CN100534422C - Personal hygiene additive for preventing STD origin of viral and chlamydia - Google Patents

Abstract

A personal medicine in the form of aqueous solution, colloid, or suppository for preventing the sexual transmission diseases (such as AIDS and HSV2) caused by virus (HIV for example) and chlamydia by applying it in vagina is prepared from Myramistin. It has high safety to human body.

Description

Prevent the Personal hygiene agent of the sexually transmitted disease (STD) of viral and chlamydia origin

Technical field

The invention belongs to the medicine and pharmacology field.The present invention can be used for developing and making the various preparations that can be used for hygiene and disease prevention.

Background technology

Herpesvirus, human immunodeficiency virus (HIV), human papillomavirus and some other virus can cause the sexually transmitted disease (STD) that people know.Some chlamydia (Chlamydia spp.) has the approach of same intrusion human tissue, and with all virus the same showed cell endoparasitism phenomenon.

The Personal hygiene reagent that the known HIV that is used to prevent the do as one likes contact transmission infects comprises: naphthalene sulfonate polymer (Profy A.T., Sonderfan A.J., Bernstein D.I., Chancellor T., Kern E.R., Stanberry L.R., 1998.PRO 2000gel, be used to prevent the potential topical germicide of HIV, it can block infection (a potential topical microbicide for HIVprevention, can block infection by other sexually transmitted disease pathogens) // the 12nd international AIDS seminar (12 of other sexually transmitted disease (STD) pathogen ^ThWorld AIDS Conference) (Geneva, Switzerland, 28 days～July 3 June in 1998): summary N 33146.P.620-621), Nonoxynol-9, benzalkonium chloride, sodium cholate (Colin P., Thibodeau L., Tremblay C., Sauriol C., 1998. one kind is prevented the outer anti-HIV-1 of potential novel vagina antibacterial F-5 colloid of STD disease and the performance of killing the virus (the In vitro anti-HIV-1 and HIV-2 virucidal preperties of the F-5 gel of HIV-2, a potentiallynew vaginal microbicide for STD prevention) // the same, summary N 60370, P.1067) and mixture (the Malamud D. of C14-alkanamine oxide and C16-alkyl betaine, Howett M.K., Wigdahl B, .Christensen N., Krebs F., Weisz J., the antiviral activity of antibacterial in the Kreider J. mode system (Anti-viral activity of microbicides in model systems) // the same, summary N 33149.P.621).

Summary of the invention

About the virus that can be used as defence trafficability characteristic contact transmission and the Personal hygiene medicament of chlamydiaceae (Chlamydia spp.), also not having can be for the information of utilizing.

According to proposal, this class reagent may be that myristamide propyl-dimethyl benzylamine chloride (Myramistin) and above-mentioned Myramistin are dissolved in the concentrated aqueous solution of pharmaceutical solvents-pure water or cellulose derivative and the solution of 0.01 to 0.5% (weight) that forms.

In early time, Myramistin is known as preventing traditional sexually transmitted disease (STD) such as syphilis, gonorrhea and trichomoniasis (USSRAuthorship Certificate No 1832496,1978).

The reagent that is proposed (molecular formula 1) can enlarge scope such as herpes, chlamydiosis and the life-threatening AIDS of sunitizing prevention of sexually-transmitted diseases.

Myristamide propyl-dimethyl benzylamine chloride

(molecular formula 1)

The activity of the anti-HIV of myristamide propyl-dimethyl benzylamine chloride (Myramistin) can be described by the following examples.

The specific embodiment

Embodiment 1.Myramistin is to the research of the cultured cell that infected by the HIV-1 cell culture and the influence of their upgrowth situations.

What this test was used is lymphocyte MT-4 and Jurkat-tat cell.Cultured tissue is cultivated in following growth medium: RPMI-1640 culture medium (the Flow Labs that has replenished 10% hyclone, Scotland), glutamine 2mmol/L (Sigma Chem.Comp., USA), and antibiotic: penicillin 100 μ g/mL and streptomycin 100IU/mL (or gentamycin 50IU/mL).Cell culture carries out in 37 ℃ of air that contain 5% carbon dioxide.

Virus:

HIV (human immunodeficiency virus) strain (HIV-1 IIIB and HIV-1 BRU) obtains from two Russia collection units, that is, and and the Russian Academy of Medical Sciences virus product institute and Russian Academy of Medical Sciences virusology Ivanovsky institute (Moscow, Russia).HIV LAI strain is to infect control virusology system of institute (Stockholm, Sweden) from Switzerland to obtain.

Step:

With the MT-4 cell is object of study.The culture fluid (inoculum) that will have virus joins density and is about in the cell suspension of 500,000 cell/mL.Myramistin with the equal volume debita spissitudo mixes with freshly prepared inoculum.Infected cells and mentioned reagent were hatched 10 minutes in the room temperature vibration.After with growth medium cell being cleaned 3 times, its density is adjusted to 500,000 cell/mL.With the cell after cleaning with same concentration and not infected cells mixing.Infected cells and not the final ratio of infected cells be 1: 2 to 1: 3.Cell mixture was hatched in above-mentioned growth medium 30 days.

Distinguish living cells and dead cell with the trypan blue dye test.Determine the level of HIV related antigen in the culture fluid by the ELISA test.Use the ELISA test (ELISA/s) of improvement herein, can determine HIV-1 antigenic compound (Marennikova S.S., Matsevich G.R., Sheluhina E.M. etc., 1989.Voprosy virusologii，No 3.P.305-308)。(indirect fluorescent antibody test IFA) is calculated the quantity that has this antigenic cell to adopt indirect immunofluorescence assay.

Myramistin is to the result of the test of infected impact cell:

The Myramistin of 10 μ g/mL has shown the appreciable impact to the HIV-1 that carries in the MT-4 cell: hatching the 4th day, and living cells less than 20%, the average titer of the HIV-1 related antigen in the culture fluid of determining by ELISA/s is 1: 81 to 1: 243; Closed titre (closed titre) level has appearred in the positive control (not handling with Myramistin).The HIV-specificity that the Myramistin of 30 μ g/mL has promoted tissue culture to postpone change and culture fluid in the increase of antigen titre, although the concentration of IFA-positive cell is the highest at the 4th day.Though virus antigen appeared in the culture fluid afterwards, the concentration of living cells and IFA-positive cell but descends thereupon.After the Myramistin with 50 μ g/mL handled, above-mentioned variation postponed for 2 week at least.

The research of the outer particulate influence of HIV-1 of embodiment 2.Myramistin pair cell

Used T cell (Jurkat-tat cell) and the HIV-1LAI that concentrates in the culture fluid in these tests.After the Myramistin with ascending-dose handles, the inactivation situation of assessment HIV-1.The result who handles with the Myramistin of 100 μ g/mL is to reach the HIV-specificity that tissue culture did not take place in 30 days and change (viewing duration).

After the Myramistin with 50 μ g/mL handled for 2 weeks, will join in the not infected cultured cell after the dilution of the viral suspension in the culture fluid.According to the data in the table 1, the Myramistin aqueous solution under this concentration promotes extracellular HIV-1 infection activity to reduce by 1000 times.

After the outer HIV-1 of table 1Myramistin aqueous solution (50 μ g/mL) pair cell handles, to the value-added influence in the Jurkat-tat cell of this virus

Embodiment 3.Myramistin and other common fungicide anti-HIV specific activitys are

Various disinfectant can make the Cmin of HIV inactivation be shown in table 2

Table 2 is according to data in literature, the anti-HIV activity of some disinfectant

Antibacterial	The Cmin (%) that the HIV inactivation is required	Document
			Ethanol	50.00	[1]
Sodium hypochlorite (NaOCl)	0.10	[1]
			Paraformaldehyde	0.50	[1]
Hydrogen peroxide	0.30	[1]
			Nonidet P40 (NP-40)	1.00	[1]
Polysorbas20	Greater than 2.50	[1]
			Chlorhexidine (Chlogexidine)	0.02-0.05	[2]
Nonoxynol-9	0.05	[3]
			Myramistin	0.01

Note:

1.Martin L.S., Mc Dougal J.S., Laskoski J.L. to the sterilization of HIV and inactivation (Desinfection and inactivation of HIV) //J.Infect.Dis.1985 Vol.152.P.400-403.

2.Harbison M.A., the HIV inactivation that Hammer S.M. povidone iodine and chlorhexidine cause (Inactivation of HIV by betadine products and chlorhexidin) //J.AIDS.1989 Vol.2.P.16-20.

3.Polsky B., Baron P.A., Gold J.W.M., Smith J.L., Jensen R.H., ArmstrongD. contain the external inactivation (In vitro inactivationof HIV-1by contraceptive sponge containing nonoxynol-9) of the caused HIV-1 of contraceptive sponge of Nonoxynol-9 //Lancet.1988.Vol.1.P.1456.

According to these data, Myramistin makes the HIV inactivation with minimum concentration.And most of mentioned reagent produces stimulation to the genitals mucosa after application, thereby can not be as the Personal hygiene agent.

Thus, Myramistin has significant anti-HIV activity, is considered to the Personal hygiene agent of the HIV infection of preventative contact transmission.

Embodiment 4.Myramistin is to the research of the preventive effect of mouse experiment vagina herpes infection

End user's herpeslike virus 2 (HSV2) reproduces experimental herpes infection in adult white mouse (female): with 0.1ml virus-culturing fluid suspension (about 2.5 * 10 ⁷Individual granule/mL) import vagina with tuberculin syringe.The result has produced the specificity encephalitis, and all animals are all death in the 6th to 10 day after inoculation.Two class Myramistin preparations are used to prevent experimental herpes, that is, and and Myramistin 0.2% colloid and Myramistin 0.5% suppository.It is sulfo-cellulose (thilose) that colloid forms thing, and it is a cellulose derivative.The intravaginal in preceding 5 minutes of matched group 1 mouse inoculation is imported into the thilose solution that 0.1ml does not contain Myramistin.Experimental group 1 and group 2 mices are imported Myramistin 0.2% colloid or Myramistin 0.5% suppository in the same manner respectively.Experimental result is shown in table 3.

Table 3 Myramistin is to the preventive effect of mouse experiment vagina herpes infection

Group (10 mice/groups)	The feature of HSV2 infection animal	Dead animal quantity in the group	Preventive effect (%)
				Experimental group 1	Myramistin 0.2% colloid before the inoculation	0/10	100
Experimental group 2	Myramistin 0.5% suppository before the inoculation	0/10	100
				Matched group 1	The Thilose solution that does not contain Myramistin before the inoculation	8/10	20
Matched group 2	Be untreated	10/10	0

Note:

This part research work carries out in Sweden infection control institute (Stockholm), and it is subjected to the support of international fund (project INTAS UA 95-55).

According to data shown in the table 3,8 dead mouses are arranged in the matched group 1, two experimental grouies there is no dead mouse.Therefore, the Myramistin preparation of test has shown very high preventive effect under these experiment conditions.

The experimentation of 5. pairs of Myramistin anti-chlamydia activities of embodiment

According to the data of many research worker, the inoculation tissue culture is the best chlamydia infection experimental model that is used for testing anti-chlamydia reagent, although lack the strong dependency with clinical data, and this model is tested some technical complexity.

At first should assess the toxicity of Myramistin to cultured cell (chick embryo fibroblast).For this reason, handle 1-2 days cultured cell 10 to 30 minutes with the Myramistin of various dosage.The summary result of these experiments is shown in table 4.

Table 4.Myramistin determines the toxicity of chick embryo fibroblast tissue culture of former generation

The final concentration of Myramistin (%)	Processing time (minute)	Myramistin handles the feature of back cell
			0.0075	10 to 30	Degenerate fully
0.0005	-“-	A large amount of cells form cavity and degeneration
			0.00025	-“-	Complete monolayer is not seen degeneration

Therefore, Myramistin concentration is 0.00025% o'clock, and chick embryo fibroblast is cultivated does not have toxicity, this concentration to can be used for anti-chlamydia activity research.

Use two cell strains to assess the anti-chlamydia activity of Myramistin: in yolk sac through cultured cells strain C.psittaci B and the cell strain C.psittaci K (in tissue culture, growing) of going down to posterity several times.The inoculum of two cell strains all is equivalent to the LD of rat ₅₀4-6 doubly.

Preceding 30 minutes of step 1. inoculation joins the monolayer chick embryo fibroblast with Myramistin, and its final concentration is 0.00025%.After adding the chlamydia be suspended in the yolk material, tissue culture 37 ℃ hatch 2 hours after, clean cell monolayer, will not contain again in the cell after antibiotic fresh " 199 " culture medium joins cleaning.

Step 2.Myramistin mixes with " tissue " strain K and " yolk " strain B respectively, and its final concentration is 0.00025%, hatches respectively 1 and 2 hour at 37 ℃ then.Then clean cell monolayer, after covering with fresh culture, hatch under the same conditions again.After 24 to 120 hours, fixedly have the coverslip of cell monolayer, the anti-chlamydial fluorescent antibody (being combined with acridine orange) of reuse is handled.Microscope slide fluorescence microscope after observing dyeing under the UV light.Use two different cell monolayers in contrast: the nonvaccinated cell monolayer of handling through the Myramistin 1) cell monolayer and 2 of the inoculation of not handling with Myramistin).10 experiments have been carried out in this way.

Have been found that after two strains are hatched 72 to 120 hours to have only utmost point individual cells to have the chlamydia form (L-form) of degeneration.In the cell of (contrast) that is untreated, found typically to be in the chlamydia in different maturation period; Chlamydial quantity is different because of cell strain and inoculum density.Therefore, under employed concentration, Myramistin has the effect that chlamydia is polluted that suppresses.

These experiments show that together with the data of the broad spectrum antibiotic activity of known relevant Myramistin Myramistin is convenient feasible conscientiously as the Personal hygiene agent of the chlamydia disease of the preventative propagation of men and women.

Claims (2)

1. myristamide propyl-dimethyl benzylamine chloride is used to prepare the purposes of the preparation of the hygiene of the sexually transmitted disease (STD) that the prevention chlamydia causes and disease prevention, it is characterized in that, described preparation comprises: pharmaceutical solvents or solid pharmacy substrate, and concentration is the myristamide propyl-dimethyl benzylamine chloride of 0.01-0.5 weight %, wherein

Described myristamide propyl-dimethyl benzylamine chloride is shown in following molecular formula:

2. purposes according to claim 1, wherein, described pharmaceutical solvents is a pure water; Described solid pharmacy substrate is suppository.