U.S. patent application number 13/112122 was filed with the patent office on 2011-09-15 for spermicidal and virucidal properties of various forms of sophorolipids.
Invention is credited to Gustavo F. Doncel, Richard A. Gross, Vishal Shah.
Application Number | 20110223239 13/112122 |
Document ID | / |
Family ID | 34994409 |
Filed Date | 2011-09-15 |
United States Patent
Application |
20110223239 |
Kind Code |
A1 |
Gross; Richard A. ; et
al. |
September 15, 2011 |
SPERMICIDAL AND VIRUCIDAL PROPERTIES OF VARIOUS FORMS OF
SOPHOROLIPIDS
Abstract
A method for inactivating spermatozoa, neutralizing or
inactivating a virus, and neutralizing or inactivating HIV using
sophorolipids having spermicidal and/or antiviral properties
produced by synthesizing the sophorolipid by fermentation of
Candida bombicola in a fermentation media to form a natural mixture
of lactonic sophorolipids compounds and non-lactonic sophorolipids
compounds and utilizing the natural mixture as a spermicidal and/or
antiviral agent, and/or separating the lactonic sophorolipids from
the natural mixture to form a lactonic fraction and mixing all
remaining fractions to form a non-lactonic fraction and utilizing
the lactonic fraction and/or the non-lactonic fraction as an
spermicidal and/or antiviral agent, and sophorolipid compounds for
use as spermicidal and/or antiviral agents.
Inventors: |
Gross; Richard A.;
(Plainview, NY) ; Shah; Vishal; (Oakdale, NY)
; Doncel; Gustavo F.; (Norfolk, VA) |
Family ID: |
34994409 |
Appl. No.: |
13/112122 |
Filed: |
May 20, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10804778 |
Mar 19, 2004 |
|
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13112122 |
|
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60456208 |
Mar 20, 2003 |
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Current U.S.
Class: |
424/450 ;
514/25 |
Current CPC
Class: |
A61P 15/18 20180101;
A61P 31/18 20180101; C12P 19/44 20130101; C07H 15/24 20130101; A61P
31/12 20180101; A61K 31/704 20130101; A61K 36/062 20130101; A61K
45/06 20130101; A61P 15/00 20180101; A61K 36/062 20130101; A61K
2300/00 20130101; A61K 31/704 20130101; A61K 2300/00 20130101; A61P
15/16 20180101 |
Class at
Publication: |
424/450 ;
514/25 |
International
Class: |
A61K 31/7028 20060101
A61K031/7028; A61K 9/127 20060101 A61K009/127; A61P 15/16 20060101
A61P015/16; A61P 31/18 20060101 A61P031/18 |
Claims
1. A method for inactivating spermatozoa by treating the
spermatozoa with an effective amount of a
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate sophorolipid compound.
2. The method as claimed in claim 1, wherein the
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate sophorolipid compound is selected from the group
consisting of:
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-
-9-octadecenoate-6',6''-diacetate; Hexyl 17-L[(2'-O-.beta.-D
glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-octadecenoate;
Ethyl 17-L[(2'-O-.beta.-D
glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-octadecenoate;
and combinations thereof.
3. The method as claimed in claim 1, wherein the sophorolipid
compound is in the form selected from the group consisting of a
gel, a film, a foam, a suppository, a pessary, a liposomic
formulation, a liquid imbibed in a sponge, and a liquid being
released from an intravaginal or intrauterine delivery system.
4. The method as claimed in claim 1, further comprising combining
the
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate sophorolipid compound with a spermicidal agent.
5. A method for neutralizing or inactivating a virus by treating
the virus with an effective amount of a
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate sophorolipid compound.
6. The method as claimed in claim 5, wherein the
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate sophorolipid compound is selected from the group
consisting of:
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-
-9-octadecenoate-6',6''-diacetate; Hexyl 17-L[(2'-O-.beta.-D
glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-octadecenoate;
Ethyl 17-L[(2'-O-.beta.-D
glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-octadecenoate;
and combinations thereof.
7. The method as claimed in claim 5, wherein the sophorolipid
compound is in the form selected from the group consisting of a
gel, a film, a foam, a suppository, a pessary, a liposomic
formulation, a liquid imbibed in a sponge, and a liquid being
released from an intravaginal or intrauterine delivery system.
8. The method as claimed in claim 4, further comprising combining
the
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate sophorolipid compound with an antiviral agent.
9. A method for neutralizing or inactivating HIV by treating the
HIV with an effective amount of a
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate sophorolipid compound.
10. The method as claimed in claim 9, wherein the
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate sophorolipid compound is selected from the group
consisting of:
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-
-9-octadecenoate-6',6''-diacetate; Hexyl 17-L[(2'-O-.beta.-D
glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-octadecenoate;
Ethyl 17-L[(2'-O-.beta.-D
glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-octadecenoate;
and combinations thereof.
11. The method as claimed in claim 9, wherein the sophorolipid
compound is in the form selected from the group consisting of a
gel, a film, a foam, a suppository, a pessary, a liposomic
formulation, a liquid imbibed in a sponge, and a liquid being
released from an intravaginal or intrauterine delivery system
12. The method as claimed in claim 9, further comprising combining
the
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate sophorolipid compound with an antiviral agent.
Description
STATEMENT OF RELATED APPLICATIONS
[0001] This application is a divisional and claims the benefit of
U.S. patent application Ser. No. 10/804,778 having a filing date of
19 Mar. 2004, which is a nonprovisional patent application that is
based on and claims the benefit of U.S. Provisional Patent
Application No. 60/456,208 having a filing date of 20 Mar.
2003.
BACKGROUND OF THE INVENTION
[0002] 1. Technical Field
[0003] The present invention relates generally to the field of uses
for sophorolipids and more specifically to the field of uses of
sophorolipids as spermicidal and antiviral agents.
[0004] 2. Prior Art
[0005] First described in 1961, sophorolipids occur as a mixture of
macrolactone and free acid structures that are acetylated to
various extents at the primary hydroxyl position of the sophorose
ring. Gorin, P. A. et al., Can. J. Chem., vol. 39, p. 846 (1961).
Careful examinations have revealed that at least eight structurally
different sophorolipids are produced. Davila, A. M. et al., J.
Chromatogr., vol. 648, p. 139 (1993). The main component of
sophorolipids is 17-hydroxyoctadecanoic acid and its corresponding
lactone. Tulloch, A. P. et al., Can. J. Chem., vol. 40, p. 1326
(1962) and Tulloch, A. P. et al., Can J. Chem., vol. 46, p. 3337
(1968).
[0006] Work has been carried out to tailor sophorolipid structure
during in vivo formation, mainly by the selective feeding of
different lipophilic substrates. Zhou, Q.-H., et al., J. Am. Oil
Chem. Soc., vol. 72, p. 67 (1995). Also unsaturated C-18 fatty
acids of oleic acid may be transferred unchanged into
sophorolipids. Rau, U. et al., Biotechnol. Lett., vol. 18, p. 149
(1996). However, while physiological variables during fermentation
have provided routes to the variation of sophorolipid composition,
this has not led to well-defined pure compounds.
[0007] Existing data suggests that glycolipids may be useful in
treating very severe immune disorders. For example, glycolipids
have been reported to be of interest for in vivo cancer
treatment/antitumor cell activity, treatment of autoimmune
disorders, in vivo and in vitro antiendotoxic (septic) shock
activity, regulation of angiogenesis, and apoptosis induction, all
by cytokine activity. See, e.g., U.S. Pat. No. 5,597,573 to Massey,
U.S. Pat. No. 5,514,661 to Piljac, U.S. Pat. No. 5,648,343 to
Carlson, and the references cited in notes 9-13 of Bisht, K. S. et
al., J. Org. Chem., vol. 64, pp. 780-789 (1999).
[0008] At the turn of the millennium, the Joint United Nations
Programme on HIV/AIDS (UNAIDS) and the World Health Organization
(WHO) estimated that 34.3 million adults and children were living
with HIV/AIDS. More than 18 million had already died of the
disease. An estimated 95% of all people living with HIV/AIDS live
in developing countries where limited resources and cultural
factors make containment of the epidemic especially difficult.
[0009] Most HIV infections are transmitted through heterosexual
intercourse, and in many areas women are disproportionately
affected. About 55% of adults living with HIV/AIDS in sub-Saharan
Africa, for example, are women.
[0010] The impact of the current level of HIV seroprevalence is
enormous in terms of mortality, resource depletion, and human
suffering. There is clearly an unmet need for treatment of those
already infected. However, there is also a desperate need to
prevent further infection.
[0011] Efforts to prevent HIV transmission have centered around
three approaches: behavioral change (safer sex), the development of
a vaccine, and development of a microbicide. In the context of
prevention of sexually transmitted diseases (STDs), microbicides
are compounds that, when applied topically, protect the body's
mucosal surfaces from infection by STD-causing pathogens.
[0012] The world's population is increasing at a sustained rate.
But population growth is disproportionately high in developing
countries with limited resources, those that are especially exposed
to infectious diseases such as AIDS. Fertility control and
prevention of sexually transmitted diseases are high-priority
issues in the public health agenda of developing nations.
[0013] Thus, there exists an urgent need to develop new and
improved methods for fertility control and prevention of unwanted
pregnancies and sexually transmitted infections. It is to the
development of sophorolipids for pharmaceutical and industrial
purposes, and in particular as microbicides and spermicides or
vaginal contraceptives, that the present invention is directed.
BRIEF SUMMARY OF THE INVENTION
[0014] Sophorolipids are microbial extracellular glycolipids
produced by resting cells of Candida bombicola. The chemical
composition of sophorolipid is constituted by a disaccharide sugar
viz. sophorose and a fatty acid or an ester group. Candida
bombicola produces the sophorolipids as a mixture of macrolactones
and free acid structures that are acetylated to various extents at
the primary hydroxyl sophorose ring positions (FIG. 1). Bisht, K.
S. et al., J. Org. Chem., vol. 64, pp. 780-789 (1999).
[0015] For this invention, a natural mixture of sophorolipids was
synthesized by fermentation of Candida bombicola. Lactonic
sophorolipid was separated from the crude mixture. A lactonic
fraction was collected separately and all other fractions were
mixed to form a non-lactonic sophorolipid mixture. Ethyl
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate then was synthesized and then further treated to
obtain Ethyl 17-L[(2'-O-.beta.-D
glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-octadecenoate
6',6''-diacetate. Hexyl
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate also was synthesized.
[0016] A quantity of sperm was treated with sophorolipids and the
ability of the sophorolipids to immobilize and kill the sperm was
measured. All forms of sophorolipids were highly effective as
spermicidal agents. Also, sophorolipids were evaluated as virucidal
agents using the human immunodeficiency virus (HIV) as model
organism. All the sophorolipids tested showed virucidal activity.
The ethyl ester form of sophorolipid was highly effective as an
antiviral agent. Howsoever, other forms of sophorolipids were also
effective.
[0017] Thus, the present invention relates to the application of
sophorolipids in the field of medicine to prevent and treat viral
infections. Additionally, the present invention relates to the
application of sophorolipids in the field of medicine as
contraceptive agents. Further, while sophorolipids can find
applications in diverse fields, the present invention relates to
sophorolipids as antiviral and spermicidal contraceptive
agents.
[0018] These features, and other features and advantages of the
present invention, will become more apparent to those of ordinary
skill in the relevant art when the following detailed description
of the preferred embodiments is read in conjunction with the
appended drawing and tables.
BRIEF DESCRIPTION OF THE FIGURE
[0019] FIG. 1 contains representative structures of sophorolipids
produced by Candida bombicola, with FIG. 1a showing a lactonic
sophorolipid and FIG. 1b showing an open-ring sophorolipid.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
1. Sophorolipid Fermentation
[0020] Sophorolipids were synthesized by fermentation of Candida
bombicola. The fermentation media was composed of glucose 100 g,
yeast extract 10 g, urea 1 g and oleic acid 40 g in 1000 ml of
water. After 7 days of fermentation, sophorolipid was extracted
thrice using ethyl acetate. The extracts were pooled and the
solvent then was removed. The obtained product was then washed with
hexane to remove the residual fatty acids. This was "natural"
sophorolipid. The sophorolipid was dried in a vacuum
desiccator.
2. Preparation of Lactonic Sophorolipid
[0021] Column chromatographic separations were performed over
silica gel 70 (Aldrich Chemical Co.) to separate lactonic
sophorolipid from the crude mixture. 50 g of silica gel was used to
pack a glass column (5 cm.times.50 cm) in the eluent
(CHCl.sub.3/MeOH mixture). 200 ml of eluent was run through the
column before the natural mixture (dissolved in a minimal volume of
eluent) was loaded onto the top of the column matrix. Different
fractions were subsequently eluted (1 mL/min). A lactonic fraction
was collected separately and all other fractions were mixed to form
non-lactonic sophorolipid mixture.
3. Synthesis of Ethyl 17-L[(2'-O-.beta.-D
glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-octadecenoate
6',6''-diacetate
[0022] Ethyl
17-L-[(2'-O-.beta.D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-oc-
tadecenoate was synthesized by adding 2 g of dry crude sophorolipid
and 2.5 mL 0.021 N sodium ethoxide in methanol solution to a 100 mL
round-bottomed flask equipped with a reflux condenser. The reaction
assembly was protected from atmospheric moisture by a CaCl.sub.2
guard tube. The reaction mixture was refluxed for 3 hr, cooled to
room temperature (30.degree. C.), and acidified using glacial
acetic acid. The reaction mixture was concentrated by
rotoevaporation and poured with stirring into 100 mL of ice-cold
water that resulted in the precipitation of the sophorolipid
ethylester as a white solid. The precipitate was filtered, washed
with ice-water, and lyophilized.
[0023] The synthesized Ethyl
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate (500 mg) was dissolved in 20 mL of dry tetrahydrofuran
(THF). To this solution were added vinyl acetate (2 mL) and Novozym
435 (1 g), and the suspension was stirred magnetically at
35.degree. C. for 96 hr. The enzyme was filtered off, the solvent
was evaporated, and the product was purified by column
chromatography (eluent CHCl.sub.3/MeOH, 9:1) to give 490 mg of
Ethyl 17-L[(2'-O-.beta.-D
glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-octadecenoate
6',6''-diacetate.
[0024] The synthesis of other related compounds, such as methyl-
and butyl-based compounds, can be accomplished by substituting
sodium methoxide or sodium butoxide respectively for the sodium
ethoxide, resulting in sophorolipid methylester and sophorolipid
butylester, respectively. The amount of dry natural sophorolipid
and the amount and normality of the sodium (CH.sub.2).sub.noxide
can be varied appropriately by those of ordinary skill in the art
without undue experimentation. Other types of suitable
sophorolipids also can be synthesized by those of ordinary skill in
the art without undue experimentation.
4. Synthesis of Hexyl 17-L[(2'-O-.beta.-D
glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-octadecenoate
[0025] Hexyl
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate was synthesized by adding 2 g of dry natural
sophorolipid and 2.5 mL 0.021 N sodium hexanoxide in hexanol
solution to a 100 mL round-bottomed flask equipped with a reflux
condenser. The reaction assembly was protected from atmospheric
moisture by a CaCl.sub.2 guard tube. The reaction mixture was
refluxed for 3 hr, cooled to room temperature (30.degree. C.), and
acidified using glacial acetic acid. The reaction mixture was
concentrated by rotoevaporation and poured with stirring into 100
mL of ice-cold water that resulted in the precipitation of the
sophorolipid ethylester as a white solid. The precipitate was
filtered, washed with ice-water, and lyophilized.
5. Spermicidal Study
[0026] Motility and viability time-responses were studied with a
sophorolipid testing concentration of 0.3 mg/mL. At this
concentration no effect of the solvent was observed. All sperm used
for this experiment and throughout the study were obtained from
human semen samples through a one hour swim-up separation technique
in BWW medium+0.1% HSA.
[0027] 100 uL of swim-up sperm were incubated with 1 uL of compound
for the time points of 2 min, 30 min, 2 hrs, and 4 hrs and assessed
for progressive motility ("real-time" assessment). n=5 for all time
points. Co-incubation samples were diluted with 1 mL of BWW
medium+0.1% HSA. The tubes were centrifuged, the supernatant
removed, and the sperm resuspended in the original volume of 100 uL
BWW+0.1% HSA and incubated at 37.degree. C., 5% CO.sub.2. At 30 min
post-washing, motility was again assessed. After the 30 min
post-wash assessment, 900 uL of HOS medium was added to the sample
and tubes were incubated for 30 min at 37.degree. C.
Post-incubation, samples were assessed for viability. Motility and
viability were quantified "manually" under dark field microscopy
(Nikon E-800).
6. Viral Inactivation Study Using Cell-Free HIV
[0028] Pre-titered concentrated virus (HIV-1 RF) stock (10 .mu.L)
was mixed with 90 .mu.L of the test agent formulation in
microtubes. At the end of the two-minute exposure period, serial
10-fold dilutions were carried out with a multichannel pipettor so
that the sample dilutions were performed simultaneously. Aliquots
(100 .mu.L) of the serial dilutions were then transferred
simultaneously to another 96-well plate which had been pre-seeded
with MT-2 cells in RPMI medium supplemented with FBS. Four wells
were used for each dilution of virus. The cultures were incubated
at 37.degree. C. in a humidified atmosphere of 5% CO.sub.2. The
cultures were scored microscopically for virus-induced cytopathic
effect and agent-induced cytotoxicity on day 4 and 7 of incubation,
and day 7 reported. Cultures were routinely maintained 2 to 3 days
after initial analysis, and observed visually and microscopically
for abnormalities.
7. Results and Discussion
[0029] As shown in Table 1, all four sophorolipid compounds tested
immobilized almost 100% of spermatozoa in the shortest time studied
(2 min.). This effect was irreversible since motility did not
recover after washing off the compounds and further incubating the
sperm for 30 min. in fresh medium. Such immotility may be due to a
severe alteration of the membrane selective permeability, since the
sperm viability test revealed that almost 100% of spermatozoa had
their membrane integrity compromised.
[0030] As shown in Table 2, all four sophorolipid compounds tested
displayed some degree of virucidal activity. Our criterion to
consider a result as "positive" indicates a minimum reduction in
virus titer of 2 logs. The best selectivity index was displayed by
Ethyl
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate, which showed high activity and reduced
cytotoxicity.
[0031] Thus, the invention claims the use of natural mixture,
lactonic, 6',6''-diacetate, Ethyl ester, Hexyl ester sophorolipids
as spermicidal and virucidal agents. The applications in field of
medicine, particularly in the areas of contraception and infectious
diseases, would be tremendous, providing new means for "safe sex"
as well as to treat and prevent viral infections, apart from other
applications.
8. Utilizing the Natural Mixture, the Lactonic Fraction, and/or the
Non-Lactonic Fraction of Sophorolipids as a Spermicidal and/or
Antiviral Agent
[0032] The present invention also is a method for producing
sophorolipids having spermicidal and/or antiviral properties and
using either the natural mixture, the lactonic fraction of the
mixture, the non-lactonic fraction of the mixture, and/or
combinations of these as antiviral agents or spermicidal
agents.
[0033] One such method for producing sophorolipids having
spermicidal and/or antiviral properties comprises the steps of (a)
synthesizing the sophorolipid by fermentation of Candida bombicola
in a fermentation media to form a natural mixture of lactonic
sophorolipids and non-lactonic sophorolipids; (b) utilizing the
natural mixture as a spermicidal and/or antiviral agent; (c)
separating the lactonic sophorolipids from the natural mixture to
form a lactonic fraction and mixing all remaining fractions to form
a non-lactonic fraction; (d) utilizing the lactonic fraction as an
spermicidal and/or antiviral agent; and (e) utilizing the
non-lactonic fraction as a spermicidal and/or antiviral agent.
[0034] Another such method for producing sophorolipids having
spermicidal and/or antiviral properties comprises the steps of: (a)
synthesizing the sophorolipid by fermentation of Candida bombicola
in a fermentation media to form a natural mixture of lactonic
sophorolipids and non-lactonic sophorolipids; and (b) utilizing the
natural mixture as a spermicidal and/or antiviral agent.
[0035] Another such method for producing sophorolipids having
spermicidal and/or antiviral properties comprises the steps of: (a)
synthesizing the sophorolipid by fermentation of Candida bombicola
in a fermentation media to form a natural mixture of lactonic
sophorolipids and non-lactonic sophorolipids; (b) separating the
lactonic sophorolipids from the natural mixture to form a lactonic
fraction and mixing all remaining fractions to form a non-lactonic
fraction; and (c) utilizing the lactonic fraction as an spermicidal
and/or antiviral agent.
[0036] Another such method for producing sophorolipids having
spermicidal and/or antiviral properties comprises the steps of: (a)
synthesizing the sophorolipid by fermentation of Candida bombicola
in a fermentation media to form a natural mixture of lactonic
sophorolipids and non-lactonic sophorolipids; (b) separating the
lactonic sophorolipids from the natural mixture to form a lactonic
fraction and mixing all remaining fractions to form a non-lactonic
fraction; and (c) utilizing the non-lactonic fraction as an
spermicidal and/or antiviral agent.
[0037] Preferably, these methods use a
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate based sophorolipid. Even more preferably, the
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate based sophorolipid is selected from the group
consisting of
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate-6',6''-diacetate, Hexyl 17-L[(2'-O-.beta.-D
glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-octadecenoate,
and Ethyl 17-L[(2'-O-.beta.-D
glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-octadecenoate.
9. Delivery Mechanisms
[0038] The sophorolipid compounds disclosed herein can be delivered
in many different forms. Illustrative examples of the delivery
forms include a gel, a film, a foam, a suppository, a pessary, a
liposomic formulation, as a liquid imbibed in a sponge, and as a
liquid being released from an intravaginal or intrauterine delivery
system. Those of ordinary skill in the art can chose other delivery
systems and formulate the novel sophorolipid into the delivery
system chosen without undue experimentation.
10. Combination Systems
[0039] The novel sophorolipids disclosed herein also can be
combined in various forms and with other antiviral agents or
spermicidal agents to result in alternative novel antiviral and
spermicidal agents. For example, one such method of combining the
novel sophorolipids is to produce a sophorolipid synthesized by
fermentation of Candida bombicola in a fermentation media to form a
natural mixture of lactonic sophorolipids and non-lactonic
sophorolipids and to combine this with at least one sophorolipid
selected from the group consisting of
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate-6',6''-diacetate, Ethyl
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate, Hexyl
17-L-[(2'-O-.beta.-D-glucopyranosyl-.beta.-D-glucopyranosyl)-oxy]-cis-9-o-
ctadecenoate, and combinations thereof. These combinations can be
used as antiviral agents and as spermicidal agents.
[0040] Further, the novel sophorolipids disclosed herein can be
made and/or used in combination with one or more known antiviral
agents or one or more known spermicidal agents to produce
alternative antiviral agents and spermicidal agents. Those of
ordinary skill in the art can choose the appropriate or desired
known antiviral agents or spermicidal agents to combine with the
novel sophorolipids to result in an alternate antiviral agent or
spermicidal agent, respectively, without undue experimentation.
[0041] The foregoing detailed description of the preferred
embodiments and the appended FIGURE and tables have been presented
only for illustrative and descriptive purposes. They are not
intended to be exhaustive nor to limit the scope and spirit of the
invention. The embodiments were selected and described to best
explain the principles of the invention and its practical
applications. One skilled in the art will recognize that many
variations can be made to the invention disclosed in this
specification without departing from the scope and spirit of the
invention. For instance, sophorolipids have been tested against HIV
but will most likely be active against other retrovirus, herpes
virus, papillomavirus, and other viruses as well.
TABLE-US-00001 TABLE 1 Motility and Viability Time Response
Post-Dilution Initial Conc Real-Time Post-Dilution Viability
Compound Solvent Time (mg/mL) Motility (%) Motility (%) (%) n SL-1
DMSO 2 min 0.3 3.26 .+-. 3.36 3.10 .+-. 6.66 3.07 .+-. 6.86 5 30
min 0.3 0.00 .+-. 0.00 0.00 .+-. 0.00 0.50 .+-. 1.11 5 2 hour 0.3
0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 5 SLET DMSO 2 min 0.3
0.73 .+-. 0.81 3.37 .+-. 3.87 2.36 .+-. 5.29 5 30 min 0.3 0.00 .+-.
0.00 0.00 .+-. 0.00 1.00 .+-. 2.24 5 2 hour 0.3 0.00 .+-. 0.00 0.00
.+-. 0.00 0.00 .+-. 0.00 5 SLHEX DMSO 2 min 0.3 1.26 .+-. 1.22 1.55
.+-. 2.18 2.27 .+-. 3.22 5 30 min 0.3 0.00 .+-. 0.00 0.00 .+-. 0.00
1.10 .+-. 2.46 5 2 hour 0.3 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-.
0.00 5 SLETDA DMSO 2 min 0.3 0.00 .+-. 0.00 0.00 .+-. 0.00 0.40
.+-. 0.90 5 30 min 0.3 0.00 .+-. 0.00 0.00 .+-. 0.00 0.20 .+-. 0.45
5 2 hour 0.3 0.00 .+-. 0.00 0.00 .+-. 0.00 0.00 .+-. 0.00 5 DMSO
1:100 -- 2 min 0.3 99.57 .+-. 1.22 99.27 .+-. 1.12 99.49 .+-. 0.78
5 30 min 0.3 99.28 .+-. 0.85 99.66 .+-. 0.50 99.40 .+-. 0.80 5 2
hour 0.3 99.19 .+-. 1.27 99.27 .+-. 1.18 99.20 .+-. 0.73 5 *Values
expressed as Mean .+-. Standard Deviation
TABLE-US-00002 TABLE 2 Cell-Free Inactivation Assay TCTD50 Initial
Percent of Agent Cyto- Log Reduction Drug Solvent During Exposure
toxicity in Virus Titer SL-1 DMSO 0.3 1.5 1.7 0.09 1.5 1.7 0.03
.ltoreq.0.5 1.7 SLET DMSO 0.3 1.5 4.3 0.09 .ltoreq.0.5 3.3 0.03
.ltoreq.0.5 2.0 SLETDA DMSO 0.3 1.5 .gtoreq.4.5 0.09 1.5
.gtoreq.4.5 0.03 .ltoreq.0.5 1.7 SLHEX DMSO 0.3 1.5 4.0 0.09
.ltoreq.0.5 3.0 0.03 .ltoreq.0.5 0 DMSO 1:10 dil .ltoreq.0.5 0 1:32
dil .ltoreq.0.5 0 1:100 dil .ltoreq.0.5 0 1:320 dil .ltoreq.0.5 0
Virus Control = TCID50/0.1 mL 6.0 - log10 TCID.sub.50: The minimum
drug concentration that inhibits HIV-induced cytopathic effect by
50%, calculated by using a regression analysis program for semilog
curve fitting. TCTD.sub.50: The minimum drug concentration that
reduces cell viability by 50%
* * * * *