CN103893167A - Podophyllotoxin preparation resisting condyloma acuminata relapse and HPV latent infection - Google Patents
Podophyllotoxin preparation resisting condyloma acuminata relapse and HPV latent infection Download PDFInfo
- Publication number
- CN103893167A CN103893167A CN201310489910.6A CN201310489910A CN103893167A CN 103893167 A CN103893167 A CN 103893167A CN 201310489910 A CN201310489910 A CN 201310489910A CN 103893167 A CN103893167 A CN 103893167A
- Authority
- CN
- China
- Prior art keywords
- podophyllotoxin
- hpv
- nano
- preparation
- lipid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Abstract
The invention relates to a podophyllotoxin preparation resisting condyloma acuminata relapse and HPV latent infection, which is mainly used for treating condyloma acuminate of a cervix uteri, a vagina, a urethra, an anal tube mucosa and skin, HPV latent infection and the like. The podophyllotoxin preparation disclosed by the invention takes a nanostructured lipid carrier as a drug carrier material; the packaged pharmaceutical active component is podophyllotoxin; the mass-to-volume ratio of podophyllotoxin to the nanostructured lipid carrier is 0.5%. The podophyllotoxin preparation disclosed by the invention has good pro-epidermal targeting property and high physical stability and is capable of thoroughly clearing HPV infection, controlling drug release, preventing a medicine from being degraded, increasing the drug-loading capacity, etc.
Description
Technical field
The invention belongs to pharmaceutical sanitary field, relate to a kind of podophyllotoxin preparation, especially relate to the podophyllotoxin preparation of a kind of anti-condyloma acuminatum recurrence and anti-HPV latent infection, be mainly used in treating condyloma acuminatum and the HPV latent infection etc. of cervix uteri, vagina, urethra, mucous membrane of anal canal and skin.
Background technology
Condyloma acuminatum (CA) is that human papillomavirus (HPV) infects one of modal sexually transmitted disease (STD) (STD) that anus, external genitalia position cause, rank first in American-European countries, in China, CA sickness rate is only second to gonorrhea, occupies the 2nd of sexually transmitted disease (STD).It is 100 many types of that the genotype of HPV reaches, and low risk comprises 6,11,40,42,43,44,54,61,70,72,51,12 kinds of ep6108 etc., and to become benign lesions such as (CIN1) relevant with tumor in external genitalia condyloma acuminatum and the low squamous epithelial cancer of cervix uteri; High-risk-type has 15 kinds, respectively HPV16,18,31,33,35,39,45,51,52,56,55,59,68,73 and 82, closely related with cervical cancer and cervix uteri height intraepithelial neoplasia (CINII, CINIII), especially HPV16,18 types, but still have some genotype due to epidemic data very limited and sort out on not yet reach common understanding.Epidemiological study shows that HPV has high infection rate, and property is enlivened in crowd, and the people of 75%-80% will infect HPV in its a certain period in life.In HPV infection population, women is far more than male.According to Cancer center of Britain investigation report, its cervix uteri of women or the vagina in the whole world approximately 1/5 carry HPV virus.
At present, the optimal method that treatment HPV infects is direct for virus or by the cell of viral infection, and virus is thoroughly removed from cell and tissue.But, owing to lacking effective antiviral means, and be difficult to thoroughly remove by the cell of viral infection, all there is higher relapse rate in traditional Therapeutic Method.It is reported, treat at present the recent cure rate of traditional means that HPV infects between 20%-94%, but the relapse rate for the treatment of in latter 3 months is generally 25%.
New Therapeutic Method, for example HPV vaccine or photodynamic therapy, injected new vitality for the control that HPV infects, and still, still there are many problems in these methods.The HPV vaccine of having succeeded in developing belongs to preventative vaccine, but can not prevent all types HPV to infect, the current two kinds of vaccines that have in U.S.'s listing, HPV tetravalent vaccine is (for HPV 6, 11, 16, 18) and HPV bivalent vaccine (for HPV 16, 18), they infect and invalid (the Smith G D of associated diseases existing disease, Travis L. Getting to know human papillomavirus (HPV) and the HPV vaccines[J]. J Am Osteopath Assoc, 2011, 111 (3 Suppl 2): S29-S34.), because HPV is easy to variation, therefore therapeutic vaccine is difficult to prove effective.Local light motivation therapy is making remarkable progress aspect treatment HPV infection, Nucci etc. think that this is a kind of efficient, the treatment anus of safety, method (the Nucci Vm of external genitalia condyloma acuminatum, Torchia D, Cappugi P. Treatment of anogenital condylomata acuminata with topical photodynamic therapy:report of 14 cases and review[J]. Int J Infect Dis, 2010, 14 Suppl 3:e280-e282.), but it is expensive, easily cause mucosa to fester and secondary infection, light power is studied still at heuristic process for the treatment of vagina and urethra HPV infection at present simultaneously.
Podophyllotoxin (Podophyllotoxin, POD) is from podophyllin lignans, to separate the remarkable Cytotoxic natural active matter that has obtaining.Be currently applied to clinical podophyllotoxin preparation, it is mainly tincture, ointment, gel etc., to the cure rate of condyloma acuminatum all in 90% left and right, but its relapse rate is all higher, (once anti-in 40% left and right, Li Guofeng, Xu Chongyuan, Deng, the double blind random controlled trial [J] of podophyllotoxin liposome Ointment in Treatment condyloma acuminatum, No.1 Military Medical Univ.'s journal, 1998, 18 (3): 246.), its local irritation is large, it is large that system absorbs toxicity, adverse reaction rate is at 40%-60% left and right (Zhang Wei, Qian Yihong, Zhang Gang, Deng, the efficacy analysis [J] of 0.5% podophyllotoxin treatment condyloma acuminatum, clinical department of dermatologry magazine, 2001 (3): 172-173.), thereby can not be applied to vagina, cervix uteri, urethra, the mucosal sites such as anal canal.Therefore,, although podophyllotoxin is the specific drug for the treatment of condyloma acuminatum, traditional podophyllotoxin preparation uses and is subject to great restriction in significant points such as vagina, cervix uteri, urethra, anal canals.
Solid lipid nanoparticle (SLN) is early 1990s a kind of active component carrier newly developed, is mainly the solid-state structure that solid grease solidifies in cooling or phase separation, crystallization forms, and active component is covered by lipid conformation.Research shows, it is few that POD-SLN has local untoward reaction compared with tincture, untoward reaction mild degree, the advantage (Zhang Min such as untoward reaction occurs late, and skin targeting is good, once anti-, Li Guofeng, etc., the safety comparison [J] of podophyllotoxin-solid lipid nanoparticle and the external of podophyllotoxin tincture percutaneous, China's Tissue Engineering Study and clinical rehabilitation, 2008 (1): 103-107.).Clinical trial finds that POD is after nanoparticle parcel, cure rate and conventional formulation are close, but relapse rate and adverse reaction rate obviously reduce, and respectively in 10%, 20% left and right (Xie Fangming, once anti-, Chen Zhiliang, Deng, the randomized controlled research [J] of Podophyllotoxin in Solid Lipid Nanoparticles gel for treating recurrent condyloma acuminatum, Nanfang Medical Univ's journal, 2007,27 (5): 657-659.).Name is called the Chinese invention patent application (application number: 200510111606.3 of " solid lipid nanoparticle is as the novel form of Podophyllotoxin and its derivatives carrier "; Publication number: CN1981755A) application using solid lipid nanoparticle as Podophyllotoxin and its derivatives drug carrier material disclosed.But the drug level of the podophyllotoxin solid lipid described in this application only has 0.05%, far can not meet the clinical practice of condyloma acuminatum, because being the World Health Organization (WHO), 0.5% podophyllotoxin recommends one of first-line drug for the treatment of condyloma acuminatum.And, adopt simple solid lipid to prepare SLN, gained nanoparticle may form crystallization fine and close as " brick wall ", and the space of nanoparticle medicine carrying is very limited, therefore the drug loading of podophyllotoxin solid lipid is lower, and medicine is easily squeezed leakage in storage process.Therefore, even the podophyllotoxin drug level of above-mentioned patent documentation is brought up to 0.5%, be still not suitable for the mucosa targeting preparation as POD, be not suitable for the treatment difficulty for solving the position such as vagina and cervix uteri condyloma acuminatum.
Summary of the invention
The object of the present invention is to provide a kind of novel podophyllotoxin preparation, this novel formulation has good close epidermis targeting and thoroughly removes the ability that HPV infects, break through the limitation that existing preparation can only be used for skin, can be applied to the mucosal sites such as vagina, cervix uteri as targeting preparation, be particularly useful for anti-condyloma acuminatum recurrence and anti-HPV latent infection, this is the not available new feature of existing podophyllotoxin preparation and new effect.
The podophyllotoxin preparation of anti-condyloma acuminatum recurrence of the present invention and anti-HPV latent infection, is taking nano-lipid carrier as drug carrier material, and its active constituents of medicine of sealing is podophyllotoxin; The mass volume ratio of podophyllotoxin and nano-lipid carrier is 0.5%.
According to the further feature of podophyllotoxin preparation of the present invention, described nanoparticle mean diameter is 180 ± 20nm, and average envelop rate is (82.9 ± 2) %, and pH is 6.20 ± 0.04.
According to the further feature of podophyllotoxin preparation of the present invention, described podophyllotoxin preparation is to be prepared as suspension.
According to the further feature of podophyllotoxin preparation of the present invention, the suspension of described podophyllotoxin preparation is prepared by following steps:
A. by podophyllotoxin, solid-state lipid, liquid lipid and emulsifiers dissolve in organic solvent, form oil phase; Surfactant dissolves, in water, is formed to water;
B. distinguish, continue to stir oil phase, make it fully dissolve post-heating to 74 ± 1 DEG C;
C. the product of abovementioned steps is at the uniform velocity added in the water of 74 ± 1 DEG C, stir 3～4 hours;
D. mixture of ice and water is added rapidly in the product of abovementioned steps, stop stirring, set to 0 in DEG C ice-water bath ultrasonic 30 minutes, adopt filtering with microporous membrane, obtain the suspension of described podophyllotoxin preparation.
According to the further feature of podophyllotoxin preparation of the present invention, the mixing speed in described step b and c is 1200-1500r/min.
Compared with the Podophyllotoxin in Solid Lipid Nanoparticles preparation of reporting; nano-lipid carrier (the Nanostructured lipid carriers that the present invention adopts; NLC) be the second filial generation lipid nanoparticle growing up at the beginning of 21 century on solid lipid nanoparticle basis, it is to have physiological compatibility and biodegradability, dystectic natural or synthesis of solid lipid and liquid fatty as the made nano medicament carrying system of framework material.The present invention is taking mixing lipid as carrier, pharmaceutical pack is wrapped in lipid film, there is the drug release of control, avoid the advantages such as drug degradation and good targeting, and, the present invention joins sour sad the liquid lipid under room temperature/certain herbaceous plants with big flowers triglyceride in solid-state lipid glyceryl monostearate, thereby the randomness of crystal increases, and makes carrier have higher crystal defect, thereby can improve drug loading, the physical stability of medicine, reduce medicine and in storage process, squeezed the possible of leakage.Therefore, the novel formulation of podophyllotoxin-nano-lipid carrier of the present invention has broken through the limitation that existing preparation can only be used for skin, especially can be applied to the treatment of condyloma acuminatum and the HPV latent infection of the mucosal sites such as vagina, cervix uteri.
Brief description of the drawings
Below in conjunction with drawings and Examples, podophyllotoxin preparation of the present invention is described in detail.
Fig. 1 is the preparation technology of podophyllotoxin preparation of the present invention (0.5% podophyllotoxin-nano-lipid carrier).
Fig. 2 is the outward appearance of podophyllotoxin preparation of the present invention (0.5% podophyllotoxin-nano-lipid carrier) suspension.
Fig. 3 is the Electronic Speculum picture of podophyllotoxin preparation of the present invention (0.5% podophyllotoxin-nano-lipid carrier).
Fig. 4 shows the particle size distribution of podophyllotoxin preparation of the present invention (0.5% podophyllotoxin-nano-lipid carrier).
Fig. 5 is the local mucous membrane irritant experiment result of podophyllotoxin preparation of the present invention (0.5% podophyllotoxin-nano-lipid carrier).
Fig. 6 shows the distribution of podophyllotoxin preparation of the present invention (0.5% podophyllotoxin-nano-lipid carrier) at pig cervical mucosal layer.
Fig. 7 shows the distribution of 0.5% podophyllotoxin tincture at pig cervical mucosal layer.
Fig. 8 shows the blood drug level of podophyllotoxin preparation (0.5% podophyllotoxin-nano-lipid carrier) suspension of the present invention and 0.5% podophyllotoxin tincture.
Fig. 9 shows the impact of podophyllotoxin preparation of the present invention (0.5% podophyllotoxin-nano-lipid carrier) on VK2/E6E7 cell proliferation.
Figure 10 shows podophyllotoxin preparation of the present invention (0.5% podophyllotoxin-nano-lipid carrier) and the impact of podophyllotoxin tincture on VK2/E6E7 cell cycle.
Figure 11 show 0.5% podophyllotoxin tincture and podophyllotoxin preparation of the present invention (0.5% podophyllotoxin-nano-lipid carrier) induce respectively VK2/E6E7 apoptosis form (Hoechst33342, dyeing, 400 ×).
Figure 12 shows podophyllotoxin preparation of the present invention (0.5% podophyllotoxin-nano-lipid carrier) and the apoptosis induction effect of POD to VK2/E6E7 cell.
Detailed description of the invention
Embodiment 1: the preparation of podophyllotoxin preparation of the present invention
The present embodiment will use orthogonal design optimization of C/C composites, adopt emulsifying evaporation-low-temperature setting legal system for 0.5% podophyllotoxin-nano-lipid carrier suspension, as shown in Figure 1.
Experiment material: podophyllotoxin (Sigma company, content 98.8%).
Concrete grammar is as follows:
Take 100mg lecithin and be dissolved in 20mL dehydrated alcohol, be placed in constant temperature blender with magnetic force, temperature is 74 ± 1 DEG C, is stirred to abundant dissolving, and mixing speed is 1200～1500r/min.Take 50mg podophyllotoxin, 100mg glyceryl monostearate and 400 μ L sad/certain herbaceous plants with big flowers acid triglyceride are dissolved in 10mL acetone, until completely dissolved, gained alcoholic solution are mixed to formation oil phase with acetone soln.
Take 200mg Brj(ionic surface active agent) be dissolved in 40mL tri-distilled water, be placed in constant temperature blender with magnetic force, temperature is 74 ± 1 DEG C, is stirred to abundant dissolving, mixing speed is 1200～1500r/min, forms water.
Oil phase is heated to, after 74 ± 1 DEG C, it at the uniform velocity be injected to the water of 74 ± 1 DEG C, continues to stir 3～4h, mixing speed is that 1200～1500r/min evaporates organic solvent completely, and makes solution be concentrated into about 5mL, forms light blue nano-emulsion.
The mixture of ice and water of 0 DEG C of 5mL is added in the nano-emulsion of gained fast, the ultrasonic 30min of ice-water bath, the filtering with microporous membrane of employing 0.22 μ m, obtaining concentration is 0.5% podophyllotoxin-nano-lipid carrier suspension.
Observe the outward appearance of this podophyllotoxin-nano-lipid carrier suspension, present translucent colloid (as shown in Figure 2) homogeneous, with light blue opalescence, under Electronic Speculum, podophyllotoxin-nano-lipid carrier is spherical or elliposoidal (as shown in Figure 3) substantially.
Podophyllotoxin-nano-lipid carrier the suspension obtaining by the method for the present embodiment, it is positioned under 4 DEG C of conditions, and sample still keeps translucent, light lacteous, homogeneous and be with light blue opalescence outward appearance within June, has no layering, precipitation and drug crystallization and separates out.Be positioned over the sample of room temperature, in 3 months, occur without floccule, precipitation, layering; 6 months time, there is a small amount of precipitation, layering, after vibration, can recover homogeneous outward appearance.There is not mould bacterial plaque in sample standard deviation.
Podophyllotoxin-nano-lipid carrier the suspension obtaining by the method for the present embodiment, detecting podophyllotoxin-nano-lipid carrier particle diameter through Malvern particle size analyzer is 180 ± 20nm, polydispersity index is 0.165(Fig. 4), distribution uniform.It is 6.20 ± 0.04 that PH instrumentation is determined podophyllotoxin-nano-lipid carrier suspension PH.
Podophyllotoxin-nano-lipid carrier the suspension obtaining by the method for the present embodiment, has measured the envelop rate of podophyllotoxin in three parts of podophyllotoxin-nano-lipid carrier suspensions, and its average envelop rate is (82.9 ± 2) %.Because podophyllotoxin is insoluble drug, the dissolubility in liquid fatty is large compared with solid lipid, and introducing liquid fatty can increase the envelop rate (in aforesaid CN1981755A patent, SLN envelop rate is 78.3%) of medicine.
Prepared podophyllotoxin-nano-lipid carrier the suspension of the present embodiment is by the experiment for following each embodiment.
Embodiment 2: podophyllotoxin preparation local mucous membrane irritant experiment of the present invention
1. experiment material
1.1 laboratory animals: 36 of SD female Tibet Mini-Pigs, at 6 monthly ages, quality is (25 scholar 3) kg, Nanfang Medical Univ's Experimental Animal Center provides.Experiment pig reaches clean laboratory animal standard.
1.2 Experimental agents:
Podophyllotoxin-nano-lipid carrier suspension: obtain by the method in embodiment 1.
Control drug podophyllotoxin tincture (self-control): take 0.05g podophyllotoxin with electronic balance, be dissolved in 10ml dehydrated alcohol, be mixed with 0.5% podophyllotoxin tincture.
2. coating method:
To test miniature pig and raise in Clean Facility, after 2 days, after miniature pig has adapted to new environment, start coating.Miniature pig sleeps after new (0.3ml/kg) anesthesia by anesthetis speed, and the fixing miniature pig of dorsal position, in operating board, is used disposable sterilized vaginal speculum to open pig vagina, chooses cervix uteri one week as coating district.36 experiment miniature pig random number, odd number is smeared 0.5% nano-lipid carrier podophyllotoxin suspension, and 0.5% podophyllotoxin tincture is smeared in even numbers, draws medicine with lml syringe, and every painting dose is 0.5ml, identical to ensure being coated with dose.Smear gently medicine approximately 20 seconds with little Glass rod, make drug distribution even.Respectively at this first 4,8,12,16,20 and 24 hours single dose coatings of label taking.
3. draw materials and film-making:
After coating finishes the last time, adopt air tap inserting method to put to death experiment pig, the mucous membrane tissue of clip coating central area, a pig cervix uteri is got two mucous membrane tissues in left and right, washes down after the left drug on specimen surface with PBS liquid, in fixative, spends the night.The mucous membrane tissue specimen that formalin solution has been fixed to 12 hours is put in dewaterer, complete after the processes such as dehydration, transparent, waxdip, embedding, with routine paraffin wax microtomy serial section, the thick 3 ~ 4mm of sheet, do not dye, after resin mounting, take under Laser Scanning Confocal Microscope and observe.
4. experimental result
Podophyllotoxin-nano-lipid carrier the suspension obtaining by the method in embodiment 1, acts on after Tibet Mini-Pigs cervical mucosa 4h, 24h, and local mucous membrane is without (referring to A and the B of Fig. 5) such as redness, blister, erosions; And the redness, vesicle, erosion etc. (referring to C and the D of Fig. 5) of cervical mucosa appear rapidly in podophyllotoxin tincture group after medication, miniature pig all survives well, flushes, and searches for food, drinks water and be normal, without phenomenons such as perpendicular hair, cough, sialorrheas.
Experimental result shows, podophyllotoxin-nano-lipid carrier is enriched in mucosa top layer, there is the feature of slow release, podophyllotoxin-nano-lipid carrier distributes few at corium, fluorescence intensity is far below tincture group, and podophyllotoxin-nano-lipid carrier shows good mucosa targeting (Fig. 6, Fig. 7).Also find, podophyllotoxin-nano-lipid carrier blood drug level is starkly lower than tincture (Fig. 8), and prompting podophyllotoxin-nano-lipid carrier system absorbs few, and safety is high compared with tincture.
Embodiment 3: the impact of podophyllotoxin preparation of the present invention on VK2/E6E7 cell proliferation and apoptosis
Condyloma acuminatum is the sexually transmitted disease (STD) that human papillomavirus (HPV) infection anus, external genitalia position cause, at present, the optimal method that treatment HPV infects is directly thoroughly to remove from cell and tissue for virus or by the cell of viral infection and by virus.But thoroughly remove by the cell of viral infection owing to lacking effective antiviral means and being difficult to, all there is higher relapse rate in traditional Therapeutic Method.Local HPV latent infection is considered to the main cause of condyloma acuminatum recurrence.
Can carry out pharmacodynamic study owing to there is no at present the animal model that maturation, generally acknowledged HPV infect both at home and abroad, people's immortalization vaginal epithelial cell (VK2/E6E7 cell) that therefore the present invention selects HPV to infect carries out experimental verification.
1. experiment material:
VK2/E6E7 cell, purchased from ATCC.With the cultivation of going down to posterity containing the Keratinocyte serum-free medium of 15% calf serum.
Control drug podophyllotoxin tincture (self-control): take 0.05g podophyllotoxin with electronic balance, be dissolved in 10ml dehydrated alcohol, be mixed with 0.5% podophyllotoxin tincture.
Podophyllotoxin-nano-lipid carrier suspension: obtain by the method in embodiment 1.
2. experimental technique:
(1) impact of POD-NLC on VK2/E6E7 cell proliferation.It is 0.0005 μ g/ml, 0.005 μ g/ml, 0.05 μ g/ml, 0.5 μ g/ml, 5 μ g/m that POD-NLC Concentraton gradient is set, and processes respectively VK2/ E6E7 24h, 48h, and after 72h, CCK8 detects each hole OD value, and calculates suppression ratio.In triplicate, each each concentration arranges 3 multiple holes in experiment.
(2) POD of different dosage form is to the comparison of VK2/E6E7 cell growth inhibition.It is 0.0005 μ g/ml, 0.005 μ g/ml, 0.05 μ g/ml, 0.5 μ g/ml, 5 μ g/m that POD tincture and POD-NLC Concentraton gradient are set, and processes respectively VK2/ E6E7 24h, 48h, and after 72h, CCK8 detects each hole OD value, and calculates suppression ratio.POD tincture and POD-NLC are compared VK2/E6E7 cell inhibitory rate.In triplicate, each each concentration arranges 3 multiple holes in experiment.
(3) comparison of the POD of different dosage form to the effect of VK2/E6E7 cell Cycle Arrest.It is POD and the POD-NLC of 0.05 μ g/ml that experimental group adds respectively concentration, matched group adds isopyknic culture medium, process VK2/E6E7 cell 24h, after 48h, centrifugal collecting cell, 70% ethanol is fixed, after PI dyeing, flow cytometer (FCM) detects, relatively the impact of POD and the retardance of POD-NLC cell cycle.
(4) POD of different dosage form is on the apoptotic impact of VK2/E6E7.Experimental group adds respectively the culture medium containing 0.05 μ g/ml POD and POD-NLC, and matched group adds isopyknic culture medium, processes after VK2/E6E7 cell 48h the 1. apoptosis form of inverted microscope observation of cell; 2. cell is carried out after Hoechst33242 dyeing observation of cell core apoptosis form under fluorescence microscope; 3. use Annexin V-FITC/PI kit detection cell apoptosis, after according to test kit description, cell being dyeed, carry out flow cytometer (FCM) and detect.
3. experimental result
Experimental result shows, podophyllotoxin-nano-lipid carrier can significantly suppress the growth of VK2/E6E7 cell in vitro, and its effect presents concentration and time dependence.The main effect significant difference of the variance analysis displaying time of repeated measure, there is statistical significance (P<0.01), the main effect significant difference of concentration, there is statistical significance (P<0.01), interaction difference between time and concentration is also remarkable, has statistical significance (P<0.01) under same treatment time conditions, with the increase of activity, A value and reducing, suppression ratio raises.Under certain concentration, under (0.05,0.5,5 μ g/mL) concentration conditions, with the prolongation of action time, suppression ratio raises.Bonferroni method compares A value between each group, find compared with matched group, in the time of 24h, concentration is that podophyllotoxin-nano-lipid carrier of 0.0005 μ g/ml produces inhibitory action to VK2/E6E7 cell, and difference has statistical significance (P<0.01) (see figure 9).
Experimental result also shows, podophyllotoxin-nano-lipid carrier the suspension obtaining by the method in embodiment 1, 0.05 μ g/mL podophyllotoxin-nano-lipid carrier and podophyllotoxin tincture are processed respectively after VK2/E6E7 cell 24h, the cell number of podophyllotoxin-nano-lipid carrier processed group G2/M phase accounts for total cell number ratio and is increased to 22.92% by 6.12% of matched group, podophyllotoxin tincture processed group G2/M phase cell proportion is increased to 11.03%, tentatively conclude that the retardation of podophyllotoxin-nano-lipid carrier cell cycle is as podophyllotoxin tincture, make cell block at G2/M phase (Figure 10).In the time that the processing time is increased to 48h, podophyllotoxin-nano-lipid carrier and podophyllotoxin tincture processed group G2/M phase cell proportion extremely significantly (86.3 ± 8.5% vs. 5.4 ± 4.6%, P<0.01 of increase compared with matched group; 60.2 ± 4.3% vs. 5.4 ± 4.6%, P<0.01), meanwhile, between podophyllotoxin-nano-lipid carrier and podophyllotoxin tincture processed group, also there is significant difference (86.3 ± 8.5%vs. 60.2 ± 4.3%; P<0.01), podophyllotoxin-nano-lipid carrier cell cycle inhibitory action is obviously better than podophyllotoxin tincture (Figure 10,12).
Podophyllotoxin-nano-lipid carrier and POD with 0.05 μ g/mL process respectively VK2/E6E7 cell 48h, observation of cell under microscope, visible obvious metamorphosis, compared with matched group, the cell of two processed group, cell rounding, cell membrane shrinkage, adherent property weakens, and has part cell come off (Fig. 2-7).Through Hoechest33342 dyeing, under fluorescence microscope ultraviolet excitation light source activation, fine and close dense the dying of visible appearance, and chunky shape nucleus, present typical apoptosis morphological feature, compared with the irregular nucleus of processed group, cellular control unit nuclear shape size all compares homogeneous (Figure 11).
In sum, podophyllotoxin-nano-lipid carrier can significantly suppress the growth of VK2/E6E7 cell in vitro, make cell block in the G2/M phase, its effect presents concentration and time dependence, and dye through Hoechest33342, under microscope, can clear view be fine and close dense dying and chunky shape nucleus to cell, present typical apoptosis morphological feature.
Drug-induced apoptotic mechanism of action is not single, but the interactional result of many factors.The inventor also proves that POD-NLC can act on VK2/E6E7 cell mitochondrial approach related apoptosis gene by experiment, also can induce the ROS level of VK2/E6E7 cell to increase, raising AIF and Cyt-C expresses, show that thus POD-NLC can be by mitochondria pathway and non-caspase Dependent induction VK2/E6E7 apoptosis, further confirm that from molecular biological angle POD-NLC can induce HPV infection cell generation apoptosis, and then successfully remove sick cell, reach the object of clinical treatment condyloma acuminatum, effective anti-condyloma acuminatum recurrence and HPV latent infection.Therefore,, than the various POD preparations of having reported, POD preparation of the present invention will have more the curative effect of anti-HPV recurrence and anti-HPV latent infection.
Claims (5)
1. a podophyllotoxin preparation for anti-condyloma acuminatum recurrence and anti-HPV latent infection, is characterized in that: taking nano-lipid carrier as drug carrier material, its active constituents of medicine of sealing is podophyllotoxin; The mass volume ratio of podophyllotoxin and nano-lipid carrier is 0.5%.
2. podophyllotoxin preparation according to claim 1, is characterized in that: described nanoparticle mean diameter is 180 ± 20nm, and average envelop rate is (82.9 ± 2) %, and pH is 6.20 ± 0.04.
3. podophyllotoxin preparation according to claim 1, is characterized in that, described podophyllotoxin preparation is to be prepared as suspension.
4. podophyllotoxin preparation according to claim 3, is characterized in that, the suspension of described podophyllotoxin preparation is prepared by following steps:
A. by podophyllotoxin, solid-state lipid, liquid lipid and emulsifiers dissolve in organic solvent, form oil phase; Surfactant dissolves, in water, is formed to water;
B. distinguish, continue to stir oil phase, make it fully dissolve post-heating to 74 ± 1 DEG C;
C. the product of abovementioned steps is at the uniform velocity added in the water of 74 ± 1 DEG C, stir 3～4 hours;
D. mixture of ice and water is added rapidly in the product of abovementioned steps, stop stirring, set to 0 in DEG C ice-water bath ultrasonic 30 minutes, adopt filtering with microporous membrane, obtain the suspension of described podophyllotoxin preparation.
5. podophyllotoxin preparation according to claim 3, is characterized in that: the mixing speed in described step b and c is 1200-1500r/min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310489910.6A CN103893167A (en) | 2013-10-18 | 2013-10-18 | Podophyllotoxin preparation resisting condyloma acuminata relapse and HPV latent infection |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310489910.6A CN103893167A (en) | 2013-10-18 | 2013-10-18 | Podophyllotoxin preparation resisting condyloma acuminata relapse and HPV latent infection |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103893167A true CN103893167A (en) | 2014-07-02 |
Family
ID=50984959
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310489910.6A Pending CN103893167A (en) | 2013-10-18 | 2013-10-18 | Podophyllotoxin preparation resisting condyloma acuminata relapse and HPV latent infection |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103893167A (en) |
-
2013
- 2013-10-18 CN CN201310489910.6A patent/CN103893167A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 王春慧等: "0.5%鬼臼毒素纳米脂质载体包封率的测定", 《皮肤性病诊疗学杂志》 * |
| 种树彬等: "鬼臼毒素纳米脂质载体的制备及体外对HPV感染的永生化人宫颈上皮细胞的作用", 《南方医科大学学报》 * |
| 韩凯等: "0.5%鬼臼毒素纳米脂质载体的制备及理化性质考察", 《皮肤性病诊疗学杂志》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2006275232B2 (en) | Use of hydroxybenzoic acid ester and analogues for the manufacture of a mendicament for the prevention and treatment of virus infection | |
| CN105031625B (en) | Lactoferrin and carragheen composition of medicine of a kind of electric charge modification and preparation method thereof | |
| CN101890030B (en) | Composite capable of preventing bacteria, viruses, oxidation and pigment deposition | |
| CN106511512A (en) | Traditional Chinese medicine external-use gel and preparation method and application thereof | |
| CN109320570A (en) | A kind of icariside I class compound, derivative, officinal salt and application | |
| CN105748506A (en) | Application of alginate sulfate in preparation of medicines and health products used for preventing and treating diseases caused by human papilloma virus | |
| CN103893167A (en) | Podophyllotoxin preparation resisting condyloma acuminata relapse and HPV latent infection | |
| WO2014107828A1 (en) | Drug for killing acid-fast (red) bacillus | |
| CN101342170A (en) | Application of Xinjiang radix macrotomiae for treating flat wart, common wart and fig wart | |
| CN110433132B (en) | Tetrandrine nasal preparation for treating post-traumatic stress disorder | |
| CN108619136B (en) | A kind of preparation of anti-HPV | |
| CN103751105A (en) | Application of ginkgolic acid nanometer liposome in treatment of gynecological diseases and venereal diseases | |
| CN102349935A (en) | Purpose of ginkgolic acid in preparing external preparation for treating venereal disease, gynopathy and perianal disease | |
| CN106177900A (en) | A kind of gel of anti-human papilloma virus (anti-HPV) and preparation method thereof | |
| CN109820901A (en) | A kind of Traditional Chinese medicinal gel preparation with sterilization | |
| CN110292621A (en) | The antitoxin antibacterial complexing agent of anti-HPV viruse infection and its preparation method of phase inversion gel | |
| CN106074564B (en) | Ursolic acid is preparing the application in anti-hydatid drugs | |
| CN102512359B (en) | Mangiferin cream with anti-herpes virus effect | |
| CN110368463A (en) | A kind of Chinese medicine composition and the preparation method and application thereof for treating human papilloma virus HPV infection | |
| CN105101969A (en) | Antiviral agent composition containing boric acid, citric acid, and zinc | |
| CN103751230A (en) | Application of gingko extract nanometer liposome in gynecological disease treatment | |
| CN115040528B (en) | Application of digitonin D and anti-tumor metastasis medicine | |
| CN103070876B (en) | The compositions that the anti-encephalitis b virus of one class infects and application thereof | |
| CN114452249A (en) | Application of toona sinensis milk gel in preparation of medicines for resisting low-risk HPV infection and preventing and treating low-risk HPV related diseases | |
| CN115813980A (en) | Application of pharmaceutical composition in preparation of pseudomonas aeruginosa drug-resistant strain biofilm inhibitor |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20140702 |