CN100364518C - Use of 1,4-naphthalene diphenol in preparing external use sperm killing contraceptive medicine of preventing and curing genital tract infection - Google Patents

Abstract

The present invention discloses the use of 1, 4-naphthalene diphenol in preparing external use spermicidal contraceptive capable of preventing and treating genital tract infection and the external use spermicidal contraceptive prepared with 1, 4-naphthalene diphenol. The external use spermicidal contraceptive possesses triple functions of diminishing inflammation, birth control and improving sexual life. The spermicidal contraceptive has powerful spermicidal effect 25 times higher than that of nonoxynol nonylphenoxy-polyethoxyethanol, no bacteriostasis on normal lactobacillus inside vagina and effect of defending partial STD pathogens. It has the features of safety, high efficiency and convenience.

Description

1, the 4-naphthalenediol is used to prepare the external use sperm killing contraceptive medicine purposes of preventing and treating reproductive tract infection

Technical field

The invention belongs to pharmaceutical sanitary field (family planning contraceptive category), be particularly related to 1,4-naphthalenediol (1,4-Naphthohydroquinone/1,4-Dihydroxynaphthalene, N) be used to prepare the purposes of the external use sperm killing contraceptive medicine of preventing and treating reproductive tract infection, and this 1, a kind of external use sperm killing contraceptive medicine of preventing and treating reproductive tract infection---the female hundred health contraceptive creams (KBK) (preparation) of 4-naphthalenediol preparation.

Background technology

In existing various contraceptives, though differ from one another, no matter be intrauterine device, Unterbindung des Eileiter and vasoligation, or hormones oral contraceptive etc., all existed wound property or life-time service side effect big, use inconvenient; Both may upset the human body normal physiological processes, have many potential dangerous paathogenic factors, particularly teratogenesis, carcinogenecity, STDs propagation etc. again.Though male contraceptive pill is expected to become promising contraceptives, seek a kind of effectively, safety, easily accept, reusable male contraceptive agent is more difficult than female contraceptive agent.In recent years condom for man (MC) and condom (FC) though use we can say on be the important breakthrough of contraceptive techniques because its sensory properties is poor, use inconvenient, uncomfortable, or too expensive and in the ordinary people lives, be difficult to be promoted.

In numerous methods of birth control, contraceptive killing sperm in vagina is the most ancient a kind of contraceptive device, in recent years since it not only to have a whole body side reaction little, local side effects is light, cheap, it is convenient to use, and usage is appropriate, advantages such as contraceptive effect is also satisfied, and can effectively prevent unexpected gestation, prevention STDs propagate and be more suitable in the adult and premenopause the women use and obtain again paying attention to.

Nonoxynolum (NP-9) for World Health Organization (WHO) in the whole world unique a kind of external use sperm killing contraceptive of promoting the use of, though its spermicide effect is very strong, but manyly in recent years studies have shown that there is the circulation of liver-intestinal in vivo in it, has body accumulation, all toxic to liver, kidney, and do not have the effect that prevents and treats reproductive tract infection.2002, Cochrane Library published all meta-analysis that can obtain to study, and its conclusion is: NP-9 may be harmful to, because it increases the incidence rate of reproductive tract infringement, thereby may increase the probability that STDs propagates.WHO and CONRHD sound a warning: the spermicide that contains NP-9 can not play a protective role to HIV, gonorrhea or chlamydial propagation, often uses the women of these products may increase the danger of suffering from STDs.Just moderate is effective to contraception to use NP-9 separately, and the high-risk women that should advise the HIM infection is with the contraception of NP-9 spermicide, unless can not obtain other contraceptive device.

In addition, though also have other reports of the sub-contraceptive of many external use sperm killings both at home and abroad, as the Chinese herbal medicine phytolaccatoxin, the Radix Achyranthis Bidentatae Saponin, Fel Sus domestica (ZDZ), Fructus Mume, Fructus Cnidii, matrine and Western medicine phenylmercuric acetate, Septin, 47319 dexpropranolol, sodium morrhuate, phytohemagglutinin, phenolic aldehyde, phenolic ketone, phenolic acid compound, benzalkonium chloride (BZN), hibitane (CH), sulfonated polystyrene and miconazole etc., but all be in the experimentation, and owing to reasons such as the big or smart extremely effectiveness of its toxic and side effects are too low, not appearing in the newspapers as yet, it is clinical to be applied to.And known killing sperm in vagina agent class contraceptive has certain damage effect to women's vaginal epithelial cell, more also can influence intravaginal normal flora.

In a word, because China's number of women pregnant before marrige, ratio of induced abortion, reproductive tract infection rate and birth defect rate are rapid ascendant trend in recent years.And the married women of child-bearing age's of China main contraceptive device is male sterilization (accounting for 7.9%) and female sterilization (accounting for 38.1%) and placement of intrauterine devices (accounting for 45.6%), and these three kinds of contraceptive devices are well-known for the influence and the side effect thereof of people's healthy reproduction.Therefore, can develop a kind of novel spermicide contraceptive of vagina better tolerance, perhaps if two or more such medication combined application can be made the compound compatibility preparation, not only can strengthen the effect of its spermicide effect and prevention STDs, but also can reduce stimulation to vaginal mucosa, thereby, improve life quality and be of great practical significance and high economic results in society improving the healthy reproduction level.

Summary of the invention

An object of the present invention is, provide 1, the 4-naphthalenediol is used to prepare the purposes of the external use sperm killing contraceptive medicine of preventing and treating reproductive tract infection;

Another object of the present invention is, with 1, the 4-naphthalenediol is prepared into a kind of external use sperm killing contraceptive medicine of preventing and treating reproductive tract infection, and this medicine has antiinflammatory, contraception, improves the sexual life triple functions.

In order to realize above-mentioned task, the present invention takes following technical solution:

The applicant is to 1, the 4-naphthalenediol has carried out clinical preceding materia medica, pharmacokinetics, pharmacological effect toxicologic study, prove according to result of study, 1, the 4-naphthalenediol can be used in the external use sperm killing contraceptive medicine purposes of preparation control reproductive tract infection, and the spermicide effect is higher more than 25 times than nonoxynolum.

A kind of external use sperm killing contraceptive medicine of preventing and treating reproductive tract infection is characterized in that, it is 1 that this drug main is wanted the raw material the effective elements of the medicine, the 4-naphthalenediol, and its structural formula is:

Chemical molecular formula: C ₁₀H ₈O ₂, molecular weight: M=160.

C wherein ₁₀H ₈O ₂Labelled amount in medicine＞98.0%; Mp:192 ℃-194 ℃; Residue on ignition＜0.1%; Moisture content＜0.05%; Dissolubility is 0.04 ‰ in water.

This medicine and cream base are made oil-in-water type half viscous fluid sample preparation, and the volume of every minimum package unit is 3ml, and medicament contg wherein is 2mg, and all the other are cream base.

Described cream base is that tristerin, stearic acid, liquid Paraffin, white vaseline, lanoline and triethanolamine, sanlose (CMC-NA), polypropylene carboxylic acids macromolecular compound (PA) and an amount of distilled water constitute.

The present invention is first with 1, the 4-naphthalenediol is prepared as the sub-contraceptive of the external use sperm killing that has anti-reproductive tract infection concurrently, it is strong to have the spermicide effect, under drug use concentration to the normal lactobacillus unrestraint of vagina effect, the part venereal bacteria that spreads through sex intercourse is had defense reaction, and tool safety, efficiently, characteristics easily.This medicine can form the molecular film structure in intravaginal in addition, not only mucosa is played the mechanical barrier protective effect, can block N by mucosa absorption, and can limit sperm motility and effectively kill sperm fast.

Description of drawings

The preparation process flow chart of Fig. 1 female hundred health contraceptive creams (KBK) for the present invention prepares.

Below in conjunction with accompanying drawing and main materia medica, pharmacodynamics and pharmacology, the present invention is described in further detail in the toxicology test report.

The specific embodiment

The applicant is according to national I kind new medicine registration management way, and with 1, the 4-naphthalenediol is prepared into a kind of external use sperm killing contraceptive medicine that is used to prevent and treat reproductive tract infection---female hundred health contraceptive creams (KBK).

Below be the detailed content of female hundred health contraceptive creams (KBK):

(1) female hundred health contraceptive cream preparation raw material medicines 1, the quality standard of 4-naphthalenediol

1, the 4-naphthalenediol

Naierfen

1，4-Dihydroxynaphthalene

Structural formula:

Chemical equation is: C ₁₀H ₈O ₂Molecular weight is: M=160

This product is 1, and the 4-naphthalenediol (1,4-Dihydroxynaphthalene), press dry product and calculate, contain (C ₁₀H ₈O ₂) must not measure and be less than 98.0%.

[character] this product is light grey to colourless crystallization, odorless or odorless almost.Meet light or in air color gradually dark, often show taupe, solution shows the faintly acid reaction.

This product is soluble in methanol, hot ethanol, ether and acetic acid, is slightly soluble in water.

Index of refraction: index of refraction should be 1.422 (two appendix VIF of Chinese Pharmacopoeia version in 2005).

Fusing point: fusing point should be 192～194 ℃ (two appendix VIC of Chinese Pharmacopoeia version in 2005).

[discriminating]

(1) this product retention time in the gained chromatogram under the assay item should be consistent with the retention time of reference substance.

(2) get this product,, absorption maximum is arranged at 280nm and 320nm place according to ultraviolet spectrophotometry (two appendix IVA of Chinese Pharmacopoeia version in 2005) test.

(3) infrared absorption pattern of this product is consistent with the standard diagram of contrast.

[inspection] acidity: get this product, add the injection water and be dissolved into and make the solution that contains 1mg among every 1ml, measure (two appendix VIH of Chinese Pharmacopoeia version in 2005) in accordance with the law, pH should be 4.0～4.5.

The operation of [assay] lucifuge is measured according to ultraviolet spectrophotometry and/or high performance liquid chromatography (two appendix VD of Chinese Pharmacopoeia version in 2005).

[classification] external applied contraceptive

[storage] shading, sealing is preserved

(2) crude drug 1, the synthesis route of 4-naphthalenediol

[synthesis route flow process]:

A. reaction equation:

1, raw material: 4-nitro-1-naphthols, zinc powder, glacial acetic acid, hydrochloric acid, ethanol

2, temperature: 94 ℃-105 ℃

3, pressure: normal pressure

4, the time: 3 hours

5, Reducing agent: zinc powder

6, operating procedure:

With zinc powder, glacial acetic acid, hydrochloric acid add in the three-necked bottle, add 4-nitro-1-naphthols in batches, be heated to backflow,, reacted three hours by observing change color, sufficient reacting is complete, add hydrochloric acid, filtered while hot, decompression steams the part glacial acetic acid to there being crystallization to separate out, mass crystallization (brown) is separated out in cooling.

B. reaction equation:

1, raw material: 4-amino-1-naphtholate hydrochlorate, water, sulphuric acid, K ₂Cr ₂O ₇

2, temperature: room temperature

3, pressure: normal pressure

4, the time: half an hour

5, oxidant: K ₂Cr ₂O ₇

6, operating procedure:

In flask, put into 4-amino-1-naphtholate hydrochlorate and water, 30 ℃ are stirred to dissolving fully, handle with concentrated sulphuric acid, heated and boiled, allow the sulfate that is precipitated out all till the dissolving, solution is pale red slightly, hot solution was poured into rapidly in a big funnel that once heat was crossed on steam bath the round-bottomed flask (in K is arranged ₂Cr ₂O ₇Aqueous solution), shaking flasks makes wherein that material fully mixes, yellow in a large number acicular crystal, be chilled to room temperature, sucking filtration, washing, drying, (dark yellow, m.p.124-125 ℃, the fusing back is a dark brown liquid to obtain crude product.)

7, process for purification:

Crude product and ether are steam heated to dissolving, added decolorizing with activated carbon 10 minutes, sucking filtration, reclaim ether to beginning to form crystal, cool off sucking filtration, a small amount of ether is washed crystallization, in mother solution, add decolorizing with activated carbon again, repeat said process repeatedly, final light yellow crystallization (m.p.124 ℃～125 ℃, the fusing back is a yellow transparent liquid).

C. reaction equation:

1, raw material: 1,4-naphthalene diquinone, glacial acetic acid, acetic anhydride, anhydrous sodium acetate, zinc powder

2, temperature: 110 ℃

3, pressure: normal pressure

4, the time: one hour

5, acylating agent: acetic anhydride

6, operating procedure:

In flask, add 1,4-naphthalene diquinone, glacial acetic acid, acetic anhydride, anhydrous sodium acetate stirs and makes whole dissolvings, add zinc powder in batches, control temperature be no more than 40 ℃, add zinc powder after, heating makes temperature rise to 110 ℃, keeps half an hour, and solution colour transfers brownish red to by yellow, take off into colourlessly at last gradually, kept this temperature afterwards again 10 minutes, the heat filter, wash with acetic acid, merge washing liquid, concentrating under reduced pressure reclaims acetic acid and acetic anhydride, residue is poured in the frozen water, fully stir, promptly separate out crystallization, sucking filtration, be washed to neutrality, promptly get crystallization.

7, process for purification:

Crude product adds decolorizing with activated carbon with 5 times of ethyl alcohol recrystallizations, and the heat filter is chilled to below 10 ℃, makes crystallization complete, sucking filtration, and washing, drying promptly gets elaboration.

D. reaction equation:

1, raw material: diacetyl naphthols, glacial acetic acid, sodium hydroxide, hydrochloric acid

2, temperature: 60 ℃

3, pressure: normal pressure

4, the time: 2 hours

5, catalyst: sodium hydroxide

6, operating procedure:

With the diacetyl naphthols, glacial acetic acid adds in the flask, heated and stirred adds sodium hydroxide and transfers pH to 9 to dissolving fully, and (observe color and become dark brown by white) reacts completely, reactant liquor is poured in the water, transfer to pH=4-5 with hydrochloric acid, separate out crystallization, the sucking filtration washing, drying gets crude product (m.p.192-194 ℃)

7, process for purification:

Crude product adds decolorizing with activated carbon with 5 times of recrystallizing methanol, and the heat filter is chilled to room temperature, and crystallization is separated out, sucking filtration, and washing, drying promptly gets elaboration (m.p.192-194 ℃).

(3) female hundred health contraceptive cream preparation quality standard and preparation were establisheds

1, female hundred health contraceptive cream preparation quality standards:

Female hundred health contraceptive creams

Kunbaikang biyun rugao

Kunbaikang Cream

This product contains 1,4-naphthalenediol (C ₁₀H ₈O ₂) should be in the labelled amount 90.0%～110.0%.

[character] this product is the white or the milky emulsifiable paste (cream) of emulsion-type substrate.

Retention time in the chromatogram of [discriminating] (1) this product gained solution under the assay item should be consistent with the retention time of reference substance.

(2) this product gained solution under the assay item has absorption maximum according to spectrophotography (two appendix IVA of Chinese Pharmacopoeia version in 2005) test at 280nm and 320nm place.

(3) infrared absorption pattern of this product gained solution under the assay item is consistent with the standard diagram of contrast.

[inspection] acidity: measure (two appendix VD of Chinese Pharmacopoeia version in 2005), pH should be 5.5～6.0 in accordance with the law.

The operation of [assay] lucifuge is measured according to high performance liquid chromatography and/or ultraviolet spectrophotometry (two appendix VD of Chinese Pharmacopoeia version in 2005).

The preparation of reference substance solution and algoscopy: the lucifuge operation, precision takes by weighing and be dried to 1 of constant weight in sulphuric acid desiccator, and accurate the title, decide behind an amount of 0.7mg of 4-naphthalenediol standard substance, and dissolve with methanol is settled to 25.0ml, according to high effective liquid chromatography for measuring.

The preparation of need testing solution and algoscopy: the lucifuge operation, get the about 1.0g of ointment, the accurate title, decide, and uses the 20.0ml dissolve with methanol, and supersound process 20min filters, and is settled to 25.0ml, according to high effective liquid chromatography for measuring.

[classification] external applied contraceptive

[specification] 3ml/ props up, 10/box

[storage] shading, sealing is preserved

[preparation] cream

2, female hundred health contraceptive cream preparation process thereof routes:

[prescription] 1,4-naphthalenediol 666.7mg

The cream base surplus

Make 1000g

[method for making]

With oil-phase component (tristerin 35g, stearic acid 120g, liquid Paraffin 60g, white vaseline 10g, lanoline 50g) and water-phase component (triethanolamine 4ml, sanlose and polypropylene carboxylic acids macromolecular compound be 300g altogether, distilled water is an amount of) be heated to 80 ℃ respectively, constantly stir down, fused oil phase is added aqueous phase, temperature is reduced to about 40 ℃, gets 1, and the 4-naphthalenediol dissolves with small amount of ethanol, add in the above-mentioned substrate under stirring, stir evenly fill, packing, promptly.

[process chart]

Referring to Fig. 1.

The about 1.0g of ointment is got in the operation of [assay] lucifuge, and accurate the title decides, and uses the 20.0ml dissolve with methanol, and supersound process 20min filters, and is settled to 25.0ml, according to high effective liquid chromatography for measuring.

(4) crude drug 1, the toxicity research of 4-naphthalenediol and female hundred health contraceptive cream preparations:

1, crude drug 1, the acute toxicity testing result of 4-naphthalenediol mice oral administration: LD ₅₀(95% credibility interval is: 136.22-166.08mg/kg) for 150.42mg/kg.

2, crude drug 1, and the acute toxicity testing result of 4-naphthalenediol rabbit vagina administration: (1.67mg/kg) do not see any animal dead and toxic reaction under the concentration of clinical using dosage 50 times (MTD).

3, the acute toxicity testing result of female hundred health contraceptive cream preparation rabbit vagina administrations: (1.67mg/kg) do not see any animal dead and toxic reaction under the concentration of clinical using dosage 50 times (MTD).

4, press the safety regulations of medicine toxicity and effectiveness dose relationship, this product should be the avirulence medicine under clinical working concentration.

(5) the stimulation test result of study of female hundred health contraceptive cream preparations:

1, rabbit (4) cornea stimulation test result: administration after 6 hours irritant reaction scoring be 0.5, visible conjunctiva mild hyperaemia under the slit lamp, corneal transparency, sclera are normal; Irritant reaction scoring until 7 days after 24 hours is 0, and visible conjunctiva does not have hyperemia, corneal transparency, normal, the no secretions of sclera under the slit lamp.Compare zero difference with the excipient matched group.In accordance with regulations, experiment conclusion should be the preparation nonirritant.

2, rabbit (5) vaginal irritation experimental result: vaginal mucosa does not have hyperemia, pathological phenomenons such as edema.In accordance with regulations, experiment conclusion should be the preparation nonirritant.

3, rabbit (3) skin irritation test result: 1, irritant reaction scoring in 24,48,72 hours is 0.33.Compare zero difference with the excipient matched group.In accordance with regulations, experiment conclusion should be the preparation nonirritant.

(6) crude drug 1, the experimental result of the female hundred health contraceptive cream main pharmacodynamics of 4-naphthalenediol and preparation thereof:

1, new synthetic crude drug 1, experimental result is consistent repeatedly is 0.005mg/ml-0.008mg/ml for the killing sperms in vitro least concentration of 4-naphthalenediol (in 20 seconds).Experimental result is consistent repeatedly is 0.003mg/ml-0.006mg/ml for the killing sperms in vitro least concentration (in 20 seconds) of the female hundred health contraceptive creams of preparation.

2, the disease-resistant protozoa of new synthetic crude drug and preparation spectrum and effect are with former 1, and the sterilization experiment result of 4-naphthalenediol (N) reports unanimity.As shown in table 1 below:

Table 1 N is to the external MIC and the MBC table of reference culture

Bacterial strain	MIC(mg/ml)	MBC(mg/ml)
			Escherichia coli ATCC25922 strain	0.2500	0.2500
Escherichia coli 44825 strains	0.2500	0.5000
			Staphylococcus aureus ATCC25925 strain	0.0625	0.0625
Staphylococcus aureus 26113 strains	0.0625	0.0625
			Bacillus pyocyaneus ATCC27853 strain	＞2000.0	＞2000.0
Bacillus pyocyaneus 10103 strains	＞2000.0	＞2000.0
			Bacillus proteus 49010 strains	0.5000	0.5000
Bacillus proteus 49080 strains	0.3125	0.3125

Candida albicans 85021 strains	0.0078	0.0156
			Gonococcus 29016 strains	0.0018	0.0036
Gonococcus 29017 strains	0.0018	0.0036
			Streptococcus faecalis ATCC10541 strain	0.5000	0.5000
Lactobacillus 1.555 strains	＞2000.0	＞2000.0

(7) the killing sperms in vitro experimentation of female hundred health contraceptive creams

7.1 summary

Use female hundred health contraceptive creams (KBK) (preparation) and main component-1 thereof, 4-naphthalenediol (1,4-naphthohydroquinone/1,4-Dihydroxynaphthalene, N) do the killing sperms in vitro experiment, the result shows that N has the stronger smart effect of killing, and it makes the minimal effective concentration of the inactivation of moment in the sperm 20 seconds is 0.005mg/ml-0.008mg/ml; It is 0.003mg/ml-0.006mg/ml that KBK makes the minimal effective concentration of the inactivation of moment in the sperm 20 seconds.And use the nonoxynolum of generally acknowledging and make comparisons, prove that the smart effectiveness of killing of N and KBK is higher more than 25 times than nonoxynolum.

7.2 material and method

7.2.1 medicine and preparation thereof

7.2.1.1 medicine

N and KBK provide by the medicine chemical preparation chamber preparation of department of pharmacy of Xi'an Jiaotong University Medical College; Nonoxynol-9 (nonoxynol-9 is available from Orthopharmaceutical Company).

7.2.1.2 preparation

The N drug powder is a certain amount of with accurate weighing, add the dilution of 0.9% normal saline and be mixed with 1.000mg/ml, 0.100mg/ml, 0.050mg/ml, 0.010mg/ml, 0.005mg/ml and six kinds of variable concentrations solution of 0.001mg/ml, KBK is made into the drug solns that contains of 1.000mg/ml, 0.100mg/ml, 0.050mg/ml, 0.010mg/ml, 0.005mg/ml and six variable concentrations of 0.001mg/ml with 0.9% sodium chloride solution by the prescription volume ratio.The nonoxynolum that positive control uses also is to be made into 0.1mg/ml, 0.2mg/ml, 0.3mg/ml, 0.4mg/ml, six different concentration of 0.5mg/ml and 0.6mg/ml with normal saline.

7.2.2 instrument

Powerful Light Microscope; Calorstat; Analytical balance; Stopwatch; Micropipette etc.

7.2.3 specimen

The used seminal fluid of this test all picks up from healthy reproductive male population, after ascetic 5-6 days with friction adopt fresh semen, and meet the requirements do routine examination, standby in 35 ℃ of insulations of calorstat.Sperm count 〉=60 * 10 ⁶/ ml, activity ratio 〉=60%, propulsion sperm percentage rate 〉=75%, teratospermia percentage rate≤20%, liquefying time≤30min.

7.2.4 experimental technique

It is one of important indicator that spermicide is renderd a service between the comparison different pharmaceutical that medicine makes the minimal effective concentration of sperm moment inactivation.The detection that spermicide is renderd a service is improved according to the smart experimental technique Sander-Cramer method of killing of International Programme birth fund meeting (IPPF) approval, promptly get qualified seminal fluid 15 μ l and medicinal liquid 15 μ l and drop in rapid mix homogeneously on the same microscope slide, timing, under 100 times of the microscopes that the constant temperature glass carrier bench is arranged and 400 times of visuals field, observe immediately, in 20 seconds, no sperm motion at least five visuals field, promptly all the sperm inactivations are terminal point, kill smart valid density with this as this medicine minimum.The medicinal liquid on the slide is washed off with 0.9% normal saline immediately, motile sperm is not seen in observation under the mirror again.With the slide natural drying,, find not ruined a little sperm all coloring simultaneously, show whole size degradations of sperm or death with Eosin Y dyeing.

This tests with 0.9% normal saline as the blank group, and with use at present both at home and abroad the most extensive and as the main component of vaginal contraception agent spermicide--the normal saline solution of the variable concentrations of Nonoxynol-9 is made positive controls, what draw medicine then minimumly kills smart valid density.Each medicine is measured repeatedly with 5 parts of normal man semen specimen of difference at least, every part of specimen be divided into several parts standby, repetition test is 3 times at least.

7.2.5 statistical procedures

Medicine minimal effective concentration data represent with x ± s, the significance of difference adopt in pairs or not pairing data carry out the t check analysis, the P value with 0.05 as significance limit.

7.3 result

Experimental group:, at last measured result is handled with statistical method through repetition test.Every group of sample regarded as under the situation that meets normal distribution, drawn the minimum smart valid density (x ± s), reach that kills of each medicine when α=0.05, i.e. 95% confidence interval.The results are shown in Table 2.

Matched group: 0.9% normal saline is to not influence of motility of sperm.

Table 2: each medicine is to the external comparison (n=10, x ± s, α=0.05) of killing smart usefulness of people's seminal fluid

Medicine	The minimum smart valid density (mg/ml) of killing
			Mean (x ± s)	95% puts the letter district
	N	0.006±0.003			0.005，0.008
KBK			0.004±0.003	0.003，0.006
	NP-9	0.280±0.060			0.240，0.320

7.4 discuss:

To the killing sperms in vitro Evaluation on effect of medicine, the method that International Programme birth fund meeting (IPPF) is recommended is the Sander-Cramer method of improvement, and promptly an amount of medicinal liquid and seminal fluid are prior to mix homogeneously in vitro, and insulation was vibrated 1 hour, observed in microscopically again.But because the method needs dose many, need specimen amount very big, and elapsed time is long, a specimen can't be carried out parallel assay to several medicines simultaneously, especially is not suitable for for some medicine that contains the easy oxidation discoloration of two phenolic hydroxyl groups.So in the experiment, the method is improved, is adopted direct slide method.Through overtesting, it is 0.28 ± 0.06mg/ml that measured reference substance NP-9 minimum kills smart valid density, with the 0.25mg/ml there was no significant difference of bibliographical information.So as seen this test method is reliable.

7.5 conclusion:

New synthetic crude drug 1, the killing sperms in vitro least concentration of 4-naphthalenediol (in 20 seconds) is 0.005mg/ml-0.008mg/ml.The killing sperms in vitro least concentration (in 20 seconds) of the female hundred health contraceptive cream KBK of preparation is 0.003mg/ml-0.006mg/ml.Spermicide intensity surpasses nonoxynolum more than 25 times.

The bactericidal assay report of (eight) 1,4-naphthalenediols:

8.1 summary:

This test determination 1, the 4-naphthalenediol is to the minimal inhibitory concentration and the minimal bactericidal concentration of the type strain and the clinical separation strain of colon bacillus, staphylococcus aureus, Pseudomonas aeruginosa, staphylococcus epidermidis, streptococcus faecalis, gonococcus, Bacillus proteus, lactobacillus, saccharomyces albicans.The result shows 1, and the 4-naphthalenediol can suppress, kill common urinary tract infection antibacterial effectively, but lactobacillus is not had obviously inhibition and lethal effect in experimental concentration.

8.2 material and method

8.2.1 test drug: 1, the 4-naphthalenediol.Synthetic voluntarily by this seminar, department of pharmacy of Xi'an Jiaotong University Medical College provides.Be mixed with 2mg/ml with physiological saline solution.

8.2.2 test strain:

8.2.2.1 reference culture is totally 11 strains:

Colon bacillus ATCC25922 strain

Staphylococcus aureus ATCC25923 strain

Blue pus organism ATCC27853 strain

Staphylococcus epidermidis ATCC26069 strain

Staphylococcus epidermidis 49080 strains

Streptococcus faecalis ATCC10541 strain

Gonococcus 20106 strains

Gonococcus 20107 strains

Bacillus proteus 49010 strains

Saccharomyces albicans 85021 strains

Lactobacillus 1.555 strains

The lactobacillus freeze-drying lactobacillus is available from the Chinese Academy of Agricultural Sciences lactobacillus research department; Other bacterial strain freeze-drying lactobacillus are identified institute available from Chinese Academy of Medical Sciences's Beijing pharmaceutical biological product.Strain was recovered in October, 2004, and this chamber is 4 ℃ of preservations in glycerin bouillon.The test forward pass is for standby.

8.2.2.2 clinical separation strain is totally 387 strains:

The golden 001-of Portugal gold Portugal 100 is numbered in staphylococcus aureus 150 strains;

Staphylococcus epidermidis 150 strains, the 001-of number table Portugal shows Portugal 050;

Streptococcus faecalis 30 strains, numbering excrement chain 001-excrement chain 030;

Gonococcus 25 strains numbering gonorrhea 001-gonorrhea 025;

Candida albicans 32 strains numbering is read the white thought 032 of 001-in vain;

Above clinical separation strain is by No.1 Hospital Attached to Medical College, Xi'an Communication Univ, second Affiliated Hospital, and clinical laboratory of The People's Hospital provides.All clinical isolates strains are all identified through the full-automatic digital Bacteria Identification instrument of Taket32.By this chamber stored frozen.Recovery before the experiment, it is standby to go down to posterity.

8.2.3 test organisms condition of culture:

Colon bacillus, staphylococcus aureus, staphylococcus epidermidis, Pseudomonas aeruginosa, Bacillus proteus, streptococcus faecalis are inoculated in the MH culture medium, put 37 ℃ of common incubators, cultivate in 24 hours.

Gonococcus is inoculated in Colombia's (chocolate) culture medium, and the 5%CO2 incubator was cultivated in 37 ℃, 48 hours.

Candida albicans is inoculated in the husky Ruo Shi of guarantor, and (Sabouraud, SB) culture medium are put 37 ℃ of common incubators, were cultivated in 48 hours.

Lactobacillus is inoculated in modified MRS culture medium, and 37 ℃ of anaerobism were cultivated 48 hours.

All culture medium are by " modern microbiological culture media and the preparation of reagent handbook.

8.2.4 test apparatus: CO2 incubator, common incubator, anaerobism incubator, colony count instrument etc.

8.2.5 test method: with reference to " antimicrobial drug in-vitro antibacterial test principle " and " microbiological Test technology " in " Ministry of Public Health bureau of drug administration new drug preclinical study guideline ", adopt micro-doubling dilution to measure MIC, dull and stereotyped infection protocol is measured MBC.

8.2.6 experiment content:

8.2.6.1 the mensuration of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)

With 1, the 4-naphthalenediol is that starting point is carried out continuous two times of gradient dilutions with culture fluid by 2.0mg/ml, and each gradient dilution liquid is added to 96 porocyte culture plates, 0.2ml/ hole successively.Adding concentration is the test organisms liquid 0.01ml of 106CFU/ml.Set up the contrast of antibacterial and culture medium simultaneously.4 ℃ act on 12 hours, put 37 ℃ of incubators, cultivate observed result in 48 hours.The little drug level of no bacterial growth Kongzui is minimal inhibitory concentration (MIC).To not see successively that again the culture in each hole of bacterial growth draws the 0.1ml dibbling respectively in Agar Plating, cultivate in 37 ℃, 48 hours.The minimum drug level of no bacterial growth inoculation area relative is minimal bactericidal concentration (MBC).

8.2.6.2 condition of culture is to the influence of MIC

Choose staphylococcus epidermidis ATCC26069 strain, gonococcus 29106 strains, change condition of culture, mensuration is subjected to the reagent thing to being tried the MIC of bacterium respectively.

8.2.6.3 pH value influence test:

With reference to MIC assay method in the test one, the pH of culture fluid is adjusted to 5.7,7.2,8.5 respectively, determine MIC separately respectively.

8.2.6.4 bacterial load influence test:

With reference to MIC assay method in the test one, will test bacterial concentration and change into 106,108,1010 CFU/ml successively, every hole inoculation 0.01ml carries out MIC and measures.

8.2.6.5 the medicine killing curve is measured

With dripping sick Neisseria 29106 strains is that 105CFU/ml is inoculated in and contains in the inferior meat soups of two times of MBC concentration medicine taxi driver brother rivals, in 37 ℃ of effects with the final concentration; Draw 0.1ml respectively at effect 5 ', 10 ', 20 ', 40 ', 1h, 2h, 4h, 8h, carry out ten times of continuous gradient dilutions with physiological saline solution, draw each gradient dilution liquid 10ul dibbling in Colombia's flat board, putting 5%C02,37 ℃ cultivated 48 hours, observe cultivation results, add up each time point bacteria living number.With medicine and bacterial action time be abscissa, the logarithm of bacteria living number is a vertical coordinate, draws killing curve.

8.3 result of the test:

8.3.1 1, the 4-naphthalenediol sees Table 3 to MIC, the MBC of reference culture.

Table 31, the 4-naphthalenediol is to the MIC and the MBC of examination bacterium reference culture

Bacterial strain	MIC(μg/ml)	MBC(μg/ml)
			85021 strains of colon bacillus ATCC25922 strain staphylococcus aureus ATCC25925 strain Pseudomonas aeruginosa ATCC27853 staphylococcus epidermidis ATCC26069 staphylococcus epidermidis 49080 strain streptococcus faecalis ATCC10541 strain gonococcuss 20106 strain gonococcuss 20107 strain Bacillus proteuss 49010 strain lactobacilluss 1.555 strain saccharomyces albicanses	250.0 62.5 ＞2000.0 31.25 31.25 62.5 1.8 3.6 500 ＞2000.0 62.5	500.0 125.0 ＞2000.0 31.25 62.5 125.0 7.8 7.8 1000.0 2000.0 125.0

8.3.2 1, the 4-naphthalenediol sees Table 4 to MIC, MIC50, MIC90 and the MBC of clinical isolates strain.

Table 41, the 4-naphthalenediol is to MIC, MIC50, MIC90 and the MBC of clinical isolates strain

Unit: μ g/ml

Bacterial strain	The strain number	The MIC scope	MIC50	MIC90	The MBC scope
						Golden yellow Portugal bacterium epidermis Portugal bacterium streptococcus faecalis micrococcus gonococcus is read coccus in vain	1510 50 30 25 32	31.25-125.0 15.125-60.5 62.5-250.0 3.6-15.125 31.25-125.0	150.9 139.6 179.4 5.03 187.1	193.4 164.3 221.3 13.24 223.2	62.5-250.0 31.25-125.0 62.5-500.0 6.2-31.25 62.5-500.0

8.3.3 condition of culture is to 1, the 4-naphthalenediol the results are shown in Table 5,6 to the influence of the MIC of epidermis staphylococcus A TCC26069 strain, gonococcus 29106 strains.

Table 5. pH influences result of the test to MIC

Bacterial strain	MIC(μg/ml)
				pH5.7	pH7.2	pH8.5
	29106 strains of staphylococcus epidermidis ATCC26069 strain gonococcus	31.25 -	31.25 1.8				31.25 -

Table 6. bacterial load influences result of the test to MIC

Bacterial strain	MIC(μg/ml)
				106	108	1010
	29106 strains of staphylococcus epidermidis ATCC26069 strain gonococcus	31.25 1.8	31.25 1.8				31.25 1.8

From table 5, as seen, when the acid-base value of culture fluid during at pH5.7 and pH8.5, gonococcus is not grown in 6.But do not influence the MIC of staphylococcus epidermidis ATCC26069 strain; Change bacterial load, do not influence 1, the 4-naphthalenediol is to the MIC of epidermis staphylococcus A TCC26069 strain, gonococcus 29106 strains.

8.3.4 1, the 4-naphthalenediol sees Table 7 to the killing curve of gonococcus 29016.

Table 71, the 4-naphthalenediol is to gonococcal killing curve tables of data

Action time	5’	10’	20’	40’	1h	2h	4h
								1g(CFU/ml)	5.010	5.001	5.001	4.478	4.025	0.327	0.000

1,4-naphthalenediol concentration: 15.6 μ g/ml

Gonococcus 29016 strain bacterial population: 105CFU/ml

From table 7 as seen, 1, the 4-naphthalenediol is under two times of MBC (15.6 μ g/ml) concentration conditions, mainly between effect 40 minutes to 2 hours, number of viable sharply descends around here, acts on that antibacterial is killed fully after 4 hours to gonococcal bactericidal action.

Conclusion:

This test has selected for use common antibacterial of clinical urogenital infections and fungus as test strain, adopts doubling dilution to measure 1, and the 4-naphthalenediol is to the MIC and the MBC of 11 reference cultures and 387 strain clinical separation strains.Result of the test shows, 1, and the 4-naphthalenediol all has obvious suppression and deactivation to testing the common antibacterial of selected urogenital infections except that bacillus pyocyaneus, and especially particularly outstanding to the antibacterial and bactericidal action of gonococcus, MIC reaches Gamma Magnitude.But it is not obvious to the inhibition and the deactivation of lactobacillus in the experimental concentration scope.This results suggest 1,4-naphthalenediol are when suppressing pathogenic bacteria (fungus) and killing and wounding, to no obvious lethal effects such as the normal flora bacterium of vagina such as vagina lactobacilluss.

The supporting self-destructed disposable vagina special syringe of female hundred health contraceptive cream (KBK) preparations and applicant and this medicine combines use, become disposable external applied contraceptive and utensil, so both can improve the quality and the economic benefit of external contraceptive medical instrument, and can overcome the deficiency and the defective of medical instrument in the past again.

Claims (4)

1.1 the 4-naphthalenediol is used to prepare the purposes of the external use sperm killing contraceptive medicine of preventing and treating reproductive tract infection.

2. purposes as claimed in claim 1 is characterized in that, and is described 1, and the structural formula of 4-naphthalenediol is:

Chemical molecular formula: C ₁₀H ₈O ₂, molecular weight: M=160;

Chemical molecular formula C ₁₀H ₈O ₂Labelled amount in medicine＞98.0%; Mp:192 ℃-194 ℃; Residue on ignition＜0.1%; Moisture content＜0.05%; Dissolubility is 0.04 ‰ in water.

3. purposes as claimed in claim 2 is characterized in that, and is described 1, and 4-naphthalenediol and cream base are made oil-in-water type half viscous fluid sample preparation, and the volume of every minimum package unit is 3ml, and medicament contg wherein is 2mg, and all the other are cream base.

4. purposes as claimed in claim 3, it is characterized in that described cream base is made of tristerin, stearic acid, liquid Paraffin, white vaseline, lanoline and triethanolamine, sanlose CMC-NA, polypropylene carboxylic acids macromolecular compound PA and distilled water.

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