have photosensitive reaction and can recover irradiation orthobiosis.
The mechanism of action:
Hypocrella bambusae (Bet Br). Sace class photosensitizer is through forming the concentration peak immediately in blood after the intravenous injection, and absorbed rapidly by vascular endothelial cell, this moment, epidermal layer cells and retina cell absorbed still seldom, thus photosensitizer to be distributed between vascular endothelial cell and epidermal layer cells and the retina cell formation significant concentration poor.Penetrate that table is shallow, the laser irradiation of the specific wavelength that can be absorbed by photosensitizer selectivity in the blood this moment; make photosensitizer produce phototoxicity materials such as singlet oxygen; the pathological changes capillary network that is rich in photosensitizer is destroyed by selectivity; and be covered in the online normal epidermis layer of pathological changes blood capillary and retina injury-free because of not containing photosensitizer; it is then shallow because of laser penetration to be positioned at pathological changes blood capillary normal deep tissues off the net, is difficult to reach effective booster dose and is protected.
In addition, the photobleaching effect of photosensitizer also helps the photosensitizer concentration that enlarges between vascular endothelial cell and epidermal layer cells and the retina cell poor, improves the tissue selectivity of photodynamic therapy.Because when photodynamic therapy treatment nevus flammeus and age-related macular degeneration, photosensitizer intravenously administrable and laser irradiation are carried out simultaneously, blood Chinese medicine concentration is higher.Vascular endothelial cell directly contacts blood flow, and cell surface is long-pending big, and is rapid to sensitiser absorption, and the photosensitizer that is consumed by photobleaching in the cell can obtain replenishing fast, so photobleaching generally can not weaken PDT to injury of vascular endothelial cells intensity.Epidermal layer cells and retina cell are away from lumen of vessels, and photosensitizer need could arrive epidermal area and retina by the indirect diffusion of tissue fluid, and photosensitizer replenishes speed and significantly is lower than vascular endothelial cell.If, just not having photosensitizer greater than the diffusion rate of photosensitizer in epidermal area and the retina, the photobleaching speed of photosensitizer do not exist.Therefore, when PDT treatment nevus flammeus and age-related macular degeneration, the photobleaching speed that improves photosensitizer can make epidermal layer cells and retina cell be protected more fully.
The optical dynamic therapy microvascular disease is to the requirement of photosensitizer: maximum absorption spectrum is at 450nm-550nm; Singlet state quantum yield height; Dark toxicity is low; Vascular endothelial cell absorbs fast; Photobleaching speed height; The lucifuge phase is short.
Hypocrellin class photosensitizer of the present invention comprise a, its structure of hypocrellin (HA) as shown in Figure 1, its structure of HB Hypocrellin B (HB) as shown in Figure 2, it can also be their modified derivative, b, pharmaceutically suitable carrier also can contain biosurfactant (cholesterol etc.) and/or synthetic surfactant (spans, Tweens and Pu Luonike class etc.).
Description of drawings
Fig. 1 is the structure chart of hypocrellin (HA).
Fig. 2 is the structure chart of HB Hypocrellin B (HB).
Fig. 3 is that hypocrellin (HA) is at liposome (HA-EPC) with at chloroform (HA-CHCI
3) in abosrption spectrogram.
Fig. 4 is that HB Hypocrellin B (HB) is at liposome (HB-EPC) with at chloroform (HB-CHCI
3) in abosrption spectrogram.
Fig. 5 is that hypocrellin (HA) is at liposome and the fluorescence spectrum figure in chloroform.
Fig. 6 is that HB Hypocrellin B (HB) is at liposome and the fluorescence spectrum figure in chloroform.
The specific embodiment
Embodiment 1-2
Extracting method is as follows:
1, the extraction of hypocrellin (HA)
Extracting method was rolled up the article that 252-254 page or leaf Zhao opens great " organic chemistry " with reference to 1989 9, had done some improvement on the method.Concrete grammar is as follows: 20g pulverizes Hypocrella bambusae (Bet Br). Sace and places the Soxlet extractor, extracts about 4 hours (to extracting liquid colourless) continuously with 500ml chloroform give solvent, and extracting solution extracts repeatedly with 4 * 100ml water.Tell chloroform layer, extract chloroform on rotary evaporator, the gained solid is with 8 * 25ml petroleum ether, this solid natural air drying in air, use chloroform-petroleum ether recrystallization twice then, the gained crystal is target product hypocrellin (HA), and purity is up to 98%.Utilize thin layer chromatography (silica gel G is made 1% citric acid plate, and with petroleum ether: ethyl acetate: dehydrated alcohol=30: 10: 1v/v does developing solvent) can reach the purpose that is further purified.
2, the preparation of HB Hypocrellin B (HB)
HB Hypocrellin B (HB) is obtained by the plain dehydration of first, and preparation method reference literature Zhao opens the article of great " organic chemistry " 1989 9 volumes 252-254 page or leaf.Concrete grammar is as follows: 22mg first element is dissolved in the potassium hydroxide aqueous solution of 22ml 1.5%, after lucifuge stirs 24h, with excessive slightly dilute hydrochloric acid neutralization.Use the chloroform extract product, get 21mg second element, productive rate 99% after separation is purified.
3, the purification of lecithin
The article (W S.Singleton et al., J.Am.Oil.Chem.Sci.42,53-56,1965) of method of purification list of references " U.S.'s POL chemistry meeting will " nineteen sixty-five 42 volume 53-56 pages or leaves.Concrete grammar is as follows: crude product lecithin alumina column chromatography chromatography purification, eluent are chloroform: methanol=9: 1 (v/v), purity silica gel G F thin layer chromatography (chloroform: methanol: water=65: 25: 4 v/v; Rf=0.3) detect.The lecithin of purifying is dissolved in the chloroform, charges into argon 0 ℃ of preservation.
4, the preparation of HA-and HB-liposome
Certain density HA (or HB) and lecithin (concentration ratio=1: 16 (weight ratio)) chloroformic solution are placed round-bottomed flask, and chloroform is removed in distilling under reduced pressure, and mixture forms the layer of even thin film on wall, dry 15 minutes of argon.Add a certain amount of phosphate buffered solution (6.4mmol/L Na again
2HPO4,1.4mmol/L KH
2PO4,137mmol/L NaCl and 2.6mmol/LKCl, pH7.4) hydration is 30 minutes, and prepared liposome was 4 ℃ of preservations until clarification in ultrasonic 2 hours in 4 ℃ of ice-water baths and argon shield then.
5, the envelop rate and the stability of HA-liposome and HB-liposome
In the liposome that this membrane process makes, the retention volume ratio inverse evaporation and the injection method of hypocrellin and second element improve a lot, and maximum retention volume can reach 1.1-1.6mg/ml.HA-Liposome in this concentration range and HB-Liposome 4 ℃ deposited for two weeks after, HA or HB do not precipitate, its optical density does not change basically.Easily oxidized and HA or HB illumination in phosphate buffer easily produce the characteristics of creating singlet oxygen by using based on HA-Liposome, so HA-Liposome and HB-Liposome should be at N
2Middle protection is deposited under low temperature and the lucifuge condition.
Form novel form if in HA-liposome and HB-liposome, add biosurfactant (cholesterol etc.) with synthetic surfactant (spans, Tweens and Pu Luonike class etc.), can increase the maximum retention volume of HA and HB, improve HA-liposome and HB-liposome stability, can under 4 ℃, preserve at least three months.
6, the spectral characteristic of HA-liposome and HB-liposome
The spectral quality of HA-liposome and HB-liposome such as Fig. 3, Fig. 4, Fig. 5, shown in Figure 6.
As can be seen from Figure 3: the visible absorption spectra of HA in liposome solutions kept 3 northern quinoness characteristic absorption band (Ia, IIa, IIIa).With in chloroform, compare, red shift slightly (6nm) takes place to 472nm in the Ia peak, other two peak positions not have change substantially; Whole peak type broadens and makes the IIa peak become acromion simultaneously.This phenomenon can be according to the explanation of Frank-Condon principle, and the molecule that promptly is in ground state equilibrium state S0 is subjected to be excited to Frank-Condon excited state S behind the irradiation
1', excite equilibrium state S through non-radiative relaxing towards then
1, transit to Frank-Condon ground state S through fluorescent radiation again
0', at last again by S
0' relax towards ground state equilibrium state S
0, this non-radiative relaxation mainly originates from the orientation of molecule in the solvent cage and the interaction between solvent and solute molecule, shows as the variation of peak type and position aspect absorption spectrum.HA is in after liposomal encapsulated in the hydrophilic layer, and water is less to the effect of HA, thereby can make the π π that comes from the northern quinone ring by the electrostatic interaction between dipole-dipole between HA and lecithin molecules
*The formed peak of electron transition red shift occurs and the peak type broadens.Because hydrone is less to the effect of HA, therefore derive from the absorption band IIa of HA intramolecularly proton transfer and the position of IIIa remains unchanged; Simultaneously IIa and IIIa band absorbs slightly and increases, this be since HA with limited the molecular motion of HA to a certain extent after liposome combines, and strengthened the cause of intramolecularly proton transfer.
The absorption spectrum of HB-liposome is similar to the situation of HA-liposome as shown in Figure 4.
From Fig. 5, and in chloroform, to compare, fluorescence emission spectrum peak type and the peak position of HA in liposome do not have big variation, and emission peak is at 608nm and 645nm (acromion), but obvious reduction is arranged on intensity.Blank experiment shows, lecithin does not have fluorescence peak detecting wave band, and adding lecithin in the ethanol of HA and buffering solution can quencher HA fluorescence.The reduction of HA fluorescence intensity can consider it mainly is because the influence of aqueous buffer solution like this.Because although HA is in the hydrophobic double-layer of lipoid, because the phase inversion temperature of lecithin is-17 ℃, double-layer of lipoid is in " flowable state " under the room temperature, and system Semi-polarity water has quenching effect to HA fluorescence.
7, the optical sensibilization of HA-liposome and HB-liposome
In liposome solutions, HA, HB also can photosensitively produce
1O
2, O
2 -With
OH records
1O
2The quantum yield that produces is respectively 0.80,0.76, with HA and HB in benzene
1O
2Quantum yield is close, illustrates that HA and HB have kept in liposome
1O
2The generation ability.Can detect O with the spin trapping method
2 -With
OH and DMPO,
1O
2The epr signal of the adduct that forms with TEMP.If add electron donor, the HA-liposome of illumination anaerobic or HB-liposome solutions can detect the plain hydroquinone (HAH of first with absorption spectrometry
2) and the plain hydroquinone (HBH of second
2) generation, the spin method can detect HA with disappearing
-Or HB
-Generation, but be difficult to detect HA with absorption spectrometry and EPR method
-Or HB
-Epr signal, this is owing to be unfavorable for HA in liposome solutions
-Or HB
-Generation and stability.
The present invention's HA-liposome and HB-liposome can be used as the photo-dynamical medicine of treatment specific blood vessels disease nevus flammeus and degeneration of macula.
HA-liposome and HB-liposome are as the configuration of treatment nevus flammeus and degeneration of macula pharmaceutical agent: HA-liposome of the present invention and HB-liposome buffer solution can be prepared into injection with traditional method, get the storing solution (concentration is 1 mg/ml) of certain amount of H A-liposome or HB-liposome before the use, being diluted to normal saline or phosphate buffer needs concentration as injection liquid.
Pharmacology and toxicological experiment are as follows:
One, cell pharmacological evaluation
1, experiment material
Cell is Mus pulmonary vascular endothelial cell system (1H11); Photosensitizer is HA, HB, HpD; Laser instrument is the IECu-10 type copper vapor laser that IEAS produces, output wavelength 510.6nm～578.2nm, pulse output, pulse frequency 6KHz, pulsewidth 20～40ns.
2, experimental technique
This experiment is be target cell with the Mus pulmonary vascular endothelial cell of In vitro culture system (1H11), hatches with the different photosensitizer of itself and variable concentrations, under saturated copper vapor laser dosage, with the medium casaulty dosage of mtt assay mensuration photosensitizer.
3, experimental result
Under the saturated illuminate condition of 510nm laser, the half of HA, HB, HpD kills and wounds drug level (IC50) and is respectively: HA=17.87ng/ml, HB=41.91ng/ml, HpD=1135ng/ml; Under the saturated illuminate condition of 578nm laser, the IC50 of HA, HB, HpD is respectively: HA=28.82ng/ml, HB=81.70ng/ml, HpD=2045ng/ml.This result shows that under 510.6nm and 578.2nm illuminate condition, the cell light power killing-efficiency of HA is high 63.51 times and 70.96 times than HpD, and the cell light power killing-efficiency of HB is high 27.08 times and 25.03 times than HpD.
Two, the animal pharmacology of nevus flammeus experiment
Animal: the prosperous chicken of Lay, provide by animal housing of Chinese People's Liberation Army General Hospital, hatching growth then, the cockscomb development condition is good, and body weight is about 1 kilogram.
Medicine: HB, HPD.
Method: the prosperous chicken intramuscular injection fiber crops of Lay protect quiet anesthesia (0.10～0.12mg/kg), before the irradiation under the lucifuge condition with original content intravenous injection photosensitizer.Fixing cockscomb, the labelling illumination area selects the copper vapor laser mixed light to shine, and power density is 100mw/cm2, irradiation time 20min.By optical fiber output vertical irradiation, spot diameter is 1cm, and the irradiation place does not suitably cover, 4 hot spots of every chicken irradiation.All detect output before and after the irradiation, guarantee that its fluctuation range is less than ± 5%.Each is organized in handling color and the surface condition that every day is carried out perusal, recording processing position cockscomb in the back.Cut 2 illumination area cockscombs, film-making, light microscopic and transmission electron microscope observing histo pathological change in back 3 days of processing and 14 days every animals.
Result: according to the criteria for classifying of PDT effect back cockscomb skin morphological change, in the experiment cockscomb skin target tissue and non-target tissue are divided into gently (L to the reaction of PDT, lightrecation), (M, middle recation), heavy (S, sever recation) three degree in.See the following form
Table 1 HB and HPD are to the comparison of the photosensitive injury characteristic of cockscomb skin target tissue
| Photosensitizer dosage (mg/kg) | Laboratory animal number (only) | Target tissue | |
| Change substantially | Mirror changes down | ||
| ??L????M????S | ????L????M????S | ||
| ??HB??0 ??HB??0.25 ??HB??0.5 ??HB??0.75 ??HPD?10 | ????4 ????16 ????16 ????16 ????16 | ??0????0????0 ??12???4????0 ??0????7????9 ??0????0????16 ??0????0????16 | ????0????0????0 ????8????8????0 ????0????5????11 ????0????0????16 ????0????0????16 |
Through X
2P value＜0.05 is shown in check, illustrates that there were significant differences between the different disposal condition, and the HB group is clear and definite to target tissue agent magnitude relation.
Table 2 HMME and HPD are to the comparison of the photosensitive injury characteristic of cockscomb skin non-target tissue
| Photosensitizer dosage (mg/kg) | Laboratory animal number (only) | Non-target tissue | |
| Change substantially | Mirror changes down | ||
| ?L?????M????S | ??L????M?????S | ||
| ??HB??0 ??HB??0.25 ??HB??0.5 ??HB??0.75 ??HPD?10 | ?????4 ????16 ????16 ????16 ????16 | ?0?????0????0 ?16????0????0 ?16????0????0 ?8?????8????0 ?10????6????0 | ??0????0?????0 ??16???0?????0 ??14???2?????0 ??3????11????2 ??6????10????0 |
Through X
2P value＜0.05 is shown in check, illustrates that there were significant differences between the different disposal condition, and the HB group is clear and definite to non-target tissue's agent magnitude relation.
This shows that HB HPD to the degree of injury of target tissue and 10mg/kg under 0.5-0.75mg/kg administration condition is suitable, but the degree of injury of non-target tissue significantly is lower than HPD.What this showed HB is significantly higher than HPD in the effect of body optical dynamic therapy, and tissue selectivity is good simultaneously, and non-special damage is light.
Attached: the criteria for classifying of PDT effect back cockscomb skin morphological change
1, target tissue
(1) changes target tissue substantially the reaction perusal of PDT is shown as change in color.Mild reaction: at once, the cockscomb surface is the redness of slightly deepening behind the PDT; 1～3 day, color restoration was normal.The moderate reaction: at once, the cockscomb surface is lilac behind the PDT; 1～3 day, be purple white based on white; In 1～2 week, be white in color.The severe reaction: at once, the cockscomb surface is dark darkviolet behind the PDT; 1～3 day, be purple white based on purple; In 1-2 week, be purple white based on white.
(2) mirror changes mild reaction down: 3 days papillary layer vascular endothelial cell mild swellings behind the PDT; Form recovers normal during 2 weeks.The moderate reaction: the papillary layer change is reacted (as follows) with severe substantially in the time of 3 days, but red cell agglutination and microthrombus are less; The papillary layer capillary network reduces during 2 weeks, and tube chamber is little, visible new capillary vessel.Severe reaction: in the time of 3 days all visible capillary lumen of papillary layer dwindle, locking, intracavity has coagulation, cracked erythrocyte and microthrombus; The papillary layer capillary network reduces in a large number during 2 weeks, and tube chamber is little, and intracavity has more thrombosis.
2, non-target tissue
(1) changes non-target tissue substantially the reaction perusal of PDT is shown as hydroderma, ulceration, incrustation and cicatrization.Mild reaction: Mild edema.Moderate reaction: in 1～2 week behind the PDT, have to be dispersed in incrustation on a small quantity.Severe reaction: behind the PDT 1～3 day, the cockscomb ulceration; 1～2 week, large tracts of land incrustation, cicatrization.
(2) mirror changes down mild reaction: 3 days the time, the epidermal area basal cell has the degeneration that is dispersed in behind the PDT; Each layer of skin do not have change during 2 weeks.Moderate reaction: 3 days the time, the epidermal area basal cell has degeneration, the necrosis that is dispersed in behind the PDT, lamina reticularis blood vessel and the expansion of corium deep-level blood vessel, but tube wall does not have degeneration; Epidermal area and corium deep layer do not have change, lamina reticularis vasodilation during 2 weeks.Severe reaction: behind the PDT 3 days, the epidermal area basal cell had focal degeneration, necrosis, lamina reticularis vasodilation, tortuous, and microthrombusis, the slight hyaline degeneration of tube wall, a small amount of lymphocytic infiltration, the corium deep-level blood vessel is obviously expanded; During 2 weeks, epidermal area has focal necrosis and epidermal cell proliferation, and lamina reticularis still has vasodilation, microthrombusis and tube wall degeneration, a small amount of collagen fiber degeneration, hypertrophy and lymphocytic infiltration, and the expansion of corium deep-level blood vessel has thrombosis in indivedual blood vessels.
Three, the animal pharmacology of degeneration of macula experiment
Animal: 30 of livid purple blue rabbits, body weight 2.5～3.5kg, male and female are not limit, and are provided by Chinese People's Liberation Army General Hospital.
Medicine: HB.
Method: the abundant mydriasis of compound recipe N-ethyl-N-(.gamma.-picolyl)tropamide, use fiber crops to protect clean 0.1mg/kg intramuscular injection, after the general anesthesia rabbit is fixed on slit lamp microscope before, three mirror contact lens is selected the irradiation position down.Auricular vein injection HB 0.5,1 or 1.5mg/kg give the 532nm laser irradiation, power density 50～600mW/cm behind the injection photosensitizer immediately
2, energy density 12～144J/cm
2, spot diameter 1500～2000 μ m.Treat laggard ophthalmoscope observation, fluorescence optical fundus radiography and the histology of connecing in the ranks.
The result:
1, simple laser irradiation matched group: power density 800mW/cm
2, irradiation time 500s, energy density 400J/cm
2, fundus observation, fluorescence optical fundus radiography and histology do not see that retina and choroid change under the ophthalmoscope.
2, simple medicine matched group: HB1.5mg/kg, fundus observation, fluorescence optical fundus radiography and histology do not see that retina and choroid change under the ophthalmoscope.
3, PDT treats 1 group: HB0.5mg/kg, power density 60mW/cm
2, irradiation time 240s, energy density 14.4J/cm
2, choriocapillary is not had tangible light power black-out effect.
4, PDT treats 2 groups: HB1.0 or 1.5mg/kg, power density 80～130mW/cm
2, energy density 18～30J/cm
2, ophthalmoscope is observed, behind the PDT at once retina do not have obvious color change, retina white after PDT1 days, opinion property enhancing thoroughly, below choroidal artery footpath slightly narrows down, surperficial pigmentation, idol has part detachment of retina or retinal edema; See the choriocapillary obturation under the optical microscope, the acromere arrangement disorder, vacuolation, the external granular layer attenuation that has, the cell pyknosis, the RPE cellular layer that has is arranged and is lost normal seriality; Fluorescence optical fundus radiography is observed, and after PDT1 days, low fluorescence occurs at irradiation area, and low fluorescence scope and irradiation scope area are approaching, hold high fluorescence around low phosphor region.
5, PDT treats 3 groups: HB0.5mg/kg, power density 800mW/cm
2, irradiation time 300S, energy density 240J/cm
2, retina is strong white, and FFA radiography seepage is obvious, and the illumination position is high fluorescence.The histology sees RPE layer arrangement disorder, the pyknosis of external granular layer cell, and thrombosis also appears in trunk.
Four, the animal pharmacology experiment of being carried out for treatment esophagus, the newborn venule of gastric mucosa
Animal: 20 of purebred New Zealand white rabbit, about 2 kilograms of body weight, male or female, animal housing of Chinese People's Liberation Army General Hospital provides.With the auricular vein is object of study.
Medicine: HB, HpD
Method: laboratory animal intravenous injection ketamine (10mg/kg) anesthesia, the ears depilation exposes auricular vein.Promptly shine the plan sealing blood vessels behind intravenous injection HB or the HMME with the copper vapor laser mixed light.By optical fiber output vertical irradiation, 1.5 centimetres of irradiates light spot diameters, the irradiation place does not suitably hide.Irradiation treatment back animal lucifuge is raised.
Result: the comparison photosensitizer dosage power density irradiation time blood vessel enclosed of table 3 HB and HpD sealing blood vessels situation
(mg/kg)????(mW/cm
2)???(min)????????(％)HB?????????1???????????150????????5??????????100HB?????????1???????????150????????10?????????100HpD????????20??????????150????????20?????????100HpD????????10??????????150????????20?????????50HpD????????5???????????150????????20?????????0
By table 3 as seen, under dosage 1mg/kg, 5～10 minutes condition of irradiation, HB is suitable with HpD 20mg/kg to venular sealing process, significantly is better than the effect of HpD10mg/kg and HpD5mg/kg.HpD presses the maximum that the 20mg/kg administration has surpassed clinical application, no practical value.
Five, acute toxicity testing
Animal: 10 of SD rats, about body weight 200 grams, animal housing of Chinese People's Liberation Army General Hospital provides.
Medicine: HA, HB.
Method: the rat tail vein injection concentration is the HA and the HB of the liposome of 0.8mg/ml.
The result: maximum dosage-feeding reaches 50mg/kg, because the qualification of circulation volume escalated dose again.Administration does not have the overt toxicity reaction at once and takes place, and is good through the ordinary circumstance of 24 hours observation rats, also do not have obvious acute toxic reaction.The result shows, HA and HB when 50mg/kg (optimal therapeutic dosage 83～250 times) do not have obvious acute toxicity.
Six, skin light poison experiment
Animal: 42 of NIH mices, about body weight 30 grams, male or female, animal housing of Chinese People's Liberation Army General Hospital provides.
Medicine: HB.
Method: 42 of mices are divided into 7 groups, every group 6, not administration of matched group, all the other 6 groups of tail vein injection HB1mg/kg, after administration at once, 1,2,3,5,7 day at noon 12:00～14:00 time gave the nature solar radiation 2 hours, observe the skin phototoxic reaction.
The result:
1, matched group does not have obvious restlessness performance, reactions such as that ear, four-footed and afterbody do not have is rubescent, swelling.
2, organize restlessness at once, frequently lick and lick front foot, ear, four-footed and afterbody are rubescent, swelling is obvious.
3,1 day group is uneasy, licks and licks front foot, and ear, four-footed and afterbody have rubescent, swelling.
4, organized slightly restlessness in 2 days, ear, four-footed and afterbody are slightly rubescent, swelling.
5,3 days groups do not have reactions such as obviously restless, that ear, four-footed and afterbody do not have is rubescent, swelling.
6,5 days groups do not have reactions such as obviously restless, that ear, four-footed and afterbody do not have is rubescent, swelling.
7,7 days groups do not have reactions such as obviously restless, that ear, four-footed and afterbody do not have is rubescent, swelling.
From above result, the object of the invention advantage is as can be known:
With the optical dynamic therapy method of HpD relatively, the present invention treats the microvascular disease such as nevus flammeus and has following characteristics:
1, photodynamic action is strong, and curative effect is high, thereby can make treatment time greatly shorten (estimating only to use 1/5 time), increases substantially clinical treatment efficiency and alleviates the misery of patient during treating.
2, the larger reduction of sensitising agent dosage (estimate approximately 1/40), therefore the Photobleaching in laser irradiation area is fast, tissue selectivity is good, shows as non-target tissue's damage light, after treatment, bad reaction is few.
3, metabolism is fast in vivo for sensitising agent, and the lucifuge phase is short, generally only 2～3 days.
4, degree of safety is large, shows as the safe range of sensitising agent dosage and light dosage greater than the HpD method.
5,, without allergic reaction, do not need to do hypersensitive test.
6, dark toxicity is low.
Claims (4)
1. hypocrellin class photosensitizer is as the application in the medicine of preparation optical dynamic therapy shallow-layer microvascular disease, and described hypocrellin class photosensitizer comprises a, hypocrellin (HA), HB Hypocrellin B (HB), b, pharmaceutically suitable carrier.
2. according to the application of claim 1, it is characterized in that: described shallow-layer microvascular disease is the nevus flammeus disease.
3. according to the application of claim 1, it is characterized in that: described shallow-layer microvascular disease is an agedness yellow spot degenerative disease.
4. according to the application of claim 1, it is characterized in that: described shallow-layer microvascular disease is the esophagus that brings out of portal hypertension, the newborn venule disease of gastric mucosa.
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| CN 03109776 CN1452956A (en) | 2002-04-22 | 2003-04-18 | Application of hypocrellin photosensitizer in pharmacy |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100387987C (en) * | 2004-12-29 | 2008-05-14 | 南京师范大学 | Method for determining hypocrellin A content by high efficiency liquid phase chromatographic method |
| CN102138641A (en) * | 2011-01-20 | 2011-08-03 | 江南大学 | Application of perylenequinone compound to preparation of feed for preventing and controlling virus of aquatic animals |
| CN101710070B (en) * | 2009-12-07 | 2011-09-21 | 昆明振华制药厂有限公司 | High performance liquid chromatography method for measuring content of hypocrellin A |
| CN112279757A (en) * | 2019-07-11 | 2021-01-29 | 中国科学院化学研究所 | Perylene quinone compound and preparation method and application thereof |
-
2003
- 2003-04-18 CN CN 03109776 patent/CN1452956A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100387987C (en) * | 2004-12-29 | 2008-05-14 | 南京师范大学 | Method for determining hypocrellin A content by high efficiency liquid phase chromatographic method |
| CN101710070B (en) * | 2009-12-07 | 2011-09-21 | 昆明振华制药厂有限公司 | High performance liquid chromatography method for measuring content of hypocrellin A |
| CN102138641A (en) * | 2011-01-20 | 2011-08-03 | 江南大学 | Application of perylenequinone compound to preparation of feed for preventing and controlling virus of aquatic animals |
| CN102138641B (en) * | 2011-01-20 | 2013-04-03 | 江南大学 | Application of perylenequinone compound to preparation of feed for preventing and controlling virus of aquatic animals |
| CN112279757A (en) * | 2019-07-11 | 2021-01-29 | 中国科学院化学研究所 | Perylene quinone compound and preparation method and application thereof |
| CN112279757B (en) * | 2019-07-11 | 2022-03-29 | 中国科学院化学研究所 | Perylene quinone compound and preparation method and application thereof |
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