U.S. patent application number 15/447365 was filed with the patent office on 2017-06-22 for vector delivery-based microbicides.
This patent application is currently assigned to Rush University Medical Center. The applicant listed for this patent is Rush University Medical Center. Invention is credited to Robert Anthony Anderson, JR., Calvin J. Chany, II.
Application Number | 20170172970 15/447365 |
Document ID | / |
Family ID | 37758269 |
Filed Date | 2017-06-22 |
United States Patent
Application |
20170172970 |
Kind Code |
A1 |
Anderson, JR.; Robert Anthony ;
et al. |
June 22, 2017 |
VECTOR DELIVERY-BASED MICROBICIDES
Abstract
A new class of anti-microbial agents and methods for preventing
or reducing the risk of sexually transmitted infections and/or
diseases is provided. Preferably, these anti-microbial agents are
also contraceptive and, thus, also prevent or reduce the risk of
unplanned pregnancies. The anti-microbial agents comprise a
delivery vector having anti-microbial activity (and preferably
contraceptive activity) coupled with a nitric oxide donor
moiety.
Inventors: |
Anderson, JR.; Robert Anthony;
(Brookline, MA) ; Chany, II; Calvin J.; (Asbury,
IA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rush University Medical Center |
Chicago |
IL |
US |
|
|
Assignee: |
Rush University Medical
Center
Chicago
IL
|
Family ID: |
37758269 |
Appl. No.: |
15/447365 |
Filed: |
March 2, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14965276 |
Dec 10, 2015 |
9603841 |
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15447365 |
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12063996 |
Sep 10, 2010 |
9393233 |
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PCT/US2006/031631 |
Aug 14, 2006 |
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14965276 |
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60708960 |
Aug 17, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/18 20180101;
A61K 31/4245 20130101; A61K 31/19 20130101; A61P 31/00 20180101;
A61P 15/18 20180101; A61K 31/192 20130101; A61K 31/215 20130101;
A61P 15/16 20180101; A61K 31/21 20130101; A61K 9/0034 20130101;
A61K 31/216 20130101; A61K 47/55 20170801; A61K 31/24 20130101 |
International
Class: |
A61K 31/24 20060101
A61K031/24; A61K 31/192 20060101 A61K031/192; A61K 9/00 20060101
A61K009/00 |
Claims
1-16. (canceled)
17. An anti-microbial agent for reducing risk of transmitting a
sexual transmitted disease, said anti-microbial agent comprising a
covalent adduct of a delivery vector having anti-microbial activity
and a nitric oxide donor, the nitric acid donor consisting of a
nitric acid moiety and a spacer, the nitric acid donor being
covalently bonded through the spacer to the delivery vector having
anti-microbial activity, wherein NO is released from the
anti-microbial agent during use.
18. The anti-microbial agent of claim 17, wherein the delivery
vector has contraceptive activity and anti-microbial activity.
19. The anti-microbial agent as defined in claim 17, wherein the
delivery vector is selected from the group consisting of
phosphorylated hesperidins, sulfonated hesperidins, polystyrene
sulfonates, substituted benzenesulfonic acid formaldehyde
co-polymers, H.sub.2SO.sub.4-modified mandelic acids, and cellulose
sulfates.
20. The anti-microbial agent as defined in claim 17, wherein the
NO-donor is selected from the group consisting of nitrate esters,
furoxans, ketoximes, S-nitrosothiols, nitrosohydrazines, and
hydroxylamides.
21. The anti-microbial agent as defined in claim 19, wherein the
NO-donor is selected from the group consisting of nitrate esters,
furoxans, ketoximes, S-nitrosothiols, nitrosohydrazines, and
hydroxylamides.
22. The anti-microbial agent as defined in claim 19, wherein the
delivery vector is a H2SO4-modified mandelic acid.
23. The anti-microbial agent as defined in claim 17, wherein the
NO-donor is a nitrate ester.
24. The anti-microbial agent as defined in claim 17, wherein the
anti-microbial agent is contained in an inert carrier.
25. The anti-microbial agent as defined in claim 18, wherein the
anti-microbial agent is contained in an inert carrier.
26. The anti-microbial agent as defined in claim 25, wherein the
anti-microbial agent is at a concentration greater than about 0.2
mg/g.
27. The anti-microbial agent as defined in claim 26, wherein the
anti-microbial agent is at a concentration greater than about 0.2
mg/g.
28. The anti-microbial agent as defined in claim 26, wherein the
anti-microbial agent is at a concentration of about 10 to 100
mg/g.
29. The anti-microbial agent as defined in claim 27, wherein the
anti-microbial agent is at a concentration of about 10 to 100
mg/g.
30. A method for reducing the risk of transmission and infection by
a sexually transmitted disease through sexual activity between two
or more parties, said method comprising: applying an effective
amount of an anti-microbial agent to an area of the body to be
engaged in the sexual activity of at least one of the parties prior
to the sexual activity and then engaging in the sexual activity,
wherein the antimicrobial agent comprises: a covalent adduct of a
delivery vector having anti-microbial activity and a nitric oxide
donor, the nitric acid donor consisting of a nitric acid moiety and
a spacer, the nitric acid donor being covalently bonded through the
spacer to the delivery vector having anti-microbial activity,
wherein NO is released from the anti-microbial agent after applying
to the area of the body.
Description
RELATED APPLICATION
[0001] This application is based on, and claims benefit of, U.S.
Provisional Application Ser. No. 60/708,960, filed Aug. 17, 2005,
and which is hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention generally relates to a new class of
anti-microbial agents and methods for preventing or reducing the
risk of sexually transmitted infections and/or diseases.
Preferably, these anti-microbial agents are also contraceptive and,
thus, also prevent or reduce the risk of unplanned pregnancies.
BACKGROUND OF THE INVENTION
[0003] In recent years, sexually transmitted diseases have become
an increasing medical problem and concern throughout the world. The
HIV/AIDS epidemic over the last decade or so has significantly and
dramatically underscored the threat of STDs to the human
population. Until there is a cure, or at least an effective
treatment, the best, and perhaps only realistic, approach to this
increasing problem of STDs (especially HIV/AIDS) appears to be
reducing the risk of transmission of STDs by the STD-causing
organisms and thus reducing the number of individuals who become
newly infected. Even when treatments or cures become available,
prevention of infections in the initial instance will likely remain
as the first line of defense. For economic, medical, and
psychological reasons, it is preferable to prevent the initial
infection rather than treating, and even curing, individuals with
STDs.
[0004] At present, education in regard to STDs, their modes of
transmission, and so-called "safe-sex" techniques has, at least to
some degree in the more developed countries, shown promise in
reducing the risks of STD transmission through sexual activity.
Screening of the blood supply has helped to reduce the risk of
transmission of such STD-causing organisms via blood transfusions
and related medical practices. Nonetheless, the spread of such STDs
has not been halted to a satisfactory degree even in developed
countries with active and progressive education programs. Even with
their known effectiveness in preventing STDs, current safe-sex
techniques are not always used, or are not always used properly,
for many reasons (e.g., carelessness, lack of knowledge, improper
techniques, cultural barriers, unplanned or spontaneous sexual
activity, and the like). Moreover, even when used, safe-sex
techniques (except perhaps abstinence) are not always effective.
For example, condoms are generally only about 90 percent effective
iii preventing conception when used alone; in the case of such
failures, STD-causing organisms, if present, may pass from one
sexual partner to the other,
[0005] Various birth control devices--including barrier methods and
vaginal contraceptives--are currently available. Some of these may,
in addition, also have a least some degree of anti-STD activity.
For example, condoms can help prevent the transmission of STDs so
long as they are properly used and/or they perform properly.
Nonoxynol-9, currently one of the most widely used contraceptive
agents, is reported, at least in some cases, to reduce the risk of
transmission of some STOs. Nonoxynol-9, which is a nonionic
detergent with strong surfactant properties, acts, like most other
chemical-based contraceptives, by killing or otherwise immobilizing
spermatozoa (e.g., spermicidal activity). Nonoxynol-9 is a potent
cytotoxic agent which tends to nonspecifically disrupt cell
membranes. These properties, however, give rise to some very
significant disadvantages. Because nonoxynol-9 is strongly
cytotoxic, it can injure vaginal/cervical epithelial and other
cells at concentrations as low as about 0.0005 percent. Clinical
studies have confirmed epithelial disruption of the vagina and
cervix. Nonoxynol-9 also disrupts the normal vaginal flora which
provides a protective mechanism, perhaps by maintaining a low pH,
to guard against the invasion of pathogenic microbes. Nonoxynol-9
may also partially dissolve or remove the protective glycoprotein
coating in the vagina. The cytotoxic, flora-disruptive, and
glycoprotein-removal effects of nonoxynol-9 can lead to vaginal
damage or Injury, including lesions. Some women are especially
sensitive to nonoxynol-9 and manifest these effects with only
occasional use. The disruption of these protective mechanisms by
nonoxynol-9 can actually increase the risks of STD since the
breakdown of the protective mechanisms, and especially the
occurrence of lesions, allows STD-causing organisms an easier
pathway into the cells. Thus, any anti-STD activity of the
contraceptive may be reduced or even lost (i.e., overwhelmed) by
the increased risk of infection due to physical damage from the
contraceptive. Even if such a contraceptive method provided some
degree of STD protection, it would, of course, mainly be directed
at heterosexual relationships in which pregnancy was not
desired.
[0006] More recently contraceptives having anti-STD activity have
become available. U.S. Pat. No. 5,925,621 (Jul. 20, 1999), U.S.
Pat. No. 5,932,619 (Aug. 3, 1999), U.S. Pat. No. 6,028,115 (Feb.
22, 2000), and U.S. Pat. No. 6,239,182 (May 29, 2001) provide
methods for the reduction of sexual transmitted diseases using
inhibitory agents such as phosphorylated hesperidins, sulfonated
hesperidins, polystyrene sultanates, substituted benzenesulfonic
acid formaldehyde co-polymers, H.sub.2SO.sub.4-modified mandelic
acids, and the like.
[0007] It would be desirable, therefore, to provide more effective
anti-microbial agents and methods for preventing or reducing the
risk of sexually transmitted infections and/or diseases; preferably
such anti-microbial agents would also be contraceptive and, thus,
prevent or reduce the risk of unplanned pregnancies. It would be
desirable if such anti-microbial agents, whether contraceptive or
not, and methods would not interfere with the natural and
protective vaginal mechanisms it would also be desirable if such
anti-microbial agents, whether contraceptive or not, and methods
would be relatively easy to use, have significantly fewer side
effects than currently available methods (i.e., nonoxynol-9) so
that it would more likely be used on a consistent basis, and be
effective at lower concentrations. It would also be desirable if
such anti-microbial agents, whether contraceptive or not, and
methods could be used in heterosexual, homosexual, and bisexual
relationships and for a wide range of sexual activities. It would
also be desirable if such anti-microbial agents, whether
contraceptive or not, and methods could be implemented by either
party to the sexual activity. The present invention, as detailed in
the present specification, provides such anti-microbial agents and
contraceptive anti-microbial agents and methods.
SUMMARY OF THE INVENTION
[0008] This invention generally relates to improved anti-microbial
agents and to methods for preventing STDs and/or reducing the risk
of transmission of such STDs through sexual activity using the
improved anti-microbial agents. Preferably, such anti-microbial
agents are also contraceptive. The anti-microbial agents comprise a
delivery vector having anti-microbial activity (and preferably
contraceptive activity) coupled with a nitric oxide donor moiety.
The method is suitable for use by heterosexual, homosexual, and
bisexual individuals to significantly reduce the risk of being
infected by, or of transmitting, a STD through sexual contact.
Moreover, the risk of pregnancy during heterosexual activity is
also significantly reduced in preferred embodiments. Although this
method can be used alone, it is generally preferred that it be used
in conjunction with other so-called "safe sex" techniques in order
to even further reduce the risk of STD transmission or
Infection.
[0009] The method of this invention generally comprises the
application of an effective amount of the improved anti-microbial
agent or agents to the area or areas of sexual contact (e.g.,
genitalia) of at least one (and preferably all) of the participants
prior to engaging in sexual activity. The anti-microbial agents of
this invention comprises a delivery vector component having
anti-microbial activity (and preferably contraceptive activity)
coupled with a nitric oxide donor moiety. For purposes of this
invention the "anti-microbial agent" is a compound or mixture of
compounds which can inactivate at least one major STD-causing
organisms (HIV, HSV, gonococci, papilloma virus, and/or chlamydia)
without necessarily killing them and which generates nitric oxide
in situ (i.e., at the binding site of the delivery vector
component). The nitric oxide, thus released, can kill or otherwise
inactivate the microbe; the microbe can also be killed or otherwise
inactivated by the delivery vector component. "Anti-microbial
agents" of this invention may or may not (but preferably do) have
contraceptive activity in addition to the anti-microbial activity.
Vector delivery components which are preferred in the present
invention for preparing anti-microbial agents are inhibitory agents
such as phosphorylated hesperidins, sulfonated hesperidins,
polystyrene sulfonates, substituted benzenesulfonic acid
formaldehyde co-polymers, H.sub.2SO.sub.4-modified mandelic acids,
cellulose sulfates, and the like. Thus, preferred NO-coupled
anti-microbial agents of this invention include, for example,
phosphorylated hesperidins coupled with a NO-donor, sulfonated
hesperidins coupled with a NO-donor, polystyrene sulfonates coupled
with a NO-donor, substituted benzenesulfonic acid formaldehyde
co-polymers coupled with a NO-donor, H.sub.2SO.sub.4-modified
mandelic acids coupled with a NO-donor, and cellulose sulfates
coupled with a NO-donor, and the like. Preferably the vector
delivery components as well as the NO-coupled anti-microbial agents
used in the present invention are at least partially water soluble
or water dispersable so that anti-SW formulations can more easily
be prepared. More preferred anti-microbial agents for use in this
invention include H.sub.2SO.sub.4-modified mandelic acids (SAMMAs)
coupled with a NO-donor (i.e., NO-SAMMAs). Preferred NO-SAMMAs
include those which have been fractionated so as to have a narrower
molecular weight distribution and increased activities.
[0010] In addition to anti-STD activity, these compounds may also
act as vaginal contraceptives (and preferably do act as such) and
generally have fewer side effects than conventional vaginal
contraceptives (e.g., nonoxynol-9). For example, the compounds
useful in this invention are generally not toxic (or only minimally
toxic) to natural and beneficial vaginal flora and, thus, do not
significantly upset the local microbiological balance or
significantly disrupt the protective glycoprotein vaginal coating.
Disruption of the natural vaginal flora and/or removal or
disruption of the protective glycoprotein vaginal coating using
conventional vaginal contraceptives can lead to irritation of the
vaginal wall and/or lesions on the vaginal wall which can make the
transmission of STD easier and/or more likely. In addition, the
compounds useful in this invention are generally not disruptive to
rectal tissue and should not, therefore, significantly contribute
to the formation of lesions or breaks in the rectal lining which
could increase the risk of STD transmission during anal
intercourse. Moreover, the anti-microbial agents of the present
invention, largely due to their dual activities and site-specific
delivery of nitric oxide, can be used at lower concentrations,
thereby reducing the risk of side effects or other adverse
effects.
[0011] Either party to the sexual contact can employ the method of
the present invention in order to protect him or herself and their
partners. This feature allows either party to take protective
measures without relying on the motivation or action of the other
party. Of course, the highest level of protection is obtained when
both or all parties take appropriate steps to practice the methods
of this invention in conjunction with "safe-sex" techniques.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is a plot of percentage maximal acrosomal loss of
NO7-SAMMA and NO23-SAMMA in the presence and absence of Ca.sup.2+
as well as their respective vector and NO-donor alone. The error
bars represent 90% confidence limits. The bold horizontal lines in
the chart area represent the predicted responses to NO7-SAMMA and
NO23-SAMMA, respectively, assuming independence of the equivalent
NO-donor and SAMMA responses in the presence of Ca.sup.2+.
[0013] FIG. 2 is a comparison of SAMMA, NO23-SAMMA, and
fractionated NO23-SAMMA as acrosomal loss stimuli.
[0014] FIG. 3 is a comparison of SAMMA and NO7-SAMMA for C.
trachomatis inhibition. Elementary bodies were preincubated with
agent (either SAMMA or NO7-SAMMA) for 4 hours at 0.degree. C.
before inoculation onto HeLa cells.
[0015] FIG. 4 is another comparison of SAMMA and NO7-SAMMA for C.
trachomatis inhibition. HeLa cells were preincubated with agent
(either SAMMA or NO7-SAMMA) for 4 hours at 37.degree. C. before
inoculation.
[0016] FIG. 5 is a comparison of fractionated NO23-SAMMA and
nitrooxypropanol as acrosomal loss stimuli in the absence of
Ca.sup.2+; thus, the activity of fractionated NO23-SAMMA is due to
the NO donor moiety.
[0017] FIG. 6 illustrates the inhibition of acrosomal loss induced
by fractionated NO23-SAMMA by selective protein kinase G inhibitor
KT5623.
[0018] FIG. 7 illustrates nitrite release from fractionated
NO23-SAMMA in the presence of 50 mM L-cysteine.
DETAILED DESCRIPTION OF THE INVENTION
[0019] This invention generally relates to improved anti-microbial
agents and to methods for preventing STDs and/or reducing the risk
of transmission of such STDs through sexual activity using the
improved anti-microbial agents. The anti-microbial agents comprises
a delivery vector having anti-microbial activity, and preferably
contraceptive activity, coupled with a nitric oxide donor moiety.
The vector delivery component is designed to have affinity toward
surface receptors required for target cell recognition by
spermatozoa (oocytes) and/or pathogenic microbes (susceptible
tissue). It provides a vector-mediated targeted delivery of NO, a
naturally occurring, biologically active compound with known
activities against spermatozoa and pathogenic microbes, including,
though not restricted to, HIV, HSV, and C. trachomatis. NO is
produced in situ by the NO donor covalently attached to the vector.
The anti-microbial agent is expected to bind with the targeted
surface receptors on spermatozoa and/or pathogenic microbes; once
bound, released NO can effect its known activities against the
spermatozoa and/or pathogenic microbes in a very effective manner.
Indeed, lower concentrations of the anti-microbial agent will be
effective since delivery of the NO to the target organisms combined
with the inherent activity of the vector delivery component will be
much more effective as compared to either the vector component
alone or NO donor alone.
[0020] The preferred contraceptive antimicrobial agents of this
invention are especially intended to prevent sexually transmitted
infections and unplanned pregnancies. However, they may also have
utility in preventing blood-borne pathogenic microbes from entering
surrounding tissues. NO-coupled H.sub.2SO.sub.4-modified mandelic
acids (NO-SAMMAs) are prototypes of this type of agent. They act by
multiple mechanisms. They provide a vector-mediated targeted
delivery of NO, a naturally occurring, biologically active compound
with known activities against spermatozoa and pathogenic microbes,
including, though not restricted to, HIV, HSV, and C. trachomatis.
NO is produced by a NO donor covalently attached to the vector. The
vector can be any one of several compounds with affinity toward
surface receptors required for target cell recognition by
spermatozoa (oocytes) and pathogenic microbes (susceptible tissue).
Examples of receptors on pathogenic microbes are collectively known
as adhesins. Examples of receptors on spermatozoa have affinity
toward oocyte-related proteins, such as the zone pellucida, and are
collectively known as heparin (or glycosaminoglycan) binding
proteins or lectins. The vector is a ligand for these
receptors.
[0021] Both vector and released NO contribute to its activity, each
by one or more mechanisms. The combination of these moieties on the
same molecule is more effective than either of the separate parts
used alone, or in combination. This applies to the contraceptive
and anti-microbial activities of these agents, possibly by
different mechanisms. The vector (ligand) promotes NO formation and
biological activity in responsive cells, including spermatozoa, by
a mechanism independent from that due to the NO donor. The response
is synergistic to the expected response to the NO donor and ligand
added in combination. The method of NO delivery provided by these
new agents is more effective than would be provided by the NO donor
alone. The ligand portion of the molecule binds directly to the
spermatozoon or pathogenic microbe. NO released from the agent is
in direct contact with the cell, allowing lower concentrations to
accomplish the same effect as the NO donor alone.
[0022] These new agents are expected to be more effective against
pathogenic microbes than the ligand or NO donor used alone or in
combination. Activity of NO-SAMMA against C. trachomatis supports
this contention. The ligand is classified as an entry inhibitor.
Entry inhibitors provide protection against microbial invasion of
susceptible cells, but probably have little beneficial effect
against microbial survival or replication. NO-SAMMA and similar
compounds increase the effectiveness of the ligand by providing a
means of killing or otherwise inactivating the microbe through the
release of NO. Adhesin-like receptors have also been identified on
potential target cells for microbial invasion. NO produced in these
cells in response to interaction with NO-SAMMA and related
compounds could contribute to their anti-microbial activities.
[0023] Moreover, these new agents are expected to have broad
anti-microbial activity since the basic activity of the delivery
vector component and the released NO are present even in cases
where the synergistic effects noted above may be absent. For
example, not all microbes are sensitive to NO. Thus, while NO-SAMMA
does not offer enhanced activity against N. gonorrhoeae, the
activity against this microbe due to SAMMA alone remains. In other
words, microbes do not have to be sensitive to both NO and the
adhesin receptor antagonist to be affected by the anti-microbial
compounds of this invention. Of course, sensitivity to both NO and
the adhesin receptor antagonist results in significantly increased
kill or inhibition rates.
[0024] Preferred vector delivery components which are useful in the
present invention for preparing anti-microbial agents are
inhibitory agents such as phosphorylated hesperidins, sulfonated
hesperidins, polystyrene sulfonates, substituted benzenesulfonic
acid formaldehyde co-polymers, H.sub.2SO.sub.4-modified mandelic
acids, cellulose sulfates, and the like. Thus, preferred NO-coupled
anti-microbial agents of this invention include, for example,
phosphorylated hesperidins coupled with a NO-donor, sulfonated
hesperidins coupled with a NO-donor, polystyrene sulfonates coupled
with a NO-donor, substituted benzenesulfonic acid formaldehyde
co-polymers coupled with a NO-donor, H.sub.2SO.sub.4-modified
mandelic acids coupled with a NO-donor, cellulose sulfates coupled
with a NO-donor, and the like. Preferably the vector delivery
components as well as the NO-coupled anti-microbial agents used in
the present invention are at least partially water soluble or water
dispersable so that anti-STD formulations can more easily be
prepared. Especially preferred anti-microbial agents for use in
this invention include H.sub.2SO.sub.4-modified mandelic acids
(SAMMAs) coupled with a NO-donor (i.e., NO-SAMMAs).
[0025] As noted above, preferred NO-SAMMAs include those which have
been fractionated so as to have a narrower molecular weight
distribution and increased activities (as measured against
unfractionated material). It is expected that the preparation of
other NO-coupled anti-microbial agents having narrower molecular
weight distributions will also have increased activities relative
to their unfractionated counterparts. Such narrower molecular
weight distributions can be obtained by fractionating the starting
materials (e.g., SAMMA) or the final products (e.g., NO-SAMMA)
using conventional separation techniques; generally, it is
preferred that the starting materials be fractionated. Although not
wishing to be limited by theory, it appears that the intermediate
molecular weight materials may have higher binding affinities for
relevant biological materials and thus higher activities. Of
course, the optimal molecular weight range for a given NO-coupled
anti-microbial agent can be determined by routine experimentation.
Moreover, the optimal molecular weight range of a given NO-coupled
antimicrobial agent may vary depending on the activity measured
(e.g., contraceptive, acrosomal loss, anti-HIV, anti-HSV, and/or
the like activities), the relative amount of NO coupled to the
agent, and the like.
[0026] SAMMA (i.e., H.sub.2SO.sub.4-modified mandelic acid), the
most preferred vector delivery component of this invention, is a
carboxylated oligomer (average molecular weight of approximately
1.5 KDa) with contraceptive and antimicrobial properties.
Generally, SAMMA has a distribution of carboxylated oligomers
having from 2-3 repeating units up to 20 or more repeating units;
typically the bulk of the material has 7-15 repeating units.
Although not wishing to be limited by theory, it is thought that
the reduction in the relative amounts of material having either low
or high number of repeating unit by fractionation results in
increased activities.
[0027] SAMMA is efficacious against HIV, HSV and C. trachomatis,
among other sexually-transmitted pathogens. SAMMA is active against
spermatozoa, inhibiting hyaluronidase and acrosin (two spermatozoal
enzymes required for fertilization), causes premature acrosomal
loss, and is contraceptive in the rabbit. (Zaneveld et at, "Use of
mandelic acid condensation polymer (SAMMA), a new antimicrobial
contraceptive agent, for vaginal prophylaxis," Fertil. Ster. 78:
1107-15 (2002); U.S. Pat. No. 5,925,621 (Jul. 20, 1999), U.S. Pat.
No. 5,932,619 (Aug. 3, 1999), U.S. Pat. No. 6,028,115 (Feb. 22,
2000), and U.S. Pat. No. 6,239,182 (May 29, 2001).) However, sperm
motility is unaffected by SAMMA at concentrations higher than those
required for its antimicrobial and contraceptive activities,
suggesting that it is not acting by killing spermatozoa. Similar
findings have been made regarding its antimicrobial properties,
insofar as minimal cytotoxic effects of SAMMA are seen on host
cells used for microbial infection in vitro. (Herold et at,
"Mandelic acid condensation polymer novel candidate microbicide for
prevention of human immunodeficiency virus and herpes simplex virus
entry," J. Virol. 76: 11236-44 (2002).)
[0028] Although research on its mechanisms of action is ongoing,
SAMMA's antiviral effects are thought to be mediated, at least in
part, by its ability to antagonize viral binding to target cells,
mediated by the viral adhesins gp120 (for HIV) and g52 (for HSV).
(Cheshenko et al., "Candidate Topical Microbicides Bind Herpes
Simplex Virus Glycoprotein B and Prevent Viral Entry and
Cell-to-Cell Spread," Antimicrob. Agent Chemother. 48: 2025-36
(2004); Herold et at, "Mandelic acid condensation polymer: novel
candidate microbicide for prevention of human immunodeficiency
virus and herpes simplex virus entry," J. Virol. 76: 11236-44
(2002).) SAMMA may be active against C. trachomatis by a similar
mechanism. Post-adhesion interference with viral-mediated signal
transduction at the level of the target cells remains to be
determined.
[0029] SAMMA appears to induce premature acrosomal loss (AL) by a
Ca.sup.2+-dependent mechanism. Anderson et al., "SAMMA induces
premature acrosomal loss by Ca.sup.2+ signaling dysregulation," J.
Androl. 27: 568-577 (2005). Although the initial point of
interaction of SAMMA with spermatozoa (e.g., surface receptor(s))
is unknown, the process appears to require entry of extracellular
Ca.sup.2+. Unlike the physiological acrosome reaction, Ca.sup.2+
entry is likely mediated by voltage-dependent T-type Ca.sup.2+
channels (dose-dependent inhibition by diphenylhydantoin and
Ni.sup.2+), is unaffected by antagonism of InSP3 receptors, does
not require release of intracellular Ca.sup.2+ stores (not
inhibited by 2-APB--an InSP3 receptor antagonist and blocker of
store-operated Ca.sup.2+ channels) and is not mediated by protein
kinase A (not inhibited by KT5720, a selective protein kinase A
inhibitor). SAMMA-induced acrosomal loss (SAL) requires protein
kinase G and soluble guanylate cyclase (>95% inhibited by 2
.mu.M KT5823--a selective protein kinase G inhibitor; .about.50%
inhibited by 0.1 .mu.M ODQ--a selective inhibitor of soluble
guanylate cyclase). Further, SAL is inhibited by inhibitors
selective for the endothelial isoform of nitric oxide synthase.
Taken together, these results suggest that SAL may be mediated by
nitric oxide. Nitric oxide also likely mediates the acrosome
reaction in response to physiological stimuli (e.g., progesterone
and follicular fluid; Herrero et al., "Evidence that nitric oxide
synthase is involved in progesterone-induced acrosomal exocytosis
in mouse spermatozoa," Reprod. Fertil. Develop. 9: 433-9 (1997);
Herrero et al., "Progesterone enhances prostaglandin E2 production
via interaction with nitric oxide in the mouse acrosome reaction,"
Biochem. Biophys. Res. Commun.; 252: 324-8 (1998); Revelli et al.,
"Follicular fluid proteins stimulate nitric oxide (NO) synthesis in
human sperm: a possible role for NO in acrosomal reaction," J. Cell
Physiol. 178: 85-92 (1999); Herrero et al., "Nitric oxide interacts
with the cAMP pathway to modulate capacitation of human
spermatozoa," Free Rad. Biol. Med. 29: 522-36 (2000)).
[0030] The second component of the NO-SAMMAs of the present
invention is a NO donor. NO donors alone also seem to induce AL,
consistent with the proposed mechanism by which SAL occurs.
However, unlike SAL, these reactions do not appear to require
Ca.sup.2+; the Ca.sup.2+ requirement for SAL is likely upstream
from the action of NO. Nor do they appear to require protein kinase
G (not inhibited by 2 .mu.M KT5823 or 0.35 .mu.M Rp-8-Br-PET-cGMPS;
Smolenski et al., "Functional analysis of cGMP-dependent protein
kinases I and as mediators of NO/cGMP effects," Naunyn-Schmied
Arch. Pharmacol., 358: 134-139 (1998)). These results suggest that
the same outcome (AL) is produced by the same biologically active
intermediate (NO) produced by two stimuli/sources (SAMMA and
NO-donors) by two independent mechanisms. Other work with NO-donors
suggest that AL in response to these agents may be mediated by a
cAMP-dependent mechanism. (Kurjak et al., "NO releases
bombesin-like immunoreactivity from enteric synaptosomes by
cross-activation of protein kinase A," Amer. J. Physiol. 276:
G1521-G30 (1999); Vila-Petroff et al., "Activation of distinct
cAMP-dependent and cGMP-dependent pathways by nitric oxide in
cardiac myocytes," Circul. Res. 84: 1020-31 (1999).)
[0031] NO is a highly bioactive, short-lived gaseous molecule
produced in response to various physiological stimuli. Among its
actions are smooth muscle relaxation, inhibition of platelet
aggregation and adhesion, neurotransmission, regulation of
apoptosis, and cytotoxicity. Further, NO is toxic to a number of
bacteria, viruses, and other foreign particles. (Gross et al.,
"Nitric oxide: pathophysiological mechanisms," Ann. Rev. Physiol.
57: 737-69 (1995).) Specifically, NO appear to play a key role in
the natural defense against microbes, including HIV and HSV, and
Chlamydia.
[0032] Viral enzymes (e.g., proteases, reverse transcriptases,
ribonucleotide reductase) containing cysteine residues are targets
for NO-mediated nitrosylation; viral-encoded transcription factors
are also targets. (Persichini et al., "Cysteine nitrosylation
inactivates the HIV-1 protease," Biochem. Biophys. Res Commun. 250:
575-6 (1998); Broillet, "S-nitrosylation of proteins," Cell Mol.
Life Sci. 55: 1036-42 (1999); Persichini et al., "Molecular bases
for the anti-HIV-1 effect of NO. Commentary," int. J. Mol. Med. 4:
365-8 (1999); Benz et al., "Tonal nitric oxide and health:
antibacterial and viral actions and implications for HIV," Med. Sci
Monitor. 8: RA27-RA31 (2002).) NO appears to react with and disrupt
structural proteins essential for viral replication. (Saavedra et
al., "The secondary amine/nitric oxide complex ion
R.sub.2N[N(O)NO].sup.- as nucleophile and leaving group in S(N)Ar
reactions," J. Org. Chem. 66: 3090-8 (2004) Stimulus-induced NO
production is beneficial in preventing HIV infection, and the
protective effect of low levels of NO on cell survival in the CNS
subsequent to HIV infection is recognized. (Fiscus, "Involvement of
cyclic GMP and protein kinase G in the regulation of apoptosis and
survival in neural cells," Neurosig. 11: 175-90 (2002).)
[0033] Endogenous or exogenous NO inhibits HSV-1 and HSV-2
infectivity. Agents lowering or antagonizing NO exacerbate HSV
infection. Inducible nitric oxide synthase (INOS) inhibition
increases pathology and viral titers of HSV-2-infected mice.
(Benencia et al., "Effect of aminoguanidine, a nitric oxide
synthase inhibitor, on ocular infection with herpes simplex virus
in Balb/c mice," Invest. Ophthalmol. Vis. Sci. 42: 1277-84 (2001);
Benencia et al., "Nitric oxide and HSV vaginal infection in BALB/c
mice," Virology 309: 75-84 (2003). Macrophage iNOS activation forms
part of the innate immune response to HSV. (Croen, "Evidence for
antiviral effect of nitric oxide. Inhibition of herpes simplex
virus type 1 replication," J. Clin. Invest. 91: 2446-52 (1993);
Benencia et al., "Nitric oxide and macrophage antiviral extrinsic
activity," Immunology 98: 363-70 (1999); Paludan et al.,
"Interferon (IFN)-gamma and Herpes simplex virus/tumor necrosis
factor-alpha synergistically induce nitric oxide synthase 2 in
macrophages through cooperative action of nuclear factor-kappa B
and IFN regulatory factor-1," Eur. Cytokine Netw. 12: 297-308
(2001).) Macrophage NO inhibits HSV-1 replication, and iNOS
inhibition increases HSV-1 titers. (Karupiah et al., "Inhibition of
viral replication by nitric oxide and its reversal by ferrous
sulfate and tricarboxylic acid cycle metabolites," J. Exp. Med.
181: 2171-9 (1995); Kodukula et al., "Macrophage control of herpes
simplex virus type 1 replication in the peripheral nervous system,"
J. Immunol. 162: 2895-905 (1999).) iNOS-deficient knockout mice are
more susceptible to HSV-1. (MacLean et al, "Mice lacking inducible
nitric-oxide synthase are more susceptible to herpes simplex virus
infection despite enhanced Th1 cell responses," J. Gen. Virol. 79:
825-30 (1998).) NOS inhibitors increase HSV-1-induced pathology.
(Benencia et al., "Nitric oxide and macrophage antiviral extrinsic
activity," Immunology 98: 363-70 (1999).) HSV elimination from the
CNS requires NO. (Chesler et at, "The role of IFN-gamma in immune
responses to viral infections of the central nervous system,"
Cytokine Grth. Fact. Rev. 13: 441-54 (2002).) iNOS-derived NO
inhibits viral replication, (Benencia et at, "Effect of
aminoguanidine, a nitric oxide synthase inhibitor, on ocular
infection with herpes simplex virus in Balb/c mice," Invest.
Ophthelmol. Vis. Sci. 42: 1277-84 (2001); Kodukula et al.,
"Macrophage control of herpes simplex virus type 1 replication in
the peripheral nervous system," J. Immunol. 162: 2895-905 (1999);
Adler et al. "Suppression of herpes simplex virus type 1
(HSV-1)-induced pneumonia in mice by inhibition of inducible nitric
oxide synthase (iNOS, NOS2)," J. Exp. Med. 185: 1533-40 (1997);
Fuji' et al., "Role of nitric oxide in pathogenesis of herpes
simplex virus encephalitis in rats," Virology 256: 203-12
(1999).)
[0034] NO inhibits C. trachomatis growth. (Igietseme et at,
"Inhibition of intracellular multiplication of human strains of
Chlamydia trachomatis by nitric oxide," Biochem. Biophys. Res.
Commun, 232: 595-601 (1997).) NO production by epithelial and
possibly T-cells is important for the resolution of chlamydial
infections. IFN-.gamma. prevents C. trachomatis replication and
promotes NO formation; both are inhibited by iNOS inhibitors.
(Mayer et al., "Gamma interferon-induced nitric oxide production
reduces Chlamydia trachomatis infectivity in McCoy cells," Infect.
Immun. 61: 491-7 (1993); Devitt et al., "Induction of alpha/beta
interferon and dependent nitric oxide synthesis during Chlamydia
trachomatis infection of McCoy cells in the absence of exogenous
cytokine," infect Immun 64: 3951-6 (1996).) NO donors inhibit C.
trachamatis replication in epithelial cells. (Igietseme et al.,
"inhibition of intracellular multiplication of human strains of
Chlamydia trachomatis by nitric oxide," Biochem. Biophys. Res.
Commun. 232: 595-601 (1997).) Protection against chlamydial
infection by T-cells correlates with their ability to induce NO
production. (Igietseme, "The molecular mechanism of T-cell control
of Chlamydia in mice: role of nitric oxide," Immunology 87: 1-8
(1996).) NOS inhibition increases bacterial titers of infected mice
and impairs the ability of T-cell clones to clear genital
chlamydial infection. (Igietseme, "Molecular mechanism of T-cell
control of Chlamydia in mice: role of nitric oxide in vivo,"
Immunology 88: 1-5 (1996).) This is also seen in macrophages, with
a strong correlation between NOS activity and chlamydial
inhibition, effects antagonized by NOS inhibition. (Chen et al.,
"Nitric oxide production: a mechanism of Chlamydia trachomatis
inhibition in interferon-gamma-treated RAW264.7 cells," FEMS
Immunol. Med. Microbiol. 14: 109-20 (1996); Azenabor et al.,
"Chlamydia pneumoniae survival in macrophages is regulated by free
Ca.sup.2+ dependent reactive nitrogen and oxygen species," J.
Infect. 46: 120-8 (2003).) Mouse strains that produce more NO are
more resistant to C. trachomatis. (Ramsey et al., "Role for
inducible nitric oxide synthase in protection from chronic
Chlamydia trachomatis urogenital disease in mice and its regulation
by oxygen free radicals," Inf. Immun. 69: 7374-9 (2001).)
Macrophages from chlamydia-infected, IFN-.gamma. receptor-deficient
mice have increased chlamydial titers, and no detectable NO.
(Johansson et al., "Genital tract infection with Chlamydia
trachomatis fails to induce protective immunity in gamma interferon
receptor-deficient mice despite a strong local immunoglobulin A
response," Inf. Immun. 65: 1032-44 (1997).) In iNOS-deficient mice,
IFN-.gamma. is bacteriostatic against chlamydial infection.
However, IFN-.gamma. is bactericidal in iNOS-sufficient mice and
eradicates the microbe. (Ramsey et al., "Chlamydia trachomatis
persistence in the female mouse genital tract: inducible nitric
oxide synthase and infection outcome," Infect. Immun. 69: 5131-7
(2001).)
[0035] The present invention, in an especially preferred form,
combines SAMMA and a NO donor in a single compound or molecule
which provides the benefits of both components in a synergistic
manner. As noted above, (1) SAL occurs by a Ca.sup.2+- and
NO-dependent mechanism; (2) NO release from NO donors induces AL by
a Ca.sup.2+-independent mechanism, distinct from that responsible
for SAL; and (3) NO has antiviral and antibacterial activities, and
has a key role in the natural defense against HIV, HSV and C.
trachomatis infections. It was hoped that both the contraceptive
and antimicrobial activities of SAMMA could be improved through the
covalent attachment of an NO donor. This has proven to be the case;
indeed the degree of improvement has been surprising.
[0036] The NO-SAMMA compound of the present invention was found to
have the following properties: (1) induces AL in the presence or
absence of Ca.sup.2+; (2) AL due to NO release (in the absence of
Ca.sup.2+) is effected by a lower concentration of NO-donor
equivalents present in the derivative than required for NO-donor
alone (not wishing to be limited by theory, this effect is thought
to be largely due to the fact that the source of NO would be
directed to the surface of the target cell); (3) activity of the
derivative against spermatozoa is synergistic compared with either
NO-donor or SAMMA alone (again, not wishing to be limited by
theory, this effect is thought to be due to different mechanisms by
which they induce AL); (4) antimicrobial activity due to NO release
occurs at lower concentration of NO-donor equivalents present in
the derivative than required for NO donor alone; and (5) a
synergistic antimicrobial effect compared to either NO-donor or
SAMMA alone has been found. It appears that the SAMMA moiety
inhibits microbial binding to target or host cells and the NO
produced by the NO-donor moiety kills or otherwise disrupts the
invasive cells (i.e., microbes and/or spermatozoa).
[0037] The NO-donor moieties suitable for use in the present
invention must be capable of being attached, preferably covalently,
to the vector delivery component and of releasing NO during use.
Suitable NO-donor moieties can be derived from nitrate esters,
furoxans, ketoximes, S-nitrosothiols, nitrosohydrazines,
hydroxylamides, and the like. Of course, other NO-donor moieties
can be used if desired so long as they meet the conditions required
for the present invention.
[0038] Nitrate esters may release NO by several routes, such as,
for example,
RONO.sub.2+2e.sup.-+H.sup.+.fwdarw.ROH+NO.sub.2.sup.-
NO.sub.2.sup.-+e.sup.-+H.sup.+.fwdarw.HO.sup.-+NO
NO.sub.2.sup.-+M.sup.n+.fwdarw.M.sup.(n+1)+.O+NO
where R is an alkyl group preferably having 2 to 8 carbon atoms,
and more preferably 2 to 6 carbon atoms, and M' is a metal ion
(e.g., Fe.sup.+, Cu.sup.+, Cr.sup.2+, Co.sup.2+, and the like. NO
may also be released from such nitrate esters by a thiol-activated
scheme:
##STR00001##
[0039] Furoxans may also release NO via a thiol-activated scheme,
as illustrated below (using 1,2,5-oxiadiazole-2-oxide as an
example):
##STR00002##
[0040] Conjugated ketoximes, using
(.+-.)-E-4-ethyl-2-[(E)-hydroxyimino]-5 nitro-3-hexenamide (NOR-3)
as an example, can release NO via the following scheme:
##STR00003##
NOR-3 (0.5 mM in 0.1 M PBS) has a half-life of about 30 minutes at
pH 7.4 and 37.degree. C. Likewise, imines can also be used as
NO-donor moieties. For example, 3-(4-morpholinyl)sydnomine (SIN-1)
can release NO through the following reaction scheme:
##STR00004##
[0041] S-nitrosothiols, as shown below for
S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione,
can also produce NO:
##STR00005##
NO can also be released from 2-nitroso hydrazine derivatives,
including diazeniumdiolates, such as
1-hydroxy-2-oxo-3-(3-aminopropyl)-3-isopropyl-1-triazene (NOC-5)
and -hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene
(NOC-7), as indicated below:
##STR00006##
(Z)-1-{N-[3-Aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino}-diazen--
1-ium-1,2-diolate] (shown below; spermine-NONOate) is thought to
release NO by a similar mechanism:
##STR00007##
[0042] Hydroxylamides can also be used as the NO-donor moieties.
For example, hydroxyurea reacts with hemoglobin to produce iron
nitrosyl hemoglobin, nitrite, and nitrate, thereby releasing NO.
Hydroxyurea can also release NO via the peroxidase-mediated
hydrolysis of hydroxyurea to hydroxylamine.
[0043] For contraceptive antimicrobial activity, the vector can be
any compound that blocks spermatozoal binding to the oocyte or is
otherwise contraceptive (a surrogate marker for this activity is
the ability of the vector to induce premature acrosomal loss in
vitro) and blocks microbial binding to the target (host) cells. By
itself, it should have contraceptive and antimicrobial activities.
Specifically, the vector should be an adhesin antagonist (receptor
analog). General examples include polyanionic polymers and/or
oligomers with affinity for adhesins that bind to heparan sulfate
or other glycosaminoglycans. Examples within this class include
cellulose sulfate, polystyrene sulfonate, dextran sulfate,
naphthalenesulfonic acid polymer (e.g., Pro2000; Indevus
Pharmaceuticals, Lexington, Miss.), polymethylene hydroquinone
sulfonic acid, or sulfuric acid modified mandelic acid (SAMMA).
These agents have activities against HIV-1, HSV-1, HSV-2, Chlamydia
trachomatis, and Neisseria gonorrhoeae. Based on adhesin receptor
specificities, other pathogens that should be affected by these
agents and NO, include Streptococcus spp (see, e.g., Puliti et al.,
"inhibition of nitric oxide synthase exacerbates group B
streptococcus sepsis and arthritis in mice," Inf. Immun. 72: 4891-4
(2004); Kerr et al., "Nitric oxide exerts distinct effects in local
and systemic infections with Streptococcus pneumoniae," Microb.
Pathogen. 36: 303-10 (2004); Ozturk et al., "Serum and mucosal
nitric oxide levels and efficacy of sodium nitroprusside in
experimentally induced acute sinusitis," Yonsei. Med. J. 44: 424-8
(2003); Leib et al., "Inducible nitric oxide synthase and the
effect of aminoguanidine in experimental neonatal meningitis," J.
Inf. Dis. 177: 692-700 (1998)), Staphylococcus spp (Nablo et al.,
"Nitric oxide-releasing sol-gels as antibacterial coatings for
orthopedic implants," Biomaterials 26: 917-24 (2005); Zhang et al.,
"Differential antibacterial activity of nitric oxide from the
immunological isozyme of nitric oxide synthase transduced into
endothelial cells," Nitric Oxide 7: 42-9 (2002)), Mycoplasma spp
(Hickman-Davis et al., "Cyclophosphamide decreases nitrotyrosine
formation and inhibits nitric oxide production by alveolar
macrophages in mycoplasmosis," Inf. Immun. 69: 6401-10 (2001);
Hickman-Davis et al., "Surfactant protein A mediates
mycoplasmacidal activity of alveolar macrophages by production of
peroxynitrite," Proc. Nat. Acad. Sci. USA 96: 4953-8 (1999)),
Mycobacterium spp (Smeulders et al., "S-Nitrosoglutathione
cytotoxicity to Mycobacterium smegmatis and its use to isolate
stationary phase survival mutants," FEMS Microbiol. Lett. 239:
221-8 (2004)), Mycoplasma spp (Bogdan, "Reactive oxygen and
reactive nitrogen metabolites as effector molecules against
infectious pathogens," in The innate immune response to infection
(Kaufmann et al., eds.), Washington, D.C.: ASM Press, p. 357-96
(2004)), Listeria monocytogenes (Carryn et al, "Impairment of
growth of Listeria monocytogenes in THP-1 macrophages by
granulocyte macrophage colony-stimulating factor: release of tumor
necrosis factor-alpha and nitric oxide," J. Inf. Dis. 189: 2101-9
(2004); Myers et al., "Localized reactive oxygen and nitrogen
intermediates inhibit escape of Listeria monocytogenes from
vacuoles in activated macrophages," J. Immunol. 171: 5447-53
(2003); Remer et al., "Nitric oxide is protective in listeric
meningoencephalitis of rats," Inf. Immun. 69: 4086-93 (2001)),
Helicobacter pylori (Bussiere et al., "Spermine causes loss of
innate immune response to Helicobacter pylori by inhibition of
inducible nitric-oxide synthase translation," J. Biol. Chem. 280:
2409-12. (2005): Potter et al., "Exogenous nitric oxide inhibits
apoptosis in guinea pig gastric mucous cells," Gut 46: 156-62
2000), Borrelia spp (Lusitani et al., "Borrelia burgdorferi are
susceptible to killing by a variety of human polymorphonuclear
leukocyte components," J. Infect. Dis. 185: 797-804 (2002)), and
Bordella pertussis (Canthaboo et al., "Investigation of role of
nitric oxide in protection from Bordetella pertussis respiratory
challenge," Infect. Immun. 70: 679-84 (2002); Torre et al.,
"Regulation of inflammatory responses to Bordetella pertussis by
N(G)-monomethyl-L-arginine in mice intranasally infected," Mediat.
Inflam. 8: 25-9 (1999)). Table 1 below provides a summary of
examples of contraceptive antimicrobial vector/NO donor
combinations for use in the present invention.
[0044] For non-contraceptive antimicrobial activity, the vector
should antagonize microbial adhesin binding to target cells. The
targeted microbe should be sensitive to NO. For example,
fibronectin antagonists (Ofek et al., "Adhesins, receptors, and
target substrata involved in the adhesion of pathogenic bacteria to
host cells and tissues," in Bacterial adhesion to animal cells and
tissues, Washington, D.C.; ASM Press, p. 177-405 (2003)) and NO are
effective against Borrelia spp (Lusitani et al., "Borrelia
burgdorferi are susceptible to killing by a variety of human
polymorphonuclear leukocyte components," J. Infect. Dis. 185:
797-804 (2002)), Chlamydia trachomatis (Chen et al., "Nitric oxide
production: a mechanism of Chlamydia trachomatis inhibition in
interferon-gamma-treated RAW264.7 cells," FEMS Immunol. Med.
Microbiol. 14: 109-20 (1996); Azenabor et al., "Chlamydia
pneumoniae survival in macrophages is regulated by free Ca.sup.2+
dependent reactive nitrogen and oxygen species," J. Infect. 46:
120-8 (2003); Igietseme "Molecular mechanism of T-cell control of
Chlamydia in mice: role of nitric oxide in vivo," immunology 88:
1-5 (1996); Igietseme et al., "Inhibition of intracellular
multiplication of human strains of Chlamydia trachomatis by nitric
oxide," Biochem. Biophys. Res. Commun. 232: 595-601 (1997)),
Streptococcus spp (Puliti et al., "Inhibition of nitric oxide
synthase exacerbates group B streptococcus sepsis and arthritis in
mice," Infect. Immun. 72: 4891-4 (2004); Kerr et al., "Nitric oxide
exerts distinct effects in local and systemic infections with
Streptococcus pneumoniae," Microb. Pathogen. 36: 303-10 (2004)),
Fusobacterium nucleatum (Allaker et al., "Antimicrobial effect of
acidified nitrite on periodontal bacteria," Oral Microbiol.
Immunol. 16: 253-6 (2001)), Mycobacterium spp (Bogdan, "Reactive
oxygen and reactive nitrogen metabolites as effector molecules
against infectious pathogens," in The innate immune response to
infection (Kaufmann et al., eds.), Washington, D.C.: ASM Press, p.
357-96 (2004); Yamashiro et al., "Lower expression of Th1-related
cytokines and inducible nitric oxide synthase in mice with
streptozotocin-induced diabetes mellitus infected with
Mycobacterium tuberculosis," Clin. Exp. Immunol. 139: 57-64 (2005);
Copenhaver et al., "A mutant of Mycobacterium tuberculosis H37Rv
that lacks expression of antigen 85A is attenuated in mice but
retains vaccinogenic potential," Infect. Immun. 72: 7084-95 2004)),
Porphyromonas gingivalis (Bogdan, "Reactive oxygen and reactive
nitrogen metabolites as effector molecules against infectious
pathogens," in The Innate immune response to infection (Kaufmann et
al., eds.), Washington, D.C.: ASM Press, p. 357-96 (2004)),
Salmonella enterica (Bogdan, "Reactive oxygen and reactive nitrogen
metabolites as effector molecules against infectious pathogens," in
The innate immune response to infection (Kaufmann et al., eds.),
Washington, D.C.: ASM Press, p. 357-96 (2004)), Staphylococcus spp
(Nablo et al., "Nitric oxide-releasing sot-gels as antibacterial
coatings for orthopedic implants," Biomaterials 26: 917-24 (2005);
Zhang et al., "Differential antibacterial activity of nitric oxide
from the immunological isozyme of nitric oxide synthase transduced
into endothelial cells," Nitric Oxide 7: 42-9 (2002)), and Yersinia
spp (Dykhuizen et al., "Antimicrobial effect of acidified nitrite
on gut pathogens: importance of dietary nitrate in host defense,"
Antimicrob. Agents Chemother. 40: 1422-5 (1996); Campos-Perez et
al., "Toxicity of nitric oxide and peroxynitrite to bacterial
pathogens of fish," Dis. Aquat. Org. 43: 109-15 (2000)), as welt as
the parasites Trichomonas vaginalis (Crouch et al., "Binding of
fibronectin by Trichomonas vaginalis is influenced by iron and
calcium," Microb. Pathogen. 31: 131-44 (2001); Gradoni et al.,
"Nitric' oxide and anti-protozoan chemotherapy," Parassitologia.
46: 101-3 (2004)) and Leishmania spp (Bogdan, "Reactive oxygen and
reactive nitrogen metabolites as effector molecules against
infectious pathogens," in The innate immune response to infection
(Kaufmann et al., eds.), Washington, D.C.: ASM Press, p. 357-96
(2004)). Table 2 below provides a summary of examples of
non-contraceptive antimicrobial vector/NO donor combinations
suitable for use in the present invention.
[0045] Other suitable adhesin receptor antagonists include, for
example, lactosyl- and galactosylceramides, laminin fragments,
peptidoglycans, and glycopeptides. (See, e.g., Ofek et al.,
"Adhesins, receptors, and target substrata involved in the adhesion
of pathogenic bacteria to host cells and tissues," in Bacterial
adhesion to animal cells and tissues, Washington, D.C.: ASM Press,
p. 177-405 (2004)
TABLE-US-00001 TABLE 1 Contraceptive antimicrobial vectors for
enhancement by nitric oxide: target microbes Adhesin/adhesion
molecule specificity Vector Microbe Nitric oxide effect heparan
sulfate, cellulose sulfate; HIV NO donors inhibit HIV-1 reverse
transcriptase; protective effect of NO on heparan and other
polystyrene CNS after HIV infection sulfated sulfonate; HSV NOS
inhibitors increase viral titers in mice; NO inhibits viral
replication glycosaminoglycans; dextran sulfate; Chlamydia spp NO
donors inhibit C. trachomatis replication; NOS inhibitors increase
bacterial sulfated sugars; SAMMA; titers in mice sulfated
polymethylene- Bordetella pertussis NO (iNOS) protects mice against
Bordetella pertussis infection and decreases polysaccharides;
hydroquinone mortality to Bordetella pertussis infection in vivo
glycosaminoglycans; sulfonate Borrelia spp NO kills Borrelia in
vitro hyaluronan; sulfomucin; Helicobacter pylori NO contributes to
killing H. pylori in macrophages and protects gastric cells
sulfated glycoprotein; from H. pylori-induced apoptosis sulfated
glycolipids; Listeria NO contributes to impairment of intracellular
growth of L. monocytogenes, sulfated monocytogenes retention of L.
monocytogenes by activated macrophages and protects against
glycoconjugates; listeric meningoencephalitis in rats sulfated
proteoglycans Mycobacterium spp Bactericidal action of NO donor
against M. smegmatis Mycoplasma spp iNOS-deficient mice have higher
titers than controls after infection with M. pulmonis Streptococcus
spp mortality of mice infected with Group B streptococcus increased
by NOS inhibitor; protective effect of NO against GBS-induced
meningitis in rats; direct inhibition of S. pneumoniae by NO and NO
donor Staphylococcus spp NO donors and NOS-derived NO kill Staph
and protect against Staph infections Plasmodium control of
infection in human tissue by NO-mediated mechanisms falciparum
(protozoon) Trypanosoma spp intracellular and extracellular
morphotypes of Trypanosoma killed by NO in (protozoon) vitro and in
vivo Leishmania spp NO produced by macrophages kills intracellular
Leishmania (pratozoon) Giardia (pratozoon) parasitostatic effect of
NO donors
TABLE-US-00002 TABLE 2 Noncontraceptive antimicrobial vectors for
enhancement by nitric oxide: target microbes Adhesin/adhesion
molecule specificity Vector Microbe Nitric oxide effect Fibronectin
Fibronectin Borrelia spp NO inhibits Borrella fragments (e.g.,
Chlamydia inhibition by macrophages antagonized end bacterial titer
in mice peptides 4-15 trachomatis increased by NOS inhibitors;
replicaton inhibited by NO donors residues in Streptococcus spp NOS
inhibitors increase mortality of infected mice; inhibition by
length that NO donors contain the Fusobacterium bactericidal effect
of nitrite (potential NO precursor) recognition nucleatum sequence
Mycobacterium spp NO contributes to pathogen control; reduced NO
production in Arg-Gly-Asp or diabetics impairs defense against M.
tuberculosis; inhibited by Arg-X-Asp-Ser NO donors or Leu-Ile-Gly-
Porphyromonas NO contributes to pathogen central Arg-Lys-Lys)
gingivalis Salmonella enterica NO is essential for pathogen control
Yersinia spp bactericidal effects of acidified nitrite; inhibited
by NO donors Leishmania NO is essential for pathogen control
(protozoon) Naegleria fowleri NO and/or NO donors kill
Naegleria
[0046] Although not wishing to be limited by theory, it appears
that NO-couplings to produce the agents in the present invention
will also improve the safety profile of vector units such as SAMMA,
sulfonated hesperidins, phosphorylated hesperidins, polystyrene
sulfonate, cellulose sulfate and the like, intended for use to
prevent HIV infection. This is thought to be due primarily to the
fact that NO donors inhibit production and actions of
pro-inflammatory cytokines.
[0047] Concerns have been expressed regarding the ability of
microbicides to increase production of inflammatory cytokines,
which can cause proliferation of target cells for HIV infection
(Keller et al., "Rigorous pre-clinical evaluation of topical
microbicides to prevent transmission of human immunodeficiency
virus," J. Antimicrob. Chemother. 51: 1099-1102 (2003); Keller et
al., "Topical microblcides for the prevention of genital herpes
infection," J. Antimicrob. Chemother. 55: 420-423 (2005); Stone,
"Microbicides: a new approach to preventing HIV and other sexually
transmitted infections," Nature Reviews 1: 977-85 (2002)). NO
donors as provided by the present invention are thought to be
beneficial in this context. NO donors either reduce production of
inflammatory cytokines or reduce their actions. The extent of these
actions is at least partially dependent upon the cell type and
level of NO (Proud, "Nitric oxide and the common cold," Cur. Opin.
Allergy Clin., Immunol. 5: 37-42 (2005)).
[0048] Vaginal application of the NO donor isosorbide mononitrate
to women has no effect on the production of interleukin (IL)-1,
IL-8, IL-8, IL-10, IL-15, tumor necrosis factor (TNF)-.alpha., or
monocyte chemoattractant protein-1 (Ledingham et al., "Nitric oxide
donors stimulate prostaglandin F(2alpha) and inhibit thromboxane
B(2) production in the human cervix during the first trimester of
pregnancy," Mel. Hum. Reprod. 5: 973-982 (1999)). On the other
hand, molsidomine, a precursor to the NO donor SIN-1, decreases
levels of the pro-inflammatory cytokines TNF-.alpha., IL-1beta and
IFN-gamma and increases production of the anti-inflammatory
cytokines IL-6 and IL-10 in ischemic renal cells (Rodriguez-Pena et
al., "Intrarenal administration of molsidomine, a molecule
releasing nitric oxide, reduces renal ischemia-reperfusion injury
in rats," Amer. J. Transplant. 4: 1605-1613 (2004)). Similar
beneficial effects of this NO donor have been seen in rats with
experimental allergic encephalomyelitis (Kwak et al., "Molsidomine
ameliorates experimental allergic encephalomyelitis in Lewis rats,"
Immunopharmacol. Immunotoxicol. 25: 41-52 (2003)).
[0049] The NO donor SNAP decreases production of the
pro-inflammatory cytokine, IL-12, in mouse macrophages (Xiong et
al., "Inhibition of interleukin-12 p40 transcription and NF-kappaB
activation by nitric oxide in murine macrophages and dendritic
cells," J. Biol. Chem. 279: 10776-10783 (2004)). Similar inhibitory
activity is seen in human peripheral blood mononuclear cells
(Rachlis et al., "Nitric oxide reduces bacterial
superantigen-immune cell activation and consequent epithelial
abnormalities," J. Leukocyte Biol. 72: 339-346 (2002)), in
activated human pulmonary microvascular endothelial cells (Jiang et
al., "Effects of antioxidants and NO on TNF-alpha-induced adhesion
molecule expression in human pulmonary microvascular endothelial
cells," Resp. Med. 99: 580-91 (2005)), rat basophilic leukemia
cells (Heywood et al., "Nicorandil inhibits degranulation and
TNF-alpha release from RBL-2H3 cells," Inflam. Res. 51: 176-181
(2002)), colonic tissue from colitic (induced colitis) mice (Sales
et al., "Nitric oxide supplementation ameliorates dextran sulfate
sodium-induced colitis in mice," Lab. Invest. 82: 597-607 (2002))
and in lipopolysaccharide (LPS)-induced airway inflammation in mice
(Lagente et al., "A nitric oxide-releasing salbutamol elicits
potent relaxant and anti-inflammatory activities," J. Pharmacol.
Exp. Ther. 310: 357-375 (2004).
[0050] The NO donor NOC-18 reduces the number of
IFN-gamma-secreting CD4+ T cells in patients with unstable angina
and coronary spastic angina (Soejima et al., "Preference toward a
T-helper type 1 response in patients with coronary spastic angina,"
Circulation 107: 2196-2200 (2003)), and the NO donor nitroprusside
decreases production of pro-inflammatory cytokines after
reperfusion in coronary artery bypass graft (Freyholdt et al.,
"Beneficial effect of sodium nitroprusside after coronary artery
bypass surgery: pump function correlates inversely with cardiac
release of proinflammatory cytokines," J. Cardiovasc. Pharmacol.
42: 372-378 (2003)). NO donors inhibit stimulus-induced increases
in pro-inflammatory cytokines, while having little effect on
resting levels.
[0051] NO produced in response to IL-1.beta. and TNF-.alpha.
stimulation causes increased cyclooxygenase 2 (COX-2) expression in
mesangial cells. However, NO donors decrease COX-2 in these cells,
likely through feedback inhibition (Diaz-Cazor a et al., "Dual
effect of nitric oxide donors on cyclooxygenase-2 expression in
human mesangial cells," J. Amer. Soc. Nephrol. 10: 943-952 (1999)).
NO donors exert feedback inhibition on NO production in response to
inflammatory cytokines and LPS (Galley et al., "Regulation of
nitric oxide synthase activity in cultured human endothelial cells:
effect of antioxidants," Free Rad. Biol. Med. 21: 97-101 (1996)).
IL-1beta-stimulated chondrocytes show increased activation of
NFkappaB, an effect that is abrogated by the NO donor S
nitrosocysteine ethyl ester (Clancy et al., "Nitric oxide sustains
nuclear factor kappaB activation in cytokine-stimulated
chondrocytes," Osteoarthrit. Cartil. 12: 552-558 (2004)). Similar
effects are noted in human mesangial cells (Diaz-Cazorla et al.,
"Dual effect of nitric oxide donors on cyclooxygenase-2 expression
in human mesangial cells," J. Amer. Soc. Nephrol. 10: 943-952
(1999)) and in mouse macrophages (Xiong et al., "Inhibition of
interleukin-12 p40 transcription and NF-kappaB activation by nitric
oxide in murine macrophages and dendritic cells," J. Biol. Chem.
279: 10775-10783 (2004)). Pretreatment of human mesangial cells
with the NO donors SIN-1 or nitroprusside decreases the subsequent
increase in NFkappaB binding and macrophage chemoattractant
protein-1 (MCP-1) expression in response to TNF-.alpha. or
IL-1.beta. (Lee et al., "Exogenous nitric oxide inhibits tumor
necrosis factor-alpha- or interleukin-1-beta-induced monocyte
chemoattractant protein-1 expression in human mesangial cells. Role
of IkappaB-alpha and cyclic GIMP," Nephron 92: 780-787 (2002)).
Increased permeability of microvessels making up the blood-brain
barrier in response to IL-15, IFN-.gamma., and LPS is reversed by
NO donors (Wong et al., "Cytokines, nitric oxide, and cGMP modulate
the permeability of an in vitro model of the human blood-brain
barrier," Exp. Neurol. 190: 446-455 (2004)). Further, NO donors
promote apoptosis of activated macrophages (Niinobu et al.,
"Negative feedback regulation of activated macrophages via
Fas-mediated apoptosis," Amer. J. Physiol. 279: 0504-0509 (2000)),
which are targets for HIV infection. This effect may depend on the
NO donor concentration (and by inference, the NO concentration)
used (von Knethen et al., "NF-kappaB and AP-1 activation by nitric
oxide attenuated apoptotic cell death in RAW 264.7 macrophages,"
Mol. Biol. Cell 10: 361-372 (1999)). Proliferation of T- and other
immune cells and their recruitment in response to TNF-.alpha., IL-2
or LPS is substantially reduced by several NO donors (Corinti et
al., "Regulatory role of nitric oxide on monocyte-derived dendritic
cell functions," J. Interfer. Cytokine Res. 23: 423-431 (2003);
Haider et al., "Dual functionality of cyclooxygenase-2 as a
regulator of tumor necrosis factor-mediated G1 shortening and
nitric oxide-mediated inhibition of vascular smooth muscle cell
proliferation," Circulation 108: 1015-1021 (2003): Macphail et al.,
"Nitric oxide regulation of human peripheral blood mononuclear
cells: critical time dependence and selectivity for cytokine versus
chemokine expression," J. Immunol. 171: 4809-4815 (2003)).
[0052] The above findings suggest that incorporation of an NO donor
into a topical microbicide as provided by this invention may, among
other effects, improve its safety profile. NO donors inhibit the
production and actions of inflammatory cytokines that may act to
proliferate target cells for HIV infection.
[0053] The synthesis of the NO donor/vector adduct of the adhesin
receptor antagonist can be achieved by standard organic synthetic
pathways, in which the NO donor moiety is attached to a spacer
molecule (e.g., alkane of 2-8 carbons in length) that contains a
moiety suitable for coupling to the vector (e.g., amino, carboxyl,
or hydroxyl). The spacer molecule containing the NO donor can be
linked to hydroxyl, amino or carboxyl moieties of the vector via an
ester or amide linkage. In instances where the vector contains
polyol groupings (e.g., dextran or cellulose derivatives),
regioselective attachment of the NO donor as a nitrate ester can be
effected. Representative examples of such synthetic methods are
presented below; of course, other methods can be used to prepare
the NO donor/vector adducts of this invention.
[0054] Nitrate Esters of SAMMA.
[0055] Reaction of the bromoalkanols in anhydrous acetonitrile with
silver nitrate affords the required nitrooxy-alkanols as shown in
Equation 1 for bromalkanols ranging in size from C2 to C6.
##STR00008##
Of course, values of n greater than 5 can be used if desired for
the nitrate esters in Equations 1-4. As shown in Equation 2,
substoichiometric coupling to SAMMA free acid is accomplished with
1,1'-carbonyldiimidazole (CDI) in dry DMF linking the nitrooxy
alkanol (1,3-nitrooxyphenol) by an ester linkage. (Endres et al.,
"NO-donors, part 3: nitrooxyacylated thiosalicylates and
salicylates--synthesis and biological activities," Eur. J. Med,
Chem. 34: 895-901 (1999).)
##STR00009##
A series of nitrooxyalkyl amines C2-C8 can be prepared, that can be
coupled to SAMMA by an amide linkage. Focus is placed on the C3
adduct at levels of substitution from 5-60%. Minin and Walton
("Radical Ring Closures of 4-Isocyanato Carbon-Centered Radicals,"
J. Org. Chem. 68: 2960-3 (2003)) have described the synthesis of
4-bromo-1-butylamine hydrobromide by refluxing 4-amino-1-butanol in
concentrated hydrobromic acid; this can be applied to prepare other
bromo-alkylamine hydrobromides. As shown in Equation 3, reaction of
the bromo-alkylamine hydrobromides with 2 equivalents of silver
bromide in dry acetonitrile gives the nitrooxy-alklyamines (as
their ammonium nitrate salts).
##STR00010##
Treatment of the amine salts with one equivalent of imidazole in
dry DMF provides the free amine necessary for CDI coupling to SAMMA
as shown in Equation 4.
##STR00011##
[0056] Furoxan Derivatives of SAMMA.
[0057] Furoxan derivatives (furazan oxide, 1,2,5-oxadiazole
2-oxide) release NO in the presence of thiol cofactors. (Medana et
al., "Furoxans as Nitric Oxide Donors.
4-Phenyl-3-furoxancarbonitrile: Thiol-Mediated Nitric Oxide Release
and Biological Evaluation," J. Med. Chem. 37: 4412-6 (1994);
Ferioli et al., "A new class of furoxan derivatives as NO donors:
mechanism of action and biological activity," J. Pharmacol. 114:
816-20 (1995); Schonafinger, "Heterocyclic NO prodrugs," Farmaco.
54: 316-20 (1999).) Among other therapeutic activities, furoxan
derivatives inhibit H1V-1 reverse transcriptase. (Persichini, et
al., "Nitric oxide inhibits the HIV-1 reverse transcriptase
activity," Biochem. Biophys. Res. Commun. 258: 624-7 (1999).)
Furoxans that can be linked to SAMMA are prepared with commercially
available starting materials. Nitromethane and sodium methoxide are
combined in dry DMF followed by the addition of sodium benzene
sulfinate and iodine to form phenyl nitromethyl sulfone (PNS). As
shown in Equation 5, PNS is cyclized by heating in glacial acetic
acid-nitric acid for 1 hour at 65.degree. C. (Kelley et al.,
"Synthesis of bis(arylsulfonyl)furoxans from aryl nitromethyl
sulfones," J. Heterocycl. Chem. 14: 1415-6 (1977).)
##STR00012##
The 3,4-bis(benzene sulfonyl) furoxan (BBSF) reacts with alcohols
under alkaline conditions to afford substituted furoxans. (Sorba et
al., "Unsymmetrically substituted furoxans. Part 16. Reaction of
benzenesulfonyl substituted furoxans with ethanol and ethanethiol
in basic medium," J. Heterocycl. Chem. 33: 327-34 (1996); Loll et
al, "A new class of ibuprofen derivatives with reduced
gastrotoxicity," J. Med. Chem. 44: 3463-8 (2001)) As shown in
Equation 6, treatment of BBSF with 1,3-propanediol in THF with 50%
NaOH leads to displacement of one sulfone group.
##STR00013##
The resulting molecule is ready for coupling to SAMNA. Similarly,
treatment of BBSF with ethanol then 1,3-propanediol forms the
ethoxy derivative as indicated in Equation 7. (Sorba et al.,
"Unsymmetrically substituted furoxans. Part 16. Reaction of
benzenesulfonyl substituted furoxans with ethanol and ethanethiol
in basic medium," J. Heterocycl. Chem. 33: 327-34 (1996).)
##STR00014##
Another furoxan can be prepared by the reaction of crotonaldehyde
with NaNO.sub.2 in acetic acid yielding
3-methyl-4-furoxancarbaldehyde (Fruttero et al., "Unsymmetrically
substituted furoxans. Part 11. Methylfuroxan-carbaldehydes," J.
Heterocycl. Chem. 26: 1345-7 (1989)) which is reduced, as shown in
Equation 8, to 3-methyl-4-furoxanmethanol with NaBH.sub.4 in
dioxane. (Di Stile et al., "New 1,4-Dihydropyridines Conjugated to
Furoxanyl Moieties, Endowed with Both Nitric Oxide-like and Calcium
Channel Antagonist Vasodilator Activities," J. Med. Chem. 41:
5393-401 (1998).)
##STR00015##
These can be coupled to SAMMA at different levels of substitution
(e.g., 5 to 60%).
[0058] Nitrated Cellulose Sulfate.
[0059] Nitrate ester derivatives of cellulose sulfate can be
prepared by regioselective sulfation of cellulose or cellulose
derivative (e.g., acetate, trimethysilyl ether, nitro, nitrite or
tosylate) on C2 and G3, followed by nitration at C1, as shown
below:
##STR00016##
The reaction shown below provides good selectivity at C-6.
##STR00017##
The amount of nitrating product can be controlled by reaction
temperature and reaction time.
[0060] The method of the present invention is carried out by
applying an effective amount of the NO-coupled anti-microbial agent
or agents of this invention to the area or areas expected to
undergo sexual contact during the sexual activity, especially to
those areas in which the transmission of STD-causing organisms is
more likely and which will likely be in contact with a partner's
bodily fluids which may contain the STD-causing organisms. For
purposes of this invention, an "effective amount" is an amount
sufficient to inactivate, but not necessarily kill, STD-causing
organisms on contact and/or upon release of nitric oxide. Suitable
NO-coupled anti-microbial agent or agents for use in the present
invention include, for example, phosphorylated hesperidins coupled
with a NO-donor, sulfonated hesperidins coupled with a NO-donor,
polystyrene sulfonates coupled with a NO-donor, substituted
benzenesulfonic acid formaldehyde co-polymers coupled with a
NO-donor, H.sub.2SO.sub.4-modified mandelic acids coupled with a
NO-donor, cellulose sulfates coupled with a NO-donor, and the like.
As indicated above, preferred anti-microbial agents for use in this
invention include H.sub.2SO.sub.4-modified mandelic acids (SAMMAs)
coupled with a NO-donor (i.e., NO-SAMMAs).
[0061] Generally, the NO-coupled anti-microbial agent or agents are
incorporated into conventional carriers, such as, for example,
lotions, creams, jellies, liniments, ointments, salves, oils,
foams, gels, washes, suppositories, slow-releasing polymers,
coatings, or devices, and the like so that they can be easily
applied topically in the present methods. The carriers may also
include other ingredients such as, for example, pH modifiers,
stabilizers, buffers, surfactants, moisturizers, colorants,
thickeners, flavorings, fragrances, perfumes, and the like. The
inhibitory agents of the present invention may also be used with
conventional birth-control or safe-sex devices. For example, the
NO-coupled anti-microbial agent or agents could be incorporated
into or simply used in conjunction with condoms (i.e., via
lubricants applied to the Interior and/or exterior surfaces),
diaphragms, cervix caps, or similar products. The NO-coupled
anti-microbial agent or agents of the present invention could also,
for example, be released into the vagina (or rectum in the case of
anal intercourse) by hand, via suppositories, or by using
conventional tampon or syringe techniques. The method of
administering or delivering the NO-coupled anti-microbial agent or
agents to the potential STD-transmission site is not critical so
long as an effective amount of the NO-coupled anti-microbial agent
is delivered to the site in a timely manner. Preferably the
formulations and/or method of delivering the NO-coupled
anti-microbial agent or agents allows the inhibitory agents to
remain in the appropriate area during (and even after) the sexual
activity in order to maximize the effectiveness.
[0062] Preferred inhibitory agents (i.e., the anti-microbial
component of the NO-coupled anti-microbial agents) include
phosphorylated hesperidins, sulfonated hesperidins, polystyrene
sulfonates, substituted benzenesulfonic acid formaldehyde
co-polymers, H.sub.2SO.sub.4-modified mandelic acids, and cellulose
sulfates. Preferably the inhibitory agents, as well as the
NO-coupled anti-microbial agents, used are water soluble or
dispersable (or at least partially so). Generally, the NO-coupled
anti-microbial agents are employed at a concentration of about 0.2
mg/g or higher in a suitable formulation, preferably at a
concentration of about 10 mg/g to about 100 mg/g, and more
preferably at a concentration of about 20 mg/g to about 70 mg/g
based on the total weight of inert and active ingredients. Although
it is generally preferred that such anti-STD compounds be used at
non-cytotoxic levels in order to minimize potential side effects,
these compounds can also be used, if desired, at levels at which
the STD-organisms (or a significant portion thereof) are
effectively killed rather than simply inactivated or inhibited.
[0063] In actual use, the NO-coupled anti-microbial agent in a
suitable carrier or vehicle is applied, preferably topically, to
the general area or areas of expected sexual contact (e.g., areas
in which bodily fluids are likely to be generated and/or deposited)
prior to the sexual activity. For vaginal heterosexual intercourse,
the NO-coupled anti-microbial agents could be inserted into the
vagina prior to intercourse. For anal intercourse (heterosexual or
homosexual), the NO-coupled anti-microbial agents could be inserted
into the rectum prior to intercourse. For either vaginal or anal
intercourse, the NO-coupled anti-microbial agents could be
Incorporated into the lubricant used with the condom. For added
protection it is generally preferred that the NO-coupled
anti-microbial agent be applied before intercourse or other sexual
activity and that, if appropriate, a condom be used. For even
further protection, the NO-coupled anti-microbial agents can be
reapplied after completion of the sexual activity; in such cases, a
douche or rinse with the NO-coupled anti-microbial agent in a
liquid carrier solution could be used. Using edible carriers and
suitable flavorings, the NO-coupled anti-microbial agents could
also be used to provide protection during oral sex (heterosexual or
homosexual); a mouthwash containing NO-coupled anti-microbial
agents could be used afterwards. By incorporating desirable
flavorants, scents, fragrances, and colorants, the NO-coupled
anti-microbial agents could become a "pleasing" or "desirable"
component of the sexual activity (i.e., a sex aid or toy) thereby
Increasing the probability of their use and, therefore, the degree
of protection afforded the sexual parties.
[0064] One advantage of the present method is that it can be used
for protection during a wide variety of sexual activities (vaginal,
anal, or oral) by heterosexuals, bisexuals, and homosexuals of
either gender. Another advantage of the present method of reducing
the transmission of STDs is that this method can be implemented
and/or used by either party. Thus, a woman could use the present
method to protect herself (as well as her partner) with or without
the partners knowledge of the method being used. Moreover, one
partner would not be required to rely on his or her partner's claim
of being STD-free or agreement to use condoms or other barrier
devices for protection. Either or both sexual parties could
initiate and Implement the use of the present method prior to, or
after, the sexual encounter. Preferably the method is used before
the sexual activity and most preferably both before and after the
sexual activity. Although use only after the sexual activity would
provide less protection, it would still be desirable to implement
this method afterwards if the method was not used prior to the
sexual activity for any reason (e.g., in cases of rape). Of course,
the sooner this method is initiated after the sexual activity the
better. Preferably the method is initiated within one hour, more
preferably within 15 minutes, and most preferably almost
immediately after the sexual activity. Even after periods greater
than these, however, the use of this method as soon as possible
after the sexual activity may provide at least some protection (as
compared to no treatment).
[0065] Still another advantage of the present invention is that, in
contrast to other contraceptive or protective methods which rely on
a cytotoxic compound (e.g., nonoxynol-9), the NO-coupled
anti-microbial agents used in this invention do not significantly
affect or inhibit the growth characteristics of the normal vaginal
flora or otherwise significantly irritate the vaginal tissue when
used at inhibitory, noncytotoxic, or clinical concentrations. Thus,
the beneficial components of normal vaginal flora are not disrupted
by the use of the present invention. Significant inhibition or
modifications of the vaginal flora or other irritations (such as
when nonoxynol-9 is used) can lead to increased risks of infections
(both STD and non-STD types), unusual discharges, general
discomforts, and the like, which, in turn, can lead to a reluctance
to use or fully take advantage of the protective method. Such
inhibition or modifications of the vaginal flora, irritation of
vaginal tissue, and/or lesions can actually increase the risk of
STD transmission and infection. By avoiding or reducing the
intensity of these effects, the present method is more likely to be
used on a consistent basis. By reducing the number of unprotected
sex acts (preferably to zero) and encouraging the use of the
methods of this invention both before and after each sex act, the
overall degree of protection should be significantly increased. By
avoiding or reducing vaginal irritations and especially lesions on
the vaginal walls (or rectum lining in the case of anal
intercourse), the transmission of STD should be further reduced
since transmission of STD-causing organisms is generally easier
where damage to the cell walls has occurred. Thus, improvements in
ease of use, reduction in side effects, the ability to be initiated
by either party, and the ability to be used for different and
varied sexual activities give the present invention a significant
advantage as a contraceptive and/or as an anti-STD method.
[0066] The present NO-coupled anti-microbial agents can also be
used by persons who are not at risk or significant risk of
pregnancy. For purposes of this application, the phrases "not at
risk for pregnancy" or "not at significant risk for pregnancy" are
intended to include individuals who, for any number of reasons, are
not capable of becoming pregnant or who are employing alternative
birth control methods. Such individuals not capable of becoming
pregnant include, for example, homosexual partners, men in general,
diagnosed sterile individuals (including women regardless of the
cause of sterility and men who are unable to impregnate a woman
regardless of the cause of sterility), post-menopause women, and
the like. Individuals who are employing alternative birth control
methods, for purposes of this application, are not at significant
risk of pregnancy. For example, a woman using a contraceptive pill
and/or condom would not be considered to be at risk for pregnancy
even though the effectiveness of the pill and/or condom is not 100
percent; similarly, users of other conventional birth control
methods would not be considered to be "at significant risk of
pregnancy" even though the failure rate may be higher than that for
the pill and/or condom. For purposes of this specification, the
phrase "not at risk of pregnancy" is also intended to also include
the phrase "not at significant risk of pregnancy" as that term is
used above.
[0067] All references (including patents, patent publications, and
other publications) are incorporated by references in their
entireties. Unless otherwise noted, all percentages and ratios in
the present specification are based on weight.
Examples
Example 1
[0068] NOT-SAMMA. This example illustrates the preparation of
anti-microbial agents wherein the delivery vector is derived from
SAMMA and the nitric oxide donor moiety is derived from
nitrooxypropanol. In this example, the anti-microbial agent is
about 7% substituted with the nitric oxide donor moiety.
[0069] The NO-donor (3-nitrooxy-1-propanol) was synthesized by
reacting silver nitrate with 3-bromo-1-propanol in acetonitrile.
Silver nitrate (15.7 g, 110 mmoles) was dissolved in acetonitrile
(200 mL). 3-Bromo-1-propanol (18.9 g; 100 mmoles) dissolved in 25
mL acetonitrile was added. The reaction flask was protected from
light using aluminum foil. The reaction mixture was stirred at
ambient temperatures for about 96 hours; after 2 hours, a yellow
precipitate (AgBr) was observed. The reaction mixture was filtered
through a cellite pad to remove particulate AgBr. The solvent was
removed using rotary evaporation to yield a yellow oil which was
dissolved in dichloromethane. The resulting solution was extracted
with saturated NaCl in water to remove residual silver as AgCl and
then dried over anhydrous sodium sulfate. After removal of the
solvent by rotary evaporation and then distillation at about 50 mm
Hg, 3-nitrooxy-1-propanol, a clear yellow oil, was obtained in
about 62 percent yield. Identity was confirmed using IR and
.sup.13C NMR.
[0070] NO-SAMMA was prepared using essentially the reaction scheme
shown in Equation 2 above. SAMMA (10.02 g; 74.8 acid meq; prepared
as described in Example 4 of U.S. Pat. No. 5,932,619) was dissolved
in 100 mL dimethylformamide (DMF) in a flask equipped with a drying
tube to maintain low moisture levels and then cooled to 0.degree.
C. using an ice bath. A sub-stoichiometric amount of
1,1'-carbonyldiimidazole (CDI; 4.02 g; 24.8 mmoles; coupling agent)
was dissolved in 40 mL dry DMF and added dropwise over 40 minutes
to the stirred SAMMA solution. The reaction was continued for an
additional 45 minutes with stirring at 0.degree. C.
Nitrooxypropanol (2.74 g; 22.6 mmoles) in 20 mL dry DMF was added
dropwise over a 30 minute period during which time the reaction
temperature was allowed to rise to ambient temperature. The
reaction was continued for about 4 hours with stirring at ambient
temperature. The reaction mixture was decanted into 1500 mL water
and then acidified to pH 1.9 using 6N HCl. The resulting light pink
precipitate was suction filtered, washed with water (500 and 250 mL
portions), and suction filtered. The wet precipitate was dried by
lyophilization to yield 10.15 g of NO7-SAMMA (salmon-colored
powder) in about 97 percent yield.
Example 2
[0071] NO23-SAMMA. This example illustrates the preparation of
anti-microbial agents wherein the delivery vector is derived from
SAMMA and the nitric oxide donor moiety is derived from
nitrooxypropanol. In this example, however, the anti-microbial
agent is approximately 23 percent substituted with the nitric oxide
donor moiety.
[0072] SAMMA (3.19 g; 23.8 acid meg; prepared as described in
Example 4 of U.S. Pat. No. 5,932,619) was dissolved in 500 mL dry
DMF in a flask equipped with a drying tube to maintain low moisture
levels and then cooled to 0.degree. C. using an ice bath. An excess
amount of 1,1'-carbonyldiimidazole (CDI; 9.73 g; 60 mmoles;
coupling agent) was dissolved in 40 mL dry DMF and added to the
stirred SAMMA solution. After a further addition of 30 mL dry DMF,
the reaction was continued for an additional 45 minutes with
stirring at 0.degree. C. Nitrooxypropanol (0.86 g; 7.1 mmoles; from
Example 1) in 5 mL dry DMF was added dropwise over a 2 minute
period. The reaction temperature was allowed to rise to ambient
temperature. The reaction was continued for about 15 hours with
stirring at ambient temperature. The reaction mixture was decanted
into 1000 mL water and then acidified to pH 1.6 using 6N HCl. The
resulting deep red precipitate was suction filtered, washed with
water (two 200 mL portions), and suction filtered. The wet
precipitate was dried by lyophilization to yield 2.78 g of
NO23-SAMMA in about 73 percent yield.
Example 3
[0073] Effect of NO-SAMNA on acrosomal loss (AL). Both NO7-SAMMA
(Example 1) and NO23-SAMMA (Example 2) were evaluated for their
effect on acrosomal loss.
[0074] Measurement of AL was carried out as described previously.
(Anderson et al., "Preclinical evaluation of sodium cellulose
sulfate (Ushercell.TM.) as a contraceptive antimicrobial agent," J.
Androl. 23: 426-38 (2002); Zaneveld et al., "Use of mandelic acid
condensation polymer (SAMMA), a new antimicrobial contraceptive
agent, for vaginal prophylaxis," Fert. Steril. 78: 1107-15
((2002).) Fresh human semen was collected from healthy donors by
self-masturbation. All samples were used within one hour of
collection. Spermatozoa were isolated and washed by centrifugation
through buffered Ficoll and resuspension in BWW medium. A sample
was withdrawn for motility assessment, and the sperm suspensions'
were treated with either SAMMA, 3-nitrooxypropan-1-ol, NO7-SAMMA,
or NO23-SAMMA. The concentrations of SAMMA and nitrooxypropanol
were equivalent to the concentrations of these moieties found in
either 0.075 .mu.g/mL NO7-SAMMA or 0.02 .mu.g/mL NO23-SAMMA, based
on their respective degrees of substitution. Reaction induced by
0.075 .mu.g/mL NO7-SAMMA was compared to the reactions induced by
either 0.072 .mu.g/mL SAMMA or 0.037 .mu.M nitrooxypropanol, and to
the predicted response to the two agents added in combination,
assuming independence of action. Similarly, reaction induced by
0.02 .mu.g/mL NO23-SAMMA was compared to the reactions induced by
either 0.019 .mu.g/mL SAMMA or 0.0364 .mu.M nitrooxypropanol. Ail
reactions were carried out in either the presence or absence of
added extracellular Ga.sup.2+ (1.28 mM). Fifteen minutes after
adding either SAMMA, nitrooxypropanol, NO7-SAMMA or NO23-SAMMA,
sperm motility was measured and spermatozoa were fixed in buffered
glutaraldehyde, air-dried onto slides, stained with Bismark Brown Y
and Rose Bengal and scored for the presence of acrosomes (De Jonge
et al., "Synchronous assay for human sperm capacitation and the
acrosome reaction," J. Androl. 10: 232-39 (1989)). Data are
expressed as the average % maximal response, based on the AL
induced by a maximally stimulating concentration of the calcium
ionophore A23187.
[0075] Both NO7-SAMMA and NO23-SAMMA induced AL in the absence of
added extracellular Ca.sup.2+. The response to 0.075 .mu.g/mL
NO7-SAMMA in the absence of added Ca.sup.2+ (41% maximal loss) was
over 6-fold higher than the predicted response to an equivalent
amount of the NO donor from which NO7-SAMMA was derived. In the
presence of Ca.sup.2+, the response to NO7-SAMMA was synergistic
over the predicted response to combined addition of equivalent
concentrations of NO donor and SAMMA. The increase in AL in the
presence of NO7-SAMMA, as shown in FIG. 1, was nearly 5.6-fold than
the predicted increase in NO donor-induced AL due to the addition
of SAMMA.
[0076] Even higher activity, see FIG. 1, was observed with
NO23-SAMMA. The response to 0.02 .mu.g/mL NO23-SAMMA in the absence
of added Ca.sup.2+ (about 47% maximal loss) was 6.6-fold higher
than the predicted response to an equivalent amount of the NO donor
from which NO23-SAMMA was derived. The observed synergy in this
instance was approximately the same as that observed for NO7-SAMMA,
but the concentration required for the effect is only 27% of that
required for NO7-SAMMA, likely due to increased level of
substitution on NO donor. The increase in AL in the presence of
NO23-SAMMA was nearly 21.5-fold higher than the predicted increase
in NO donor-induced AL due to the addition of SAMMA. As shown in
FIG. 2, the ED.sub.50 values of fractionated NO23-SAMMA (0.09
ng/mL: see Example 5 for details regarding fractionation) and
unfractionated NO23-SAMMA (8 ng/mL) are more than 2800 and 30
times, respectively, less than the ED.sub.50 for SAMMA (250
ng/mL).
Example 4
[0077] Effect of NO-SAMMA against C. trachomatis. Infection of HeLa
cells by C. trachomatis (serotype E/UW-5/CX) was measured as
described by Cooper et al. ("Chlamydia trachomatis infection of
human fallopian tube organ cultures," J. Gen. Microbiol. 136:
1109-15 (1990)) in the presence and absence of NO7-SAMMA, SAMMA, or
nitrooxypropanol. Approximately 1.times.10.sup.5 IFU/mL of
chlamydial elementary bodies were added to different concentrations
of either SAMMA or NO7-SAMMA, from 50 .mu.g/mL to 500 .mu.g/mL, and
incubated at 0.degree. C. for four hours, after which the mixture
was inoculated onto HeLa cell monolayers. In separate experiments,
HeLa cell monolayers were incubated with either SAMMA or NO7-SAMMA
at different concentrations, ranging from 50-500 .mu.g/mL at
37.degree. C. for one hour, after which the overlaying medium was
decanted and the monolayer was washed with fresh medium without
microbicide. This was followed by inoculation of the monolayer with
chlamydial elementary bodies. One hour after inoculation, free
microbes and/or microbicide were removed by washing, and the HeLa
cell cultures were incubated for an additional 48 hours at
37.degree. C. Chlamydia-induced inclusions were measured by
immunofluorescence, after reacting cultures with Kallsted chlamydia
culture confirmation fluorescein-conjugated monoclonal antibody.
Data are expressed as bacterial titer (IFU/mL) at each
concentration of either SAMMA or NO7-SAMMA.
[0078] A 3-log reduction of C. trachomatis occurred at a NO7-SAMMA
concentration that is approximately one order of magnitude lower
than for SAMMA (see FIG. 3: 3-log reduction for SAMMA alone was at
20 mg/mL as compared to 1.9 mg/mL for NO7-SAMMA). Further, the
Inhibitory effect of NO7-SAMMA, unlike that of SAMMA, occurred not
only at the level of direct effect on the elementary body, but also
on the target (HeLa) cells (see FIG. 4); an IC.sub.50 of 0.7 mg/mL
was found for NO7-SAMMA. Interestingly, the inhibitory effect of
nitroprusside against C. trachomatis (IC.sub.50=23 .mu.M) was about
the same as that against spermatozoa (74% maximal AL at 22 .mu.M),
suggesting that C. trachomatis and spermatozoa may have similar
sensitivity to NO.
Example 5
[0079] Fractioned NO23-SAMMA. This example illustrates the
preparation and evaluation of an anti-microbial agent, wherein the
delivery vector is derived from SAMMA that has been fractionated on
silica gel to achieve a more narrow range of molecular weights, and
the nitric oxide donor moiety is derived from nitrooxypropanol.
This fractionated material is distinguished from that described in
Example 2 wherein non-fractionated (bulk) SAMMA was used as the
delivery vector. In this example, the anti-microbial agent is
approximately 23 percent substituted with the nitric oxide donor
moiety. This fractionated material exhibited unexpectedly increased
activity as compared to the unfractionated material.
[0080] SAMMA (3.0 g; 22.4 acid meq, prepared as described in
Example 4 of U.S. Pat. No. 5,932,619) was dissolved in a minimal
volume (9 mL) of methanol. This solution was applied to a
200.times.35 mm glass column filled 2/3 with chromatographic silica
gel, 100-200 mesh (Fisher Scientific), equilibrated with methylene
chloride and topped with washed sand, and eluted (200 mL each) with
a discontinuous gradient of methylene chloride containing
increasing concentrations of methanol (2%, 10%, 20%, 30% 100%, v/v;
increasing polarity). Fractions of 200 mL were collected.
Fractionated SAMMA (to be used for the preparation of fractionated
NO23-SAMMA) was recovered in the 30% methanol (20-30% fraction)
elution. Yield: 2.55 g (85%). MALDI TOF MS showed a predominant
molecular weight distribution of the fractionated SAMMA between
700-2500, with molecular weight <600 representing a minor
(approx. 2-3%) constituent, and very low (<1%) amounts with
molecular weight equal to or greater than 2500. The 20-30% fraction
was used to prepare fractionated NO23-SAMMA using the procedure
described in Example 2 for NO23-SAMMA.
[0081] Fractionated NO23-SAMMA has the highest activity as a
stimulus of acrosomal loss of any the compounds studied (see FIG.
2). Covalent modification of SAMMA having a more dearly defined
range of molecular weights increased efficacy by about two orders
of magnitude over NO23-SAMMA. SAMMA fractionated on silica gel is
similar to unfractionated (bulk) SAMMA as a stimulus of AL (68%
maximal AL at 0.25 .mu.g/mL; essentially the same ED.sub.50 as for
unfractionated SAMMA). Although this represents a synergistic
response to equivalent concentrations of either nitrooxypropanol or
SAMMA added alone (separately), synergism cannot be quantified for
fractionated NO23-SAMMA, since equivalent concentrations of SAMMA
and NO donor are so low as to produce responses below the limit of
detection. The ED.sub.50 of fractionated NO23-SAMMA is 0.09 ng/mL.
This quantity of fractionated NO23-SAMMA contains the equivalent of
0.08 ng/mL SAMMA and 0.13 nM nitrooxypropanol; it, however,
represents an increased activity over SAMMA of nearly 2,800-fold.
For comparison, the ED.sub.50 of NO23-SAMMA (8 ng/mL) contains the
equivalent of 6.8 ng/mL SAMMA and 11.2 nM nitrooxypropanol. The
ED.sub.50 for nitrooxypropanol as a stimulus of acrosomal loss is
120 nM.
[0082] Fractionated NO23-SAMMA has essentially no effect (i.e.,
less than about 10% inhibition) on the percentage of motile
spermatozoa at concentrations up to 10 mg/mL (Control
motility=69.4.+-.0.6 (SEM) %; motility with 10 mg/mL fractionated
NO23-SAMMA=63.2.+-.2.2%; N=4). These data show that NO-SAMMA has no
effect on sperm viability.
[0083] Acrosomal loss induced by SAMMA is Ca.sup.2+-dependent
(Anderson et al., "SAMMA induces premature acrosomal loss by
Ca.sup.2+ signaling dysregulation", J. Andra 27: 568-577 (2006)).
Unless otherwise noted, acrosomal loss data were obtained from
assays that included Ca.sup.2+ in the extracellular medium. In
contrast, acrosomal loss induced by NO donors occurs independent of
Ca.sup.2+. These properties can be exploited to determine the
contributions of the SAMMA and NO donor moieties to acrosomal loss
induced by NO-SAMMA. Although not wishing to be limited by theory,
it appears that acrosomal loss in the presence of added
extracellular Ca.sup.2+ may be due to either or both moieties,
whereas acrosomal loss in the absence of Ca.sup.2+ is due entirely
to the NO donor moiety.
[0084] Fractionated NO23-SAMMA induces acrosomal loss in the
absence of Ca.sup.2+ with an ED.sub.50 of 0.37 ng/mL. Based on
nitrogen content of fractionated NO23-SAMMA (1.98.+-.0.106%), this
is equivalent to 0.53 nM equivalents of nitric oxide donor (see
FIG. 5). SAMMA at 0.25 .mu.g/mL in the absence of Ca.sup.2+ has
essentially no effect (Anderson et al., "SAMMA induces premature
acrosomal loss by Ca.sup.2+ signaling dysregulation", J. Androl.
27: 568-577 (2006)), and the ED.sub.50 for nitrooxypropanol is 0.12
.mu.M (Ca.sup.2+-independent). The effect is of fractionated
NO23-SAMMA in the absence of Ca.sup.2+ is likely due to the NO
donor moiety of NO-SAMMA, and is clearly enhanced relative to
effects of either SAMMA or NO donor added alone. Strictly speaking,
this does not represent a synergistic response, since SAMMA is
without effect in the absence of Ca.sup.2+.
[0085] Contraception in rabbits by fractionated NO23-SAMMA is
substantially more effective than contraception by SAMMA. Greater
efficacy is seen at a fractionated NO-SAMMA concentration one order
of magnitude lower than the concentration of SAMMA. Sperm
pretreatment with 5 mg/mL SAMMA reduces fertilization. However,
contraception of SAMMA is incomplete with 0.5 mg/mL SAMMA being
essentially without effect. In contrast, 0.5 mg/mL fractionated
NO23-SAMMA is essentially completely contraceptive; only 1 of 120
oocytes examined from 5 rabbits was fertilized. The contraceptive
data, reported below, were obtained using washed rabbit spermatozoa
incubated with the test compounds for about 15 minutes at
37.degree. C. prior to insemination. About 22 to 34 million
spermatozoa were used for fertilization testing. Oocytes were
harvested about 25 to 27 hours post insemination and scored for
fertilization. Average fertilization percentages per rabbit (along
with 90% confidence limits) were as follows:
TABLE-US-00003 Oocytes Examined % Fertilization Test Compound (No.
Of Rabbits Used) (90% confidence limits)* None (control) 269 (12)
90 (75.2-98.7).sup.A SAMMA 79 (3) 78 (29.0-99.7).sup.A (0.5 mg/mL)
SAMMA 182 (7) 7 (1.2-18.2).sup.B (5 mg/mL) Fractionated 120 (5) 0.7
(0-6.8).sup.C NO23-SAMMA (0.5 mg/mL) *Values with different
superscript letter values are significantly different using
Newman-Keuls multiple range test. .sup.A and .sup.B values are
different at a p value < 0.001; .sup.B and .sup.C values are
different at a p value of 0.055.
[0086] NO23-SAMMA, whether fractionated or not, appears, at least
in part, to act through release of nitric oxide. Fractionated
NO23-SAMMA-induced acrosomal loss in the absence of added
extracellular Ca.sup.2+ is inhibited by the selective protein
kinase G inhibitor KT5823 (see FIG. 6). As noted above,
SAMMA-induced acrosomal loss (SAL) is also inhibited by KT5823, as
well as by nitric oxide synthase and guanylate cyclase inhibitors,
suggesting a role of NO via the cGMP/protein kinase G pathway in
this process. Acrosomal loss in human spermatozoa in response to NO
donors is inhibited by protein kinase G inhibitors (Revelli et al.,
"Signaling pathway of nitric oxide-induced acrosome reaction in
human spermatozoa", Biol. Reprod., 64: 1708-12 (2001)). SAMMA is
ineffective in inducing acrosomal loss in the absence of added
extracellular Ca.sup.2+. By inference, acrosomal loss induced by
fractionated NO23-SAMMA in the absence of added extracellular
Ca.sup.2+ is likely mediated by NO release from the NO donor moiety
of fractionated NO23-SAMMA. Inhibition of acrosomal loss by the
selective protein kinase G inhibitor supports this contention. The
IC.sub.50 for inhibition of fractionated NO23-SAMMA-induced
acrosomal loss (Ca.sup.a+ independent) by KT5823 is 0.7 .mu.M (FIG.
6).
[0087] Fractionated NO23-SAMMA has activity against HIV and HSV.
When direct comparisons are made, IC.sub.50 values for NO-SAMMA are
slightly higher than those for SAMMA. However, concentrations of
fractionated NO-SAMMA required for 3-Log reduction in infectivity
are lower than those for SAMMA. These results suggest that at
higher concentrations, fractionated NO-SAMMA is more effective than
SAMMA against HIV and HSV; the change in relative efficacy may
reflect the contribution of nitric oxide release against these
pathogens. HIV is sensitive to inhibition by the NO donor used to
synthesize NO-SAMMA (nitrooxypropanol), although substantially less
than to NO-SAMMA. Experiments were conducted to compare the
activities of SAMMA and fractionated NO23-SAMMA against HIV-1 BaL
infected primary lymphocytes. Host cells were Inoculated with virus
(200 TCID50/2.times.1.sup.05 cells) for 2 h, and washed to remove
unbound virus, before either SAMMA or fractionated NO23-SAMMA was
added to the cultures. Viral replication was measured on day 7 of
incubation (p24 levels). In all instances, viability of the target
cells remained at approximately 98% for the duration of the
experiments. These data are presented below.
TABLE-US-00004 IC.sub.50 3-Log reduction Compound (.mu.g/mL)
(.mu.g/mL) SAMMA 66 6560 Fractionated 208 2084 NO23-SAMMA
Nitrooxypropanol 2544 1017 (80% reduction)
[0088] SAMMA and fractionated NO23-SAMMA are highly active in
preventing infection of lymphocytes by HIV-1 BaL. IC.sub.50 values
are less than 10 .mu.g/mL. Fractionated NO23-SAMMA concentrations
required to inhibit p24 values by 50% and 3-logs are about 3-fold
lower than those for SAMMA. The dose-response for fractionated
NO23-SAMMA between 1 .mu.g/mL and 10 .mu.g/mL is more responsive
than that for SAMMA, suggesting involvement of the NO-SAMMA NO
donor moiety. This possibility is likely, in view of possible
reduced binding affinity of NO-SAMMA relative to SAMMA, due to
reduced charge density of the NO donor adduct.
[0089] The ability of fractionated NO23-SAMMA to reduce infectivity
of HSV-1 (F) and HSV-2 (0) was compared with the anti-HSV
activities of the parent compound, SAMMA. Both agents are highly
effective against these laboratory strains. In contrast to results
obtained for HIV-1, IC.sub.50 values are somewhat lower for SAMMA
than for fractionated NO23-SAMMA. However, fractionated NO23-SAMMA
is more effective in nearly completely inhibiting both viruses;
3-Log reductions are observed at NO-SAMMA concentrations that are
80% to 87% lower than SAMMA concentrations required for the same
effect. Dose-responses for fractionated NO23-SAMMA are thus
delayed, but sharper as compared with SAMMA. The slightly increased
IC.sub.50 values for fractionated NO23-SAMMA suggest increased
sensitivity of HSV binding to changes in charge density caused by
the covalent attachment of the NO donor moiety.
[0090] HSV Studies: One hour after adding serial 2-fold dilutions
of either SAMMA or fractionated NO23-SAMMA to confluent human
fibroblasts (foreskin) at concentrations ranging from 2 .mu.g/mL to
256 .mu.g/mL, cells were inoculated with either HSV-1 (F) or HSV-2
(G) (ATCC; MOI=0.05). After 48 hours (35.degree. C., 5% CO.sub.2),
cells were visually examined for viral cytopathogenic effect (CPE)
in the virus control wells. Cells were fixed and blocked (PBS with
0.2% BSA and 0.05% Tween 20). Viral titers were determined by
ELISA, with HRP-conjugated polyclonal antibodies (Dako Corp,
Carpinteria, Calif.). Reaction with 3,3',5,5'-tetramethylbenzidine
(TMB) was measured spectrophotometrically (630 nm and 450 am). Data
were expressed as mean percentage of control viral incubations
(.+-.SEM) to which no microbicide was added.
[0091] HIV Studies: Primary lymphocytes and virus were incubated
with microbicide (serial 10-fold dilutions, ranging from 1 ng/mL to
1 mg/mL) for 1 hour, followed by inoculation (50
TCID50/2.times.10.sup.5 cells). After 2 hours, cells were washed to
remove virus. Incubations continued with microbicide for 7 days,
after which viral titers (p24) were measured. Data (.mu.g/well)
were expressed as mean.+-.SEM of triplicate determinations. The
data are consistent with activities of SAMMA and fractionated
NO23-SAMMA in HIV-1 BaL-infected lymphocytes and suggest a greater
contribution of the NO donor moiety of fractionated NO23-SAMMA at
higher concentrations.
[0092] These data for both the HSV and HIV studies are presented
below. The values for each dose-response curve includes the
coefficient of determination (r.sup.2), degrees of freedom (DoF),
calculated concentration of inhibitor required to reduce viral
titer by 50 percent (IC.sub.50), and concentration of inhibitor to
reduce viral titer by 99.9 percent (3-log).
TABLE-US-00005 SAMMA Fractionated NO23-SAMMA r.sup.2 IC.sub.50
3-log r.sup.2 IC.sub.50 3-log (DoF) (.mu.g/mL) (.mu.g/mL) (DoF)
(.mu.g/mL) (.mu.g/mL) HIV-1 0.9999 6.5 60 0.9999 2.5 23 BaL (6) (6)
HSV-1 0.9993 7.8 214 1.0000 22 42 (F) (6) (6) HSV-2 0.994 1.5 181
0.994 5.2 24 (G) (9) (7)
Example 6
[0093] NO-SAMMA retains many biological properties of the parent
compound, SAMMA, including the ability to inhibit hyaluronidase (a
property believed to be responsible, in part, for some
anti-microbial activity), and lack of effects on sperm motility and
growth of lactobacilli (indicators of specificity of action and
lack of general cytotoxic effects on spermatozoa and beneficial
vaginal flora. See generally, Zaneveld et al. "Method for
preventing sexually transmitted diseases," U.S. Pat. No. 5,932,619.
This example evaluates some of these properties for fractionated
NO23-SAMMA (prepared as in Example 5).
[0094] Hyaluronidase activity was measured as described in Example
8 of U.S. Pat. No. 5,932,619. Activity of fractionated NO23-SAMMA
against hyaluronidase is very similar to that of SAMMA. In
contrast, the NO donor moiety of NO23-SAMMA, nitrooxypropanol, is
nearly without effect. The following results were obtained.
TABLE-US-00006 3-Log IC.sub.50 reduction* Inhibition .+-. SEM Agent
(.mu.g/mL) (.mu.g/mL) (N = 4) Fractionated 11.1 13.6 100 .+-. 2.0%
at 15 .mu.g/mL ** NO23-SAMMA SAMMA 8.1 14.9 104 .+-. 2.0% at 15
.mu.g/mL Nitrooxypropanol -- -- 8 .+-. 1.8% at 50 .mu.M
*concentration required for 99.9% inhibition of activity ** 15
.mu.g/mL fractionated NO23-SAMMA contains 21.2 .mu.M equivalent of
nitrooxypropanol
[0095] Sperm immobilization by fractionated NO23-SAMMA was
evaluated by a modification (Anderson et al. "Evaluation of
poly(styrene-4-sulfonate) as a preventive agent for conception and
sexually transmitted diseases," J Androl 21:862-875 (2000)) of the
method of Sander and Cramer ("A practical method for testing the
spermicidal action of chemical contraceptives," Hum Fertil
6:134-137, 153 (1941)). Thirty seconds after adding different
concentrations of the test agent (2.5 to 20 mg/mL for SAMMA and
fractionated NO23-SAMMA and 1-10 mM for nitrooxypropanol), the
fraction of motile spermatozoa was determined with brightfield
microscopy (400.times.). Data are presented as the percentage of
motile spermatozoa at each concentration of test agent. When
possible, test outcomes were also reported as the concentration of
agent in the semen sample that reduces motility by 50%.
[0096] Neither fractionated NO23-SAMMA, SAMMA, nor nitrooxypropanol
can be regarded as spermicidal. Sperm motility is reduced by less
than 10 percent by concentrations of these agents that are 4-8
orders of magnitude greater than concentrations required to induce
acrosomal loss. The data are as follows.
TABLE-US-00007 % Motile Spermatozoa (at 10 mg/mL test agent) Test
Agent av .+-. SEM (n = 4) IC.sub.50 None 70 .+-. 0.6 --
Fractionated NO23-SAMMA 63 .+-. 2.2 at 10 mg/mL 30 mg/mL SAMMA 67
.+-. 0.6 at 10 mg/mL 64 mg/mL Nitrooxypropanol 66 .+-. 0.5 at 10 mM
163 mM
[0097] The effect of fractionated NO23-SAMMA on growth of L.
gasseri was determined as described in Example 9 of U.S. Pat. No.
5,932,619. Similar to SAMMA, fractionated NO23-SAMMA has no effect
on lactobacillus growth at concentrations up to 10 mg/mL. These
data are shown below.
TABLE-US-00008 Fractionated Doubling Difference NO23-SAMMA Time
from Control (mg/mL) (minutes) (Confidence Level) 0 121.1 -- 5.0
125.4 >0.1 10.0 130.8 >0.1
[0098] The release of nitric oxide from NO-SAMMA has been
confirmed. Although available instrumentation lacked sensitivity to
detect NO release in the biological systems that have been tested,
chemical-induced release of NO from relatively high concentrations
of NO-SAMMA could be quantified.
[0099] NO formation was measured by a modification of the method of
Bertlnaria et al. ("Synthesis and anti-Helicobacter pylori
properties of NO-donor/metronidazole hybrids and related
compounds," Drug Devel. Res. 60: 225-39, (2003). Fractionated
NO23-SAMMA (0.1 mg/mL) was reacted with 50 mM cysteine in 50 mM
sodium phosphate (pH 7.4 at 37.degree. C.) for up to 24 hours.
Equal volumes of reaction mixture and Griess reagent were reacted
for 15 minutes and the absorbency at 540 nm was determined. Nitrite
standards produced a linear standard curve (r.sup.2=0.9999) in the
range 1-50 .mu.M, from which nitrite formation from
nitrooxypropanol was measured. Higher concentrations could not be
evaluated, since the pink/red reaction product when combined with
the Griess reagent precipitated and could not be measured.
[0100] Nitrite formation (an indirect measure of NO formation) from
fractionated NO23-SAMMA increased over time. The results are
presented in FIG. 7. Nitrite formation when fitted to a kinetic
curve showed first order sequential formation, A.fwdarw.B.fwdarw.C,
which is similar to that describing nitrite formation from
nitrooxypropanol.
[0101] The embodiments and examples described and discussed above
are intended to illustrate the present invention and not to limit
the scope of the invention which is defined in the appended
claims.
* * * * *