Topical pharmaceutical preparations containing chitin soluble extract

There are provided pharmaceutical compositions for topical application for preventing or at least substantially decreasing infections by yeasts of the genus Candida. These are based on a chitin soluble extract (CSE), which is obtained by the extraction of chitin by water with stirring. There are used ointments or lotions on a water-base.

FIELD OF THE INVENTION 
The invention relates to pharmaceutical compositions for topical 
application for the prevention or for substantially decreasing infections 
by yeast and especially by Candida. 
BACKGROUND OF THE INVENTION 
The incidence of the infections by Candida, and especially by C. albicans, 
is a high one. Such infections exist as vaginal infections and as 
infections of the oral cavity, especially as denture stomatitis. There 
exists a wide variety of drugs for the treatment of such infections, which 
have various drawbacks. The present invention relates to compositions 
which substantially decrease adherence of Candida to epithelial cells. 
SUMMARY OF THE INVENTION 
The invention relates to pharmaceutical compositions for topical 
application to certain tissues, preventing adherence of Candida to such 
tissues, thereby substantially reducing the rate of infection by such 
yeasts. The active ingredient of the compositions is a substance defined 
herein as chitin soluble extract (CSE), prepared from commercially 
available chitin or from chitin extracted from C. albicans. The CSE 
derived from C. albicans is more active than the CSE derived from 
commercially available chitin. CSE can be advantageously prepared from 
various sources of chitin as set out in greater detail in the following 
experimental section. 
Typically a suspension of commercially available chitin can be incubated in 
PBS, and the CSE can be obtained from the supernatant, or this supernatant 
can be used as such. 
Candida chitin can be prepared from blastospores of C. albicans by boiling 
with hydrochloric acid, washing, boiling with a strong base. From the 
resulting chitin, CSE can be obtained. 
The thus obtained CSE has a high biological activity. It prevents both in 
vitro and in vivo adherance of yeasts to epithelial cells. Thus, such 
preparations can be used to prevent vaginal infections by C. albicans, or 
for the prevention of denture stomatitis caused by such yeasts and for 
similar applications. 
The activity of CSE derived from C. albicans is greater by a factor of 
about 10 to 20 times than that of CSE derived from commercially available 
chitin. 
The pharmaceutical compositions of the invention are used in the form of 
water-based ointments, as lotions, as tampons imbued with the active 
substance etc. Generally a quantity of from 1 to 5 mg/ml of CSE is 
adequate, and small quantities of such compositions are sufficient to 
prevent infection by C. albicans and similar yeasts. The substance defined 
as CSE is water soluble with a solubility greater than 25 mg per ml water, 
it is heat stable and has a MW in excess of 10,000.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
Methodology 
The active material (CSE) can be prepared from commercially available 
chitin as follows; and the thus obtained material was used for studies in 
vitro and in vivo. 
Preparation of Chitin Soluble Extract (CSE) 
A 20-percent chitin (Simga) suspension is PBS was incubated at room 
temperature for 5 h under constant shaking. The supernatant designated as 
CSE was then removed, dialysed overnight at 4.degree. C. against sterile 
water and lyophilized. The yield of CSE in most of the preparations was 
1-2 mg of lyophilized material per ml of supernatant. 
Preparation of Chitin Soluble Extract (CSE) from Candida 
Cultures of blastospores of C. albicans, harvested at the logarithmic 
growth phase were washed 3X with phosphate buffer (PBS), boiled 90 min 
with 1 N HCL, washed 3X with PBS, boiled with 1N NaOH for 90 min, washed 
3X with PBS to a neutral pH and an insoluble pellet of chitin was obtained 
which was identified by the Morgan Elson test for identification of 
amino-sugars. This was agitated for 5 hours with water at room 
temperature. The product was lyophilized to obtain chitin powder. The 
yield was about 2 mg lyophilized material per ml of supernatant. 
Induction of Experimental Canidal Vaginitis in Mice 
The in vivo adherence of Candida to mucosal surfaces was studied in 
experimental vaginitis in mice. Six-week old female ICR mice (out-bred 
white mouse strain) were inoculated intravaginally with C. albicans 
(various concentrations; see "Results") using a wooden spatula. Mice were 
inoculated at different stages of the hormonal cycle: at oestrus, 
metoestrus, diestrus and proestrus stages. Duration of the oestrus cycle 
in rodents is 4-5 days. 
Microscopic examination of Grain-stained or unstained vaginal smears taken 
from the inoculated mice was carried out in order to assess attachment of 
yeasts to exofoliated epithelial cells and to assay the development of 
hyphal elements at different times post-inoculation. In each smear, 100 
epihelial cells were counted and epithelial cells with 20 or more 
attaching yeasts were considered adhering cells. Smears revealing at least 
50 epithelial adhering cells and/or cells with hyphal elements on their 
surface were an indication of infection. 
Vaginal discharges taken at various times post-inoculation were cultured on 
Sabouraud dextrose agar. Vaginal tissue section (5 .mu.m) from infected 
mice were stained with Periodic acid Schiff (PAS) reagent and examined 
histopathologically. 
RESULTS 
Inhibition of In Vivo Adherence 
Inhibition of in vivo adherence was achieved by treating the mice with 
various inhibitors which were administered topically in varying 
concentrations as vaginal rinses (50 .mu.l, using Eppendorf tips) followed 
immediately by the inoculation of yeasts. Control animals were rinsed with 
PBS prior to inoculation of the yeasts and the control and test animals 
were compared for development of infection. 
Effect of CSE on In Vitro Adherence 
The addition of CSE (table 1) resulted in significant inhibition of the in 
vitro adherence of the yeasts. Lyophilizates prepared from the supernatant 
and added at a concentration of 2.5% W/V had an effect similar to that of 
the supernatant (table 1). 
TABLE I 
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Effect of CSE, on in vitro adherence 
of C. albicans to human vaginal epithelial cells. 
Percentage of adherence 
alone (a) or in the 
presence of substance (b) 
Percentage of 
Substance a b inhibition 
______________________________________ 
Supernatant from 
45.3 .+-. 6.6 
14.8 .+-. 6.1.sup.(1) 
67.4 
chitin suspension 
Lyophilizate of 
41.3 .+-. 2.3 
10.0 .+-. 3.6.sup.(1) 
51.6 
supernatant.sup.(2) 
______________________________________ 
.sup.(1) Adherence significantly lower than appropriate control = p 0.05 
(Student ttest). 
.sup.(2) Lyophilizate concentration 2.5% w/v. 
TABLE 2 
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The effect of pretreatment of vaginal epithelial cells with 
chitin, CSE on adherence in vitro 
Percentage Percentage 
Treatment of adherence 
of inhibition 
______________________________________ 
None 42.5 .+-. 10.6 
76.4 
Chitin.sup.(1) 
10.0 .+-. 5.6.sup.(3) 
None 44.0 .+-. 0 
72.7 
CSE.sup.(2) 12.0 .+-. 5.6.sup.(3) 
______________________________________ 
.sup.(1) Concentration 2.5% w/v. 
.sup.(2) Concentration 2.5% w/v of lyophilized material. 
.sup.(3) Percentage of adherence significantly lower (p &lt; 0.05) than 
control (Student t'test). 
Blocking of In Vivo Adherence of C. albicans to Murine Vaginal Mucosa 
The next step in our study consisted of attempts to block the in vivo 
attachment of yeasts to vaginal mucosa in the murine experimental 
vaginitis model. Mice were pretreated topically with CSE and immediately 
inoculated with C. albicans organisms. The infection rate was assessed 24 
hrs. after inoculation with yeasts and the results compared to those in 
the controls pretreated with PBS. 
Nine experiments totalling 156 animals (Table 3) were performed. Of 74 mice 
pretreated with CSE, only 7 (9.0%) developed infection, compared to 36 
(43.8%) out of 82 controls. The differences between infection rates of 
CSE-treated animals and those of controls were statistically significant 
(Student's t-test). 
TABLE 3 
______________________________________ 
Pretreatment of mice with CSE 
Infection rate* 
Control CSE 
Experiment 
No. infected No. infected 
No. total % total 
% 
______________________________________ 
1 3/10 30 1/10 10 
2 5/10 50 1/10 10 
3 5/10 50 2/10 20 
4 3/8 37.5 0/8 0 
5 4/8 50 0/8 0 
6 3/8 37.5 1/8 12.5 
7 4/10 40 1/10 10 
8 5/10 50 1/10 10 
9 4/8 50 -- -- 
Total 36/82 43.8 .+-. 7.7 
7/74 9.0 .+-. 6.5** 
______________________________________ 
*Infection was evaluated 24 hrs. postyeast inoculation by assaying the 
adherence of yeasts and hyphae to murine exfoliated vaginal epithelial 
cells (see "Methods"). 
**Infection rate significantly lower (p &lt; 0.01) than control (student 
ttest). 
As a control we also added a histological examination of vaginal tissues 
from mice pretreated with CSE or PBS and inoculated with yeasts. Sections 
from PBS-pretreated animals revealed fungal elements in the epithelium and 
an inflammatory cell reaction. The CSE-pretreated mice were devoid of such 
elements and the tissue appeared normal. No changes in the pattern of the 
effects of the various pretreatments were found during 5 days of followup. 
Initial experiments to quantitate the dose-effect of the CSE by using 
lyophilizates prepared from supernatant of the chitin suspension indicated 
that the effect appeared to be dose-dependent (Table 4). 
TABLE 4 
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Pretreatment of mice with various doses of lyophilizate 
of CSE 
Infection 24 hrs. post-yeast 
inoculation* 
Mice treated with CSE 
Controls Treatment dose (mg/ml) 
(PBS-treated) 25 10 1 
______________________________________ 
Inoculated 
6 6 6 6 
Infected 
3 0 0 1 
______________________________________ 
*See Table 3. 
The in vivo studies revealed that the chitin soluble extract was also 
inhibitory in vivo. The CSE effect was expressed both by blocking 
attachment to murine exfoliated vaginal epithelial cells and by preventing 
penetration into murine vaginal tissue. Such effects were obtained only 
when mice were pretreated with CSE prior to inoculation of the yeasts, and 
not when they were treated post-inoculation. This indicated that CSE leads 
to prevention of infection by blocking the attachment of yeasts to the 
host mucosal surfaces. 
There were prepared lotions, water-base ointments and tampons impregnated 
with agqueous CSE extract. The usual concentration was about 1 to 3 mg/ml 
and the quantity applied was about 0.5 ml per day. This application 
substantially reduced infections by C. albicans, by preventing adherence 
of these to epithelial cells.