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Description  |
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FIELD OF THE INVENTION
The present invention relates generally to medicine and more specifically
it concerns a medicinal preparation for individual prevention of venereal
diseases and treatment of urogenital trichomoniasis.
The herein-proposed medicinal preparation will find application in
dermatovenereological and gynecological practice.
BACKGROUND OF THE INVENTION
At present a number of medicines are in widespread use in medicine for
individual prevention of venereal diseases, such as silver-containing
drugs, potassium permanganate, and mercury preparations, e.g., mercury
dichloride solution, mercury subchloride ointment and Dublosan ointment.
With the purpose of preventing syphilis and gonorrhea a medicinal
preparation Oxyviridol has been suggested, consisting of 0.035 mass
percent mercuric oxycyanide and 50 mass percent green soap in glycerine
(cf. `Medical prescription reference book` compiled by M. Kh. Bergolts,
1952, Medgiz Publishers, Moscow). However, the aforementioned drug
possesses inadequate bactericidal potency against syphilis and gonorrhea
pathogens, produces a considerable irritating effect on urethral mucosa,
causes desquamation and lysis of the epithelial lining, and is therefore
unsafe in clinical uses. The medical preparations mentioned above fail to
find extensive application due to their inadequate reliability, whereas
mercury-based drugs cannot be guaranteed as safe for human organism.
There has been proposed within recent years a 0.05-0.1 percent solution of
Chlorhexidine (Hibitane) as a preventive of venereal diseases. The drug
possesses an adequate bactericidal activity in experiments in vitro and in
tests on animals (cf. `The dermatology and venereology herald`, No. 6,
1978, Moscow, On personal prevention of venereal diseases by I. M.
Ovchinnikov et al., p. 49 (in Russian). However, clinical application of
the aforesaid drug is accompanied by an irritating and withering effect
upon the skin and genital mucosa in man. Besides, industrial production of
Hibitane is a complicated and highly labour-consuming task.
Applied for topical (external) treatment of urogenital trichomoniasis are
boric acid, lapis, zinc sulphate, copper sulphate, laevomycetin,
Acetarsol, and others. However, these drugs are insufficiently efficacious
and fail therefore to find windspread application.
At the present time imidazole medicines are most efficient for treatment of
the aforesaid disease, e.g., Tinidazole, Metronidazole and Trichopol.
However, their clinical use is attended by ever increasing resistance of
trichomonads to the action of these drugs, which in turn is responsible
for relapsing of a specific process (up to 8.4 percent of all cases) and
for the onset of posttrichomonal phenomena. The drugs may be causative of
some side effects, and are contraindicated in cases of hemopoietic
disturbances, or active CNS affections.
SUMMARY OF THE INVENTION
The present invention has for its object to provide a medicinal preparation
for individual prevention of venereal diseases and treatment of
trichomoniasis that would have a convenient form of issue by selecting
such chemical compounds as an active principle that would impart high
efficacy to the present medicinal preparation and at the same time prevent
side effects upon its application.
The aforesaid object is accomplished by providing a medicinal preparation
for individual prevention of venereal diseases and treatment of urogential
trichomoniasis, comprising an active principle and a pharmaceutical
excipient, which medicinal preparation, according to the invention,
incorporates an efficient quantity of the active principle which is
essentially a synergic mixture comprised of paranitro-alpha-chlorocinnamic
aldehyde of the following formula:
##STR2##
and dimethylsulphoxide.
Chemical compounds that make up the mixture proposed as an active
principle, feature different modes of action which establish, in
combination, the potentiating synergism which results in complete
destruction of the protoplasm and cell membrane of the pathogens of
syphilis, gonorrhea and trichomoniasis, thus imparting quickly expressed
bactericidal and protistocidal effect to the medicinal preparation
involved. The pathogens are disintegrated under the effect of the drug as
fast as within the initial three minutes. The preparation possesses high
efficacy both in prevention of venereal diseases and in treatment of
urogenital trichomoniasis.
It is expedient to use polyethyleneglycol having a molecular mass of 400 as
the pharmaceutical excipient, since it is toxic-free and proves to be an
osmotically active substance promoting manifestation of the most
biological potency in the active principle.
The drug comprising such an excipient produces no local irritating effect
on skin and mucosa nor does it possess a systemically toxic or allergic
action.
It is recommended that the present medicinal preparation be applied
clinically in a liquid form with the following ratio of components (in
mass percent):
______________________________________
para-nitro-alpha-chlorocinnamic
0.3
aldehyde of the aformentioned formula
dimethylsulphoxide 5.0
polyethyleneglycol with a molecu-
to make up
lar mass of 400 100
______________________________________
Such a medicinal preparation features the most reliable preventive effect
and possesses high antitrichomonal activity. A liquid medicinal form of
the preparation enables its best contact with the pathogens that have got
on the skin and mucosa.
It is recommended that the present medicinal preparation be applied for
individual prevention of venereal diseases and treatment of urogenital
trichomoniasis, particularly in women, in a liquid form dispensed in
aerosol containers under pressure, its components being taken in the
following ratio (in mass percent):
______________________________________
para-nitro-alpha-chlorocinnamic aldehyde
0.3
of the aforementioned formula
dimethylsulphoxide 5.0
polyethyleneglycol with a molecu-
20 to 30
lar mass of 400
surfactant 3.0 to 6.0
water to make up 100
______________________________________
as well as a propellant in an amount of 3 to 10 percent of a total mass of
the components.
Such a medicinal form makes it possible not only to provide an antivenereal
effect whenever it becomes necessary but also enables female patients
wanting treatment for urogenital trichomoniasis, to carry out therapeutic
procedures by themselves.
There is also proposed a method for treatment of urogenital trichomoniasis,
comprising application of the medicinal preparation, according to the
invention, to human genital mucosa in a dose of 5 to 10 ml twice a day for
5 to 7 days.
Such a treatment method proves to be the most efficient in cases of
uncomplicated urogenital trichomoniasis, though it is also applicable
against ascending forms of trichomoniasis provided there is a necessary
access for the medicinal preparation, according to the invention, to the
affected area. Such a treatment method is attended by no side effects
whatever, including those accompanied by local-irritating, systemically
toxic or allergizing action.
DETAILED DESCRIPTION OF THE INVENTION
The herein-proposed medicinal preparation appears as a thick transparent
liquid coloured from light yellow having a specific faint odour and stable
when under storage in a light-protected place.
The drug may be dispensed in hermetically sealed 5 to 10 ml capacity
flasks, or in aerosol containers.
The medicinal preparation for prevention of venereal diseases and treatment
of trichomoniasis proposed according to the present invention is prepared
by mixing the efficient amounts of para-nitro-alpha-chlorocinnamic
aldehyde of the following formula:
##STR3##
and dimethylsulphoxide till formation of a synergistic mixture in the form
of a solution, followed by bringing the preparation to a preset
concentration using a pharmaceutical excipient, viz., polyethyleneglycol
having a molecular mass of 400, which has been selected due to its being
excellently compatible with the active principles and low toxic. However,
some other pharmaceutical excipients existing nowadays may be used for the
purpose, such as glycerine, or propyleneglycol, 1,2-propanediol.
In order to obtain the medicinal preparation, according to the invention,
in a liquid form under pressure dispensed in aerosol containers, added to
the aforedescribed solution are a surfactant (3 to 6 mass percent) and
water (to make up 100 mass percent), and the resultant solution is
saturated, in an aerosol container, with a propellent taken in an amount
of 3 to 10 percent of the total mass of the components. The amounts of the
aforesaid substances are selected so that quickly destructable foam be
produced upon application of the preparation.
The medicinal preparation, according to the invention, has been subjected
to preclinical trials including investigation of its bactericidal and
protistocidal potency in experiments in vitro and in tests with
experimentally induced syphilis in rabbits; the drug has been studied for
safety, including tests for local irritating effect, allergizing action,
influence upon the immune system, as well as for some aspects of
pharmacokinetics.
The preparation in question has been studied in concentration of 0.075,
0.15 and 0.3 percent. The concentration of the drug is determined
according to the amount of para-nitro-alpha-chlorocinnamic aldehyde of the
aforementioned formula, contained therein, with a constant quantitative
content of dimethylsulphoxide (5 percent) and an equivalently-varying
content of polyethyleneglycol having a molecular mass of 400. The drug has
been studied for safety with a concentration of 0.3 percent.
In order to study the drug for gonococcocidal properties a pure gonococci
culture is isolated from gonorrheal patients and seeded onto a great
culture medium area (in matrasses). Then the 24-h culture is washed off
from the matrasses with a sterile isotonic sodium-chloride solution, and a
suspension is prepared from the obtained washing, containing one thousand
millions of microbial bodies (according to the turbidimetric standard).
The resultant suspension is dispensed in 0.5-ml sterile test tubes,
whereupon added to each of them is 0.5 ml of the preparation under study
having a doubled concentration so as to obtain a desired concentration
under study after mixing the ingredients for 5 minutes. The mixture is
then subjected to centrifugation at 1500 to 3000 rpm for 10 to 15 minutes
to wash gonococci from the preparation. Then the supernatant liquid is
removed and 1 ml of an isotonic sodium-chloride is added to the
precipitate to obtain the initial volume, whereupon the mixture is
centrifugated once more for repeated washing-off. The supernatant liquid
is removed again, and the precipitate is seeded onto an agar-slant
nutrient medium (ten test tubes with the nutrient medium per strain of the
gonococcal culture). Then the test tubes containing the nutrient medium
are placed in a temperature-controlled cabinet at 37.degree. C. in an
atmosphere with an increase content of carbon dioxide gas. The rate of
growth of the gonococcal culture is determined every 24 hours within four
days.
Taken as the control is a mixture of equal amounts of a suspension of the
gonococcal culture and an isotonic sodium-chloride solution (0.5 ml each),
which mixture is dispensed in test tubes, two times exposed to
centrifugation (as in the test tubes of the experiment) and the
precipitate is seeded onto a nutrient medium equivalent as to composition.
Used as a nutrient medium is plain (meat-infusion) agar prepared from
rabbit meat and doped with casein hydrolysate, yeast autolysate, and
preservative free large-cattle serum, as well as a lyophilic medium.
Studies into the effect of the medicinal preparation under consideration
upon Treponema pallidum and vaginal trichomonads in experiments were
carried out as follows.
Treponema pallidum was obtained from eight- or ten-day specific orchitis in
rabbits and from patients affected by contagious forms of syphilis. There
were studied tissue cultures of Nichols's strain and No. 8 strain of the
Central dermatovenereological research institute (TsKVI). The cultures of
vaginal trichomonads were isolated from patients suffering from urogenital
trichomoniasis and grown on an artificial Johnson-Trussell medium.
The experimental procedure consisted in mixing equal volumes of a
suspension of Treponema pallidum (or a culture of trichomonads) and the
preparation under study, taken in a double concentration since the latter
is reduced twofold upon mixing the ingredients. Then the native
preparations are made from the resultant mixture and are exposed to
microscopy in a darkened (for Treponema pallidum and Trichomonas), or a
phase-contrast (for vaginal trichomonads) field of a microscope. Action of
the preparation under test was judged by the degree and rate of its effect
on the mobility and morphology of the pathogens. The effect was considered
positive when complete motionlessness of the pathogens occurred at once,
i.e., at the instant when the preparation was made, or within the
following 3 to 5 minutes for Treponema pallidum and 5 to 10 minutes for
trichomonads. The effect of the drug under test on the morphology of
Treponema pallidum and Trichomonas was considered still more positive if
the pathogens lost shape within the abovementioned period of time (i.e.,
the Treponuma pallidum pathogens became thinner, their spirals were
smoothed out, some bulging individuals occurred, as well as debris, or the
pathogens were lyzed completely; vaginal trichomonads lost their
flagellas, got disintegrated and lyzed).
Another version of studies into the treponemicidal effect of the drug under
test consisted in successively applying a droplet of the suspension of
Treponema pallidum and a droplet of the medicine under test to the stage
of a microscope, whereupon the mixture was carefully intermixed with the
pointed end of a Pasteur pipette, covered with a cover glass and was
immediately observed in a dark field of the microscope. Such a study made
it possible to reveal the effect of the drug under test on treponemes as
early as within the initial 20 to 30 seconds following the contact between
the suspension and the drug. Served as the control of these tests was a
mixture of equal volumes of a suspension of Treponema pallidum (from 20 to
30 up to 80 microorganisms in the field of the microscope, or of that of
vaginal trichomonads and an isotonic sodium chloride solution. Microscopic
specimens were examined in a dark field of the Biolam microscope and with
the microscope available from Opton Co using a darkfield and a
phase-contrast substage condenser, as well as objective lenses
(25.times.0.45; 40.times.0.6).
Studies in the preventive action of the drug in tests with experimentally
induced syphilis were carried out on sexually mature well nourished 2.5 to
4 kg male rabbits of the various races and colours (chinchilla, white and
silvery grey giant).
Before experiments all test rabbits had been under observation for at least
one month so as to rule out spontaneous spirochetosis in the test animals.
All the test rabbits were examined for syphilis with the use of a complex
of serologic tests.
Preventive effect of the drug under test has been studied in two parallel
running versions of tests with experimentally induced syphilis in rabbits.
The first version involved studies in the treponemicidal effect of the drug
upon intracutaneous administration of a mixture of the suspension of
Treponema pallidum and of the drug under test. With the purpose in view
the test rabbits were fixed in a routine way and 0.75 ml of a mixture
(equal volumes of the suspension of Treponema pallidum and the doubled
concentration of the drug under test, since the concentration of the
latter was reduced twofold upon mixing with the suspension) was injected
intracutaneously in either of the scrotal halves.
The test animals of one of the groups were given the aforesaid mixture just
after a ten-minute contact of the mixture of ingredients and their
intermixing with a glass stick, while the test rabbits of the other group
were administered the mixture after its having been centrifugalized and
upon a ten-minute contact of a quantity of a sterile isotonic NaCl
solution (washing off from the drug under test).
The aforediscussed studies were performed with a view to ruling out any
possibility of inactivation (by precipitation) of the drug in a protein
medium of the animals' organism and of destroying its bactericidal effect.
Centrifugation of the suspension of Tremponema pallidum before its
administration was aimed at determination of adequacy of the
treponemicidal effect of the drug after its ten-minute action on pallid
treponemes. The control rabbits were given intracutaneously in the scrotal
region a mixture of equal volumes of a suspension of pallid treponemes and
an isotonic NaCl solution.
The second, principal version of experiments involved those, wherein the
syphilis infecting conditions of the test rabbits to some extent
approximated routine syphilis infecting conditions in man. With that aim
the animals were infected with a suspension of Treponeme pallidum in an
isotonic NaCl solution by rubbing the suspension into the scarified
scrotal skin. Preliminarily the scrotal skin was to be wiped with cotton
wad wetted first in ethanol, then in ether. Then the scrotal skin is
stretched to a maximum extent with the fingers of the experimenter's left
hand, while the skin of the both scrotal halves was scarified by the
experimenter's right hand using fine-grained emery paper. The scarified
skin area should be 1 to 1.5 sq.cm and should appear as a rough surface
showing some tissue fluid emerging as pin-points. Bleeding must be
avoided. Then the freshly prepared suspension of the syphilitic pathogen
containing 20 to 25 treponemes in the field of a microscope, was applied,
in an amount of 0.1 ml, to the scarified areas of the scrotal skin and was
rubbed with a sterile glass stick for 1 to 1.5 minutes. Next the scrotal
skin was pinched with two Pean's forceps at a distance not shorter than
0.5 cm from the place of application of the inoculative material, and the
two forceps were crossed together, thus establishing a skin fold which
protects the treponemes against getting dried, so as to provide the best
conditions for infecting (after S. T. Pavlov). The forceps remained
crossed-together for 1 to 1.5 hours, whereupon they were removed.
The test rabbits were subjected to disinfecting treatment with the drug
under trial in different periods of time after infecting (i.e., after 15
minutes, 1, 2, 3 hours). The treatment consisted in applying 0.1 ml of the
drug under test to each of the infected scrotal skin areas and carefully
rubbing it into the skin with a sterile glass spatula for 1 to 1.5
minutes. No disinfecting treatment was given to the control rabbits.
The test animals were observed for 4 to 12 months with weekly clinical
examination and monthly serologic examination using a complex of serologic
tests for syphilis, i.e., Wassermann reaction with cardiolopin lipoid and
treponemal antigens, Kahh and Sachs-Witebsky precipitation tests,
immunofluorescence test (RIF-10 and RIF-200), and microprecipitation test
(RMP).
Reliability of preventive protection of the test rabbits against syphilis
was judged by the absence of any clinical manifestations of the disease in
the animals and by the negative serologic tests, as well as by the
negative results of transplanting the popliteal lymph nodes from the test
animals to the indicator rabbits in two successive (serial) passages.
Considered as the final evidence of the drug preventive action were the
positive results of reinoculation of the test and passage rabbits with a
homologous strain of Treponema pallidum.
In all experiments there was used freshly obtained suspension of Treponema
pallidum of Nichols's strain.
Given below are the results of experimental studies in the specific
activity of the medicinal preparation of the invention.
Bactericial effect of the drug upon Neisser gonococci
The drug was established to completely inhibit the vital activity of the
gonococcal cultures in experiments with the strains of pathogens isolated
from 7 female and 5 male patients. None of the experimental test tubes
exhibited any growth of gonococcal culture, whereas all the control test
tubes demonstrated their vigorous growth.
Treponemicidal activity of the drug
It has been established experimentally that nearly instant motionlessness
occurs in treponemes under the effect of the drug of the invention,
accompanied by destruction of the syphilitic pathogens till their complete
lysis as early as within the initial three minutes of their contact with
the drug. As fast as on the first minute of contact there are detected
sporadic fragments of the destroyed treponemes, there appear long
motionless thinned `filaments` and `shadows` resulting from smoothing out
of the helices, while the majority of the microslides exhibit aggregation
of an amorphous protein substance appearing as small fragments of the
lyzed pathogens. A similar destructive effect is exerted by the drug
involved upon the treponemes of both the Nichol's strain and No. 8 strain
of TsKVI. No differences whatever have been identified in the degree of
the treponemicidal activity of the drug against the pathogens isolated
directly from the patients suffering from infecting forms of syphilis. The
treponemicidal effect is confirmed also in tests with experimentally
induced syphilis in rabbits. A total of 120 male rabbits have been used in
such tests. 100 percent of the test animals have been protected against
syphilitic infection due to preventive treatment of the infected spot with
the preparation within 15 minutes to 3 hours after application of the
inoculative material to the scarified scrotal skin of the test rabbits. At
the same time there have been obtained positive results of syphilis
prevention upon intracutaneous infection of the test rabbits with a
mixture of a suspension of treponemes in an isotonic NaCl solution taken
in the same volumetric ratios.
Reliability of preventive protection of the test rabbits against syphilis
is judged both by the absence of clinical manifestations of the disease in
the animals and the negative serologic tests for syphilis within a
follow-up period from 4 to 12 months and by the negative results of
transplanting the popliteal lymph nodes from the test rabbits to the
indicator rabbits in two serial passages, i.e., first from the test
animals to the first-passage indicator rabbits, and then from the latter
to the second-passage indicator rabbits. The positive results of
reinoculation of both the test and passage animals with a homologous
strain of Treponema pallidum.
Prevention of the test animals against syphilis upon intracutaneous
infection gives evidence that a ten-minute exposure for contact of the
drug with the treponemes is quite sufficient for complete inhibition of
vital activity of the syphilitic pathogen. On the other hand this can be
considered as the demonstration of stability of the drug in a protein
medium, that is, the tissues of the rabbits organisms.
Effect of the drug on Trichomonas vaginalis
High protistocidal activity of the drug has been established in vitro
experiments with 16 strains of vaginal trichomonads isolated from patients
suffering from urogenital trichomoniasis (13 females and 3 males), a total
of 256 microspecimens being subjected to examination. Vaginal trichomonads
are found to get immobilized and to lose their flagellas upon being
exposed to the effect of the drug as early as on the first minute of
exposure. It is within the next 2 or 3 minutes that complete
disintegration of this protozoan occurs, and only some individual
microspecimens exhibit sporadic motionless nondisintegrated trichomonads
devoid of the flagellas.
The drug has been tested for safety by determining its acute and chronic
toxicity, local irritating effect on the skin and mucosa, allergizing and
immunogenic properties. There has been studied also pharmacokinetics of
the drug upon topical administration and application to mucosa.
Acute toxicity of the drug has been determined on four animal species.
There were used in four runs of experiments a total of 150 albino mice
having an average mass of 18.5 g. 18 albino rats (165 g), 18 rabbits (3000
g), and 12 Guinea pigs (400 g). A single dose of the drug under test was
calculated proceeding from a future drug application for practical aims
(0.07 ml per kg body mass of a conventional human being having an average
mass of 70 kg).
Taking account of the specificity of the drug practical application (i.e.,
for smearing the skin and administering into the urethra) toxicity of the
drug under consideration has been studied not only by intraperitoneal
administration to albino rats and albino mice but also in topical
application and intracavitary administration (intravaginal medication in
females and intrarectal administration in males) to albino rats, Guinea
pigs and rabbits. Visual observation of the state of the experimental
animals was carried out from the very instant of application of the drug
under test and lasted for 14 days.
Studies of the drug in question for chronic toxicity have been carried out
on two species of laboratory animals, i.e., 24 albino rats (170 to 200 g),
and on 24 rabbits of the Chinchilla race (2.5 to 3.0 kg). The drug was
administered daily for one month. A single dose of the drug was evaluated
in the same way as in studying drug's acute toxicity, with due account of
its practical application for preventive purposes.
The experimental animals of one of the groups (6 rabbits and 6 rats) were
given the drug in the aforementioned dose daily for one month, while the
animals of the other test group incorporating the same quantity of the
same species were given a subtoxic dose of the drug ten times the
therapeutic dose, i.e., 0.7 ml/kg daily within the initial two weeks;
beginning with the third week and up to the end of the month the rabbits
were given the drug in a dose of 2,1 ml/kg, whereas the rats were given
the drug in a dose of 3.5 ml/kg, the latter two doses exceeding the
therapeutic dose by 30 and 50 times, respectively. One of the groups of
rabbits and rats (six animals of each species) served as the control, the
animals of the group being given an isotonic NaCl solution instead of the
drug under study. The drug administration route in the experimental
animals was maximally approximated to the drug administration techniques
in man. Female animals were given the drug intravaginally and
intrarectally (one-third of a total dose); besides, the drug was smeared
on the dehaired skin of the inner femoral and abdominal surface
(two-thirds of a total dose) and rubbed into the skin for one minute; male
animals were given the drug intrarectally (one-third of a total dose), and
drug was rubbed into the skin of the scrotum, abdomen and inner femoral
surface (two-thirds of a total dose).
Throughout the entire period of observation over the experimental animals
there were noted their general status, behaviour, dynamics of their mass
and rectal temperature. Studies were made in the drug effect on the
functions of the CNS, cardiovasular and respiratory systems, as well as on
those of the liver, kidneys, endocrine glands, blood, including
biochemical studies into the activity of the heart, lungs, liver, kidneys,
and endocrine glands. On termination of a monthly course of the drug
adminstration the experimental animals were sacrificed, whereupon there
were performed histomorphologic examinations of the brain and spinal cord,
the heart, lungs, blood vessels, liver, kidneys, pancreas, gonads, blood,
and hemopoietic organic (spleen and lymph nodes). In addition, there were
examined those areas of the skin and vaginal and rectal mucosa to which
the drug had been administered, and the weight coefficients of the
internal organs were determined. The body mass and the rectal temperature
were measured once a week. Other physiological characteristics were
registered before and after the experiment. Biochemical examinations were
carried out three times, i.e., prior to the experiment, in two weeks and
in one month that is, after the experiment had been terminated.
Chronic toxicity in the test animals was judged by the following indices;
external appearance and behaviour; body temperature and mass dynamics;
physiological tests (the `open field` technique, hexenal-induced sleep
test, ECG); clinical urine analyses; integral morphological peripheral
blood indices; biochemical blood indices (determining total, direct and
indirect bilirubin, creatinine, urea, residual nitrogen,
aminotransferases, activity of alkaline phosphatase, activity of serum
cholinesterase, alpha-amylase, total protein, thymol turbidity test,
determining of C-reactive protein); determining the following
microelements: potassium, sodium, calcium, chlorides; pathomorphological
examinations of the internal organs of the experimental animals.
The functional activity of the CNS was studied using the `open field`
technique by taking notice of the motor activity and behavioural reaction
of the experimental rats within a 3-minute period by the number of runs,
washes and sets they perform. Besides, the functional status of the CNS
was assessed by the duration of a latent period in the hexenal-induced
sleep test (by intraperitoneal administration of a 0.7-percent hexenal
solution in a dose of 70 mg/kg).
Electrical activity of the heart was registered with the aid of
electrocardiograph ELKAR 087 in standard lead II at a tape advance speed
of 50 mm/s. Processing of electrocardiograms involved the heart rate
value, duration of a complete cardiac cycle R-R, intervals, QRS, QRST and
also the voltage value of the Q, R, S, T waves.
Water-excretory and concentratory renal functions were assessed by the
volume of the daily diuresis following a water load (5 ml of an isotonic
NaCl solution per 100 g animals' body weight) and by the specific mass of
urine. The nitrogen-excretory function of the kidneys was assessed by the
concentration of residual nitrogen, urea, as well as by the blood serum
creatinine content.
Blood specimen was taken from the several tail end of the test rats and
from the marginal auricular vein of the test rabbits. Hemoglobin was
determined photometrically according to a unified hemoglobin-cyanide
method using a standard set of reagents. The erythrocyte count was
determined photometrically with the FEK-56M apparatus; first there had
been established photometrically and by determining an absolute amount or
erythrocytes in Goryaev's counting chamber that 0.1 unit of an optic
density corresponds to 1.217 million erythrocytes per 0.01 ml blood.
Erythrocyte sedimentation rate, total leukocyte count and leukocytic
picture were determined. The blood content of bilirubin, creatinine, urea,
aminotransferases, total and C-reactive protein was determined by the
unified methods using standard sets of reagents.
Specimens of internal organs for pathomorphological examinations were taken
from decapitated test animals (rats), or from those sacrificed by means of
aeroembolism (rabbits). The sampled specimens were fixed in a 10-percent
neutral formalin solution and stained with hematoxylin-eosin.
Simultaneously there were measured the mass and weight coefficients of
internal organs.
Local irritating effect of the drug on the skin and mucosa was studied in
three runs of experiments. In the first run there were employed 35 Guinea
pigs and 30 rabbits of the Chinchilla race to which the preparation was
administered by being rubbed into the dehaired skin areas and medicated
into the conjunctival sac or both eyes (once and twice a day for 10 days).
In the second run of experiments (8 rabbits) there was studied local
irritating effect of the drug on the mucosa of the animals' eyes when
given in a concentration ten times that recommended for preventive
purposes. Likewise the first run of experiments the drug was instilled in
both eyes one or twice a day at 2 or 3-hour interval for 10 days.
In the third run of experiments (aimed at studying chronic toxicity) the
local irritating effect of the drug was studied under the conditions to a
maximum extent approximating the practical routine of its application. The
drug was administered daily to the test rabbits (12 animals) and the test
albino rats (12 animlas) by way of application to the skin and through the
vagina (in females) and the rectum (in males) within a period of one
month.
Apart from daily observation of the experimental animals' behaviour and of
the reaction on the part of the skin and mucosa, the skin and mucosal
areas that had been medicated with the drug were removed for histologic
examinations upon termination of the experiments.
The allergizing effect of the drug was studied on Guinea pigs, since these
animals proved to be the optimum subject for artificial sensitization (Ado
A. D., 1970). The animals of the experimental groups were given the drug
daily for six days either by smearing the skin with the preparation or by
injecting it with a syringe intracavitary (i.e., intravaginally to females
and intrarectally to males). The drug doses administered were either
therapeutic (0.07 ml/kg) or exceeded the therapeutic ten or fifteen times
(0.7 and 1.2 ml/kg, respectively). The challenge dose equal to the summary
sensitizing one, was administered in 21 to 23 days. A total of 24 Guinea
pigs were used in the experiments (four groups containing six animals
each, and one control group). The degree of the allergizing effect was
judged visually by such symptoms as hyperemia, microvesiculation, skin and
mucosa infiltration, inadequate behaviour of the test animals, disturbed
gait, tremor, etc.) | | |
