Note: Descriptions are shown in the official language in which they were submitted.
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Gel-Microemulsion Formulations
Field of the Invention
The present invention relates to a pharmaceutical formulation. and more
particularly to a gel-microemulsion formulation. In some embodiments. the gel-
microemulsion formulation has spermicidal activity and can be used as a
contraceptive. In other embodiments, the gel-microemulsion can act as a
formulation base for other theraputic agents. such as anti-microbial agents to
provide an anti-microbial formulation or spermicidal agents to enhance the
spermicidal effectiveness of the formulation. In yet other embodiments, the
gel-
microemulsion, having spermicidal activity. can act as a formulation base for
the
anti-microbial agents to provide a dual function contraceptive/anti-microbial
formulation.
Back;~round of the Invention
At present. all commercially available spermicidal contraceptives have
detergent ingredients that disrupt cell membranes. These include the neutral
surfactants isononyl-phenyl-polyoxyethylene (9) ether ornonoxynol-9 (N-9), p-
menthanyl-phenyl-polyoxyethylene (8,8) ether ormenfegol, and isooctyl-phenyl-
polyoxyethylene (9) ether or octoxynol-9 (O-9) (Digenis GA, et al., Pharm Dev
Technol, 1999;4:421-30; Furuse K, et al., JPharmacobiodyn, 1983;6:359-72.) The
detergent-ype vaginal spermicide, N-9, available without a prescription. is
the most
commonly used spermicidal contraceptive in the UK and USA (OTC Panel. Federal
Register, 1980:45:82014-49; Chantler E., Brit Fam Plann, 1992:1 ;':l 18-9.)
Worldwide, the cationic surfactant benzalkonium chloride and the anionic
detergent
sodium docusate (dioctyl sodium sulphosuccinate) are also used as vaginal
spermicides (Mendez F, et al., Contraception. 1986:34:353-62.) N-9. sodium
oxychlorosene, and benzalkonium chloride. have been used as gels,
suppositories,
ovules. sponges, or film. N-9 has been in use for more than 30 years in
creams,
gels. foams and condom lubricants. However, in several large studies for users
of
N-9. the average 6-month pregnancy rate is 26%. and the first-year pregnancy
rates
range from I I to 31 %. Thus. N-9 is approximately 75% effective in preventing
pregnancy (Trussell J, et al. Stud Fam Plann. 1987:18:237-83: Kulig JW, Pcd
Clinic
North Am. 1989:36:717-30: Raymond E, et al.. Obstet Gvnecol, 1999;93:896-903).
The spermicidal activities of these surfactants are associated with their
structural affinity to the membrane lipids (Schill WB. et al.. Andrologia,
3 > 1981:13:42-9: Helenious A. et al.. Biochem Biophys Acta. 1975:41 x:29-79).
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Therefore, the major drawback of using N-9 or other currently used surfactants
is
their detergent-type effect on epithelial cells and normal vaginal flora. N-9
displays
antiviral and spermicidal activities only at cytotoxic doses (D'Cruz OJ, et
al., Mol
Hum Reprod, 1999;5:421-32; D'Cruz OJ, et al., Biol Reprod, 2000;62:37-44).
Frequent use of N-9 as a vaginal contraceptive/microbicide has been associated
with
an increased risk of vaginal or cervical infection, irritation, or ulceration
(Niruthisard
SR, et al., Sex Transm Dis, 1991;18:176-79; Rekart ML, Defic Syndr, 1992;5:425-
27; Roddy RE, et al., Int JSTD & HIY 1993;4:165-70; Weir SS, et al.,
Genitourin
Med, 1995;71:78-81). Detergent-type spermicides alter vaginal bacteria or
flora,
and lead to an increased risk of opportunistic infections (Hooten TM, et al.,
JAMA,
1991;265:64-9.; Stafford MK, JAcguir Immune Defic Syndr Hum Retrovirol,
1998;17:327-31.; Rosenstein IJ, et al. J Infect Dis, 1998;177:1386-90.; Patton
DL,
et al., Sex Trans Dis, 1996;23:489-93.) Such opportunistic infections are
known to
enhance the susceptibility of the ectocervical epithelium and the endocervical
mucosa to HIV-1 infection (Augenbraun MH, et al., Infect Dis Clin North Am,
1994; 8:439-48.) Chemical irritation that disrupts the vaginal mucosa may
actually
enhance the risk of vaginal transmission of sexually transmitted diseases
(STDs)
including HIV-1, by mucosal erosion and local inflammation (Weir SS, et al.,
Genitourin Med, 1995;71:78-81.;. Kreiss J, JAMA, 1992;268:477-82.). In a
study conducted among commercial sex workers in Nairobi, in which some of the
women used N-9 containing sponges, a significantly higher rate of genital
ulceration
and HIV-1 seroconversion was found compared with those not using N-9 (Kreiss
J,
JAMA, 1992;268:477-82.).
Furthermore, recent clinical trials have shown that vaginal contraceptive
preparations containing N-9 have no effect on the transmission of HIV/AIDS and
other STDs when provided as part of an overall program to prevent heterosexual
transmission of HIV/AIDS (Hira SK, et al, Int J STD AIDS, 1997;8:243-50.;
Roddy
RE, et al., NEngl JMed, 1998;339:504-10.) Since heterosexual transmission of
HIV-1 is the predominant mode of the epidemic spread of HIV, new, effective,
and
safe vaginal spermicides lacking detergent-type membrane toxicity may offer
significant clinical advantage over the currently available detergent-type
spermicides.
Because vaginal spermicides would likely be used repeatedly over decades,
an ideal spermicide should have an established safety record and lack genital
epithelial toxicity. Moreover, it should be inexpensive and be produced from
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commonly available resources and should have a broad specificity for
solubilizing
drugs effective for prevention of sexual transmission of several STDs
including
HIV-1.
Therefore, there is a continuing need for new and better spermicidal
formulations.
Summary of the Invention
The inventors have developed novel gel-microemulsion formulations for use
as spermicides that in numerous respects overcomes many of the problems of the
commercially available detergent-type spermicides. Embodiments of the novel
gel
microemulsion spermicide formulations have been show to be very effective as
contraceptive agents with reduced levels of toxicity to subjects, and are also
useful
as formulation bases for anti-microbial agents.
Some novel pharmaceutical formulations embodying the gel-microemulsion
of the invention contain common pharmaceutical excipients as the active
ingredients, and provide for safe in vitro and in vivo spermicidal activity.
In some
embodiments, it is contemplated that drug solubilizing agents, such as
Cremophor
EL~ and Phospholipon 90G~, may be active ingredients since these agents were
spermicidal against highly motile fraction of sperm. Although, the individual
components of some such gel-microemulsion formulations alone lacked
spermicidal
activity in semen, the combined components in such gel-microemulsion
formulations containing the pharmacological excipients rapidly inactivated
sperm in
human semen. The lack of cytotoxicity of individual components of such
formulations in human semen and their synergestic spermicidal property in the
gel-
microemulsion formulation shows unique clinical potential to formulate them as
the
active ingredients for a novel and effective contraceptive, such as a vaginal
contraceptive for example. In testing, some embodiments of gel-microemulsion
formulations of the invention were significantly more effective as a
contraceptive
than a commercially-available N-9 gel formulation.
Embodiments of the gel-microemulsion of the invention can also be used as
an effective formulation base for other agents, for example anti-microbial
agents,
that can be incorporated into the formulation. Embodiments incorporating anti-
microbial agents are particularly useful to prevent the transmission of
diseases.
Additionally, such embodiments are especially useful as a dual function
spermicide/anti-microbial formulation, and can be especially useful in
inhibiting the
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transmission of sexually transmitted diseases. for example AIDS, genital
herpes,
gonorrhea and chlamydia. In yet other embodiments, additional spermicidal
agents
can be incorporated into the formulation to increase the effectiveness of the
gel-
microemulsion as a spermicide.
One aspect of the invention is directed to a pharmaceutical composition
adapted for use as a spermicide, the composition comprising a gel-
microemulsion
comprising an oil-in-water microemulsion and a polymeric hydrogel. Another
aspect is a method of using such a composition as a spermicide.
Another aspect of the invention is directed to a gel-microemulsion
pharmaceutical composition adapted for use as a formulation base for
additional
theraputic agents. Examples of additional agents include, anti-microbial
agents and
spermicidal agents. Another aspect is a method of using such a composition as
a
formulation base for additional theraputic agents. Another aspect is the use
of the
combined gel-microemulsion formulation base with additional theraputic agents
for
appropriate theraputic treatment.
Another aspect of the invention is directed to a gel-microemulsion
pharmaceutical composition that is adapted for use as both a spermicide and
formulation base for anti-microbial agents to provide a dual function
contraceptive/anti-microbial formulation. Another aspect is a method of using
such
a composition as a dual function contraceptive/anti-microbial formulation.
Brief Description of the Drawings
Figure 1 is a ternary phase diagram of one embodiment of a microemulsion
system. The non-grid area represents the single phase microemulsion region.
The
asterisk represents the microemulsion which was used for GM-4 formulation
listed
in Table ~.
Figure 2 shows the effect of individual components of GM-4 on the motility
of washed and enriched human sperm. Highly motile fractions of sperm were
incubated with increasing concentrations of listed compounds in the assay
medium,
and the percentage of motile sperm was evaluated by CASA. The plots show mean
values of two representative measurements. Cremophor EL and phospholipon 90G
were spermicidal at all concentration tested. Captex 300, PEG-200, propylene
glycol, seaspan carrageenan, viscarin carrageenan, and sodium benzoate
demonstrated little or no inhibition over the range of concentrations tested.
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Figure 3 shows the effect of individual components of GM-4 on the motility
of human sperm in semen. Aliquots of liquefied semen were mixed with an equal
volume of assay medium containing the final concentrations of components of GM-
4 formulation. At timed intervals, sperm motility was evaluated. The plot
shows
5 mean values from two representative experiments. All the components of GM-4
formulation demonstrated little or no inhibition of sperm motility in human
semen
over the entire range of time course tested.
Figure 4 is are light microscopic images of GM-4 and N-9 (4%)-treated
rabbit vaginal sections. Representative hematoxylin- and eosin-stained,
paraffin-
embedded sections of the mid vaginal region of a rabbit treated intravaginally
with
GM-4 formulation (Left panels, A and C) or 4% N-9 in GM-4 (Right panels, B and
D) for 10 consecutive days (x200). Higher magnification (x400) shows the
intactness of vaginal epithelium (VE) in a GM-4-treated rabbit (C) versus an N-
9-
treated rabbit (D) which shows epithelial cell layer disruption (arrows) and
leukcocyte influx characteristic of inflammation.
Figure 5 shows the mean body weights of 10 female B6C3Flmice with and
without intravaginal application of GM-4, 5 days/week for 13 consecutive
weeks.
Detailed Description of the Invention
Terms and Definitions
All scientific and technical terms used in this application have meanings
commonly used in the art unless otherwise specified. As used in this
application, the
following words or phrases have the following meanings, unless otherwise
indicated:
"Microemulsions'' are thermodynamically stable, transparent, dispersions of
water and oil, stabilized by an interfacial film of surfactant molecules.
Microemulsions are characterized by their submicron particle size of 0.1 qm or
below.
"Lipid" is an inclusive term for fats or fat derived materials.
"Surfactant" is any compound that reduces surface tension when dissolved in
water or water solutions, or that reduces interfacial tension between two
liquids or
between a liquid and a solid. An example of one type are emulsifying agents.
"Humectant" is a substance having affinity for water with stabilizing action
on the water content of a material.
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As used herein, the terms ''analog'' or "derivative" are used interchangeably
to mean a chemical substance that is related structurally and functionally to
another
substance. An analog or derivative contains a modified structure from the
other
substance, and maintains a similar function of the other substance. The analog
or
derivative need not be, but can be synthesized from the other substance.
As used herein, ''pharmaceutically acceptable salt" refers to a salt that
retains
the desired biological activity of the parent compound and does not impart any
undesired toxicological effects. Examples of such salts include, but are not
limited
to, (a) acid addition salts formed with inorganic acids, for example
hydrochloric
acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the
like; and
salts formed with organic acids such as, for example, acetic acid, oxalic
acid, tartaric
acid, succinic acid, malefic acid, furmaric acid, gluconic acid, citric acid,
malic acid,
ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid,
polyglutamic acid,
naphthalenesulfonic acids, naphthalenedisulfonic acids, polygalacturonic acid;
(b)
1 S salts with polyvalent metal canons such as zinc, calcium, bismuth, barium,
magnesium, aluminum, copper, cobalt, nickel, cadmium, and the like; or (c)
salts
formed with an organic cation formed from N,N'-dibenzylethylenediamine or
ethylenediamine; or (d) combinations of (a) and (b) or (c), e.g., a zinc
tannate salt;
and the like. The preferred acid addition salts are the trifluoroacetate salt
and the
acetate salt.
"Pharmaceutically acceptable carrier" means any material which, when
combined with a biologically active compound, allows the compound to retain
biological activity, such as the ability to potentiate antibacterial activity
of mast cells
and macrophages.
The term "inhibit" means to reduce by a measurable amount, or to prevent
entirely.
The term "to treat" means to inhibit or block at least one symptom that
characterizes a pathologic condition, in a mammal threatened by, or afflicted
with,
the condition.
"Mammals" means any class of higher vertebrates that nourish their young
with milk secreted by mammary glands, e.g., humans, rabbits, mice. monkeys,
etc.
"N-9" means the virucidal/spermicide, nonoxynol-9.
"Organometallic compound" is an organic compound comprised of a metal
attached directly to carbon (R-M).
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"Coordination compound'' is a compound formed by the union of a central
metal atom or ion with a nonmetal atom, ion or molecule called a ligand or
complexing agent.
"Ligand" or a "complexing agent" is a molecule, ion or atom that is attached
to the central metal atom or ion of a coordination compound.
"Monodentate ligand" is a ligand having a single donor atom coordinated to
the central metal atom or ion.
"Bidentate ligand" is a ligand having two donor atoms coordinated to the
same central metal atom or ion.
"Chelate" or "chelated compound" a type of coordination compound in
which a central metal ion is attached by chelated ligand containing two or
more non-
metal atoms in the same molecule. One or more heterocyclic rings are formed
with
the central metal atom to form the coordination compounds.
"Oxovanadium (IV) complex" is a coordination compound including
vanadium as the central metal atom or ion, and the vanadium has an oxidation
state
of +4 (IV), and is double bonded to oxygen.
"Metallocene" is an organometallic coordination compound containing
cyclopentadienyl rings attached to a transition metal or transition metal
halide.
"Vanadocene" is a metallocene including vanadium as the transition metal
ion.
"Transition metals" is any of a number of elements in which the filling of the
outermost shell to eight electrons within a period is interrupted to bring the
penultimate shell from 8 to 18 or 32 electrons. Transition metals include
elements
21 through 30, 39 through 48, 57 through 80, and from 89 on.
"Halo" is Br, C1, F, or I.
"Alkyl" is straight chained or branched chained alkyl, and includes halo-
substituted alkyl.
"Alkoxy" is straight chained or branched chained alkoxy, and includes an O
in the alkyl group.
"Aryl" refers to monovalent unsaturated aromatic carbocyclic radicals having
a single ring, such as cyclopentadieneyl or phenyl, or multiple condensed
rings, such
as naphthyl or anthryl, which can be optionally substituted by substituents
such as
halogen, alkyl, arylalkyl, alkoxy, aralkoxy, and the like.
"Carboalkoxy" is straight chained or branched chained alkoxy, and includes
carbamium carbom.
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Gel-Microemulsion Formulations
One aspect of the invention is directed to a pharmaceutical composition
adapted for use as a spermicide. The spermicidal activity of the
pharmaceutical
composition can be in vitro or in vivo. The spermicidal compositions of the
present
invention are suitable for use, for example, in mammals. The spermicidal
compositions comprise a gel-microemulsion. The gel-microemulsions comprise an
oil-in-water rnicroemulsion and a thickening agent, such as polymeric gel
thickening agent.
The microemulsion generally includes one or more lipids, one or more
surfactants, optionally one or more humectants, and water as a diluent.
Suitable lipids include those generally know to be useful for creating oil-in-
water microemulsions. Preferred examples include fatty acid glyceride esters,
preferably medium chain C6-C,Z fatty acid glyceride esters, and the like.
Preferred
C6-CIZ fatty acid glyceride esters include medium chain Cfi-C,2 monoglycerides
and
triglycerides, with the triglycerides being more preferred. Triglycerides of
caprylic/capric acid are particularly suitable for use as the lipid component
in the
composition. Suitable triglycerides of caprylic/capric acid include Captex
300~,
Captex~ 355, Captex~ 350 and Captex~ 200, which are commercially available
from Abitec Corp., (Columbus, OH.), with the most preferred being Captex 300~.
Mixtures of suitable lipids can be used.
Suitable surfactants include those generally know to be useful for creating
oil-in-water microemulsions wherein lipids are used as the oil component in
the
microemulsion, and preferably are well suited to aid in emulsifying the
particular
lipid being used. Non-ionic surfactants are generally preferred. Examples of
suitable surfactants include ethoxylated castor oil, and phospholipids. One
suitable
ethoxylated castor oil is Cremophor EL~ commercially available from BASF
Corp.,
(Mount Olive, NJ,). Preferred phospholipids include purified soy bean
phospholipid
or lecithins such as phosphatidylcholine. One suitable purified soy bean
phospholipid or lecithins is Phospholipon~ 90G commercially available from
American Lecithin (Oxford, CT.). Other suitable non-ionic surfactants include
block copolymers of ethylene oxide and propylene oxide. Suitable commercially
available block copolymers of ethylene oxide and propylene oxide include;
Pluronic
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F-68, Pluronic F-77, Pluronic F-87, and Pluronic F-88, commercially available
from BASF Corp., (Mount Olive, NJ.). Mixtures of suitable surfactants can be
used.
In some preferred embodiments, both ethoxylated caster oil and phospholipids
are
used as surfactants.
The microemulsion also optionally includes one or more humectants.
Preferred humectants include propylene glycol such as 1,2-propanediol, and
polyethylene glycol (PEG) with an average molecular weight in the range of 100
to
500, preferably in the range of 150 to 300, and more preferably in the range
of 190
to 210. Mixtures of suitable humectants can be used. Preferably both propylene
glycol and polyethylene glycol are used as humectants. Suitable propylene
glycol is
commercially available from under the name Propylene Glycol USP from Sigma
Chemical Co., (St. Louis, MO). Suitable polyethylene glycol includes Carbowax
Polyethylene Glycol 200 commercially available from Union Carbide Corporation
(Danbury, CT).
Water is used as the diluent, and preferably purified or distilled water is
used.
The microemulsions alone can be used, for example, as spermicites or in
drug delivery systems to enhance the solubility of poorly water soluble
substances,
such as some anti-microbial compounds, as will be discussed in more detail
below.
However, to enhance the usefulness of the microemulsions, especially as an
effective
spermicide and as a base formulation for anti-microbial compounds in certain
application, thickening agents are added.
Therefore, the gel-microemulsion formulation also includes one or more
thickening agents, such as a polymeric hydrogel. Generally, the hydrogel is a
hydrophilic natural or synthetic gel-forming polymer, preferably, a natural
gel-
forming polymer. Suitable examples of natural gel-forming polymers include
carrageenan, xanthan gum, gum karaya, gum acacia, locust bean gum, guar gum.
Mixtures of suitable humectants can be used. Suitable carrageenans include
Seaspen~ carrageenan and Viscarin~ carrageenan commercially available from
FMC Corporation (Philadelphia, PA). Suitable xanthan gums include
XANTURALTM 75 commercially available from Monsanto Pharmaceutical
Ingredients (St. Louis, MO).
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The formulation can also optionally include one or more additives such as
preservatives or antioxidants to help maintain and prolong the useful life of
the gel-
microemulsion. Preservatives and antioxidants that are generally known, and do
not
detract significantly from the usefulness of the gel-microemulsion for the
particular
5 purpose it is being used, can be incorporated into the gel-microemulsion
formulation. Particularly suitable preservatives include sodium benzoate,
methyl
parabens, propyl parabens, sorbic acid, and the like. Sodium benzoate is most
preferred, and is commercially available from Cultor Food Science, Inc.
(Ardsley,
NY). The prevention of the action of microorganisms in the formulation can be
10 brought about by various antibacterial and antifungal agents, for example,
parabens,
chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
If desired, other additives, such as colorants, scents, isotonic agents, for
example, sugars, buffers or sodium chloride, can be added to the gel-
microemulsion
to the extent desired, and to the extent that the usefulness of the gel-
microemulsion
is not disrupted.
The composition is formulated to provide a gel-microemulsion with a
submicron particle size, preferably in the range of 30-80 nm. Additionally,
the
viscosity of the gel-microemulsion is in the range of about 100 to about 1100
centipoise, more preferably from about 150 to about 1000, and more preferably
from
about 200 to about 1000 centipoise.
Those of skill in the art will recognize that the amounts of each of the
individual components used to produce a suitable gel-microemulsion are
dependent
upon the amounts and type of other components used. Therefore, the amounts and
types of components are interdependent.
Those of skill in the art will also recognize that suitable microemulsions can
be identified through systematic mapping of ternary phase diagrams. The
ternary
phase diagram of the microemulsion components used for the preparation of one
embodiment of the invention, GM-4, is shown in Figure 1, and discussed in the
Examples below. The non-grid area represents the single phase microemulsion
region suitable for use. The concentration of the components can be selected
from
within this region. The asterisk represents the particular concentration of
components of the microemulsion which was used for the GM-4 formulation.
Suitable gel-polymer suspensions can then be selected as additives to the
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microemulsion-based system to obtain a gel of desirable viscosity with high
thickening capability and compatibility with the microemulsion. It is
preferable that
the gel-microemulsion be stable at ambient temperature.
Representative examples of constituent concentration ranges for base
components of some gel-microemulsion formulations embodying the invention can
be found in Table l, wherein the values are given in wt. % of the ingredients
in
reference to the total weight of the formulation.
Table 1
Constituent Ranges Preferred RangesMore Preferred Ranges
Lipid 2 to 25 6 to 23 8 to 15
Surfactant 3 to 30 4 to 17 8 to 1 S
Humectant 2 to 24 3 to 12 5 to 10
Polymer Gel 0.5 to 1 to 2 1.2 to 1.8
4
Additives 0 to 0.5 0.1 to 0.3 0.1 S to 0.2
Water Balance Balance Balance
In some preferred embodiments, the formulation includes the specific
constituent concentrations for base components as found in Table 2, wherein
the
values are given in wt. % of the ingredients in reference to the formulation
weight.
Table 2
Ingredients Preferred RangesMore Preferred
Ranges
Medium Chain Tryglyceride6 to 23 8 to 15
Ethoxylated Castor 3 to 10 S to 9
Oil
Phospholipid 1.5 to 6 3 to 6
Propylene Glycol 1.5 to 6 3 to 6
PEG-200 1.5 to 6 3 to 6
Natural Polymer Gel 1 to 2 1.2 to 1.8
Preservative 0.1 to 0.2 0.15 to 0.2
Water Balance Balance
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Preparation of Gel-microemulsious:
A simple procedure allows for the preparation of a gel-microemulsion at
even a one-milliliter scale. The following generally describes such a simple
procedure: Combine surfactants, hydrophilic components, and the lipids
(preferably
medium chain tryglycerides) in an appropriate container. Mix the components
using
a stir bar with mild heat until a clear and homogeneous microemulsion is
formed.
Remove the composition from the heat, and wait until it reaches room
temperature.
Add two parts of a pre-prepared polymer dispersion to each part of
microemulsion
with continued mixing. The resulting gel-microemulsion is a dispersion with a
viscosity in the range of 200-1000 centipoise, and a submicron particle size,
preferably in the range of 30-80 nm.
Sperimicidal Use of the Gel-microemulsiou
The contraceptive compositions of the present invention are preferably
administered to a site for contacting sperm, in a dosage which is effective to
immobilize sperm. Such compositions are intended particularly for use in
mammals,
but use outside of mammals is contemplated. It is also contemplated that the
compositions may be used as sperm immobilization compositions. It is expected
that the present invention will be used by humans in most practical
applications.
Preferably, the amount of spermicide employed will be that amount
necessary to achieve the desired spermicidal results. Appropriate amounts can
be
determined by those skilled in the art.
The contraceptive compositions of the present invention may be delivered to
the vagina of a mammal by any means known to those skilled in the art. The gel-
microemulsion can be applied directly. Other typical forms for delivery of the
compositions include, for example, intervaginal devices such as sponges,
condoms,
including female condoms, suppositories, and films. In addition, the
compositions of
the present invention may be used as personal care lubricants, such as, for
example,
condom lubricants, and the like. The contraceptive compositions may be located
within or on a condom for example. Inter-vaginal devices may also be used to
aid in
the administration of the composition as described in U.S. Patent 5,069,906.
Further
details concerning the materials, ingredients, proportions and procedures of
such
delivery forms are known to those skilled in the art, and can be selected in
accordance with techniques well-known in the art.
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It is also contemplated that the formulation of the invention may be
incorporated into a spermicidal article such as a vaginal insert, a condom, or
other
such device, such that when the article is used, the spermicidal can be
delivered to
contact sperm.
Gel-Microemulsion as a Formulation Base for Other Therapeutic Agents
Another aspect of the invention is the use of the above described gel-
microemulsion formulations as formulation bases for incorporating
therapeutically
active agents. The base gel-microemulsion formulations generally include the
components and concentrations discussed above. The therapeutically active
agents
can include any generally known therapeutic agent where it would be desirable
to
administer such an agent with a gel-microemulsion formulation. Some
embodiments of the gel-microemulsions of the invention are especially suitable
as
solubilizing vehicles for poorly water soluble compounds. In some preferred
embodiments, gel-microemulsion formulations are used, for example, as
formulation bases for anti-microbial agents, spermicidal agents, or dual
function
anti-microbial/spermicidal agents.
Anti-Microbial Gel-Microemulsion Formulations
Suitable examples of anti-microbial theraputic agents, include anti-viral
agents, anti-bacterial agents, anti-fungal agents, and the like. Sucl-s agents
can be
incorporated into the gel-microemulsion formulations to provide for an anti-
microbial formulation, or a dual function anti-microbial/spermicidal
formulation.
Suitable examples of preferred anti-microbial agents include those used for
the
treatment of sexually transmitted diseases, for example, AIDS (HIV-1, HIV-2,
FIV,
SIV, etc.) genital herpes, gonorrhea. chlamydia, and the like.
Examples of Preferred Anti-Microbial Agents: Preferred examples of
anti-microbial agents, such as anti-viral agents, include those disclosed in
the
following copending patent applications; which are hereby incorporated by
reference
herein:
U.S. Patent Application No. 09/047,609, which is incorporated herein by
reference, and corresponding published PCT Application No. PCT/L1S99/06381
(International Publication Number WO 99/48902), which is incorporated herein
by
reference;
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U.S. Patent Application No. 09/450,082 which is incorporated herein by
reference, and corresponding published PCT Application No. PCT/US99/14774
(International Publication Number WO 00/00501 ) which is incorporated herein
by
reference; and
S U.S. Patent Application No. 09/107,716, which is incorporated herein by
reference.
Examples of preferred anti-microbial/anti-viral compounds disclosed in
copending U.S. Patent Application No. 09/047,609 and corresponding PCT Patent
Application No. PCT/LJS99/06381, include AZT derivatives disclosed therein.
Many of the AZT derivatives disclosed therein also have spermicidal activity.
Examples of such anti-microbial AZT derivatives include compounds of the
formula:
~H3
Rz
R3
y
where R, is H, N3, halo, CN, COOH or NH,, R, is halo (particularly C1, Br or
I, and more particularly Br) and R3 is alkoxy (particularly Cl-3 alkoxy, and
more
particularly methoxy (-OCH3)). The NHz group can be functionalized, for
example
with -CH3, -COCH3, -Ph, -COPh, and -CH,Ph. Pharmaceutically acceptable salt or
ester forms also can be used, such as sodium, potassium or ammonium salts.
The derivatives of the formula above include substitution on the AZT
pentose ring member. The derivatives of this aspect of the present invention
have
the chemical structure illustrated below:
SUBSTITUTE SHEET (R ULE 26)
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0
ii
I CH3
HN
Rz
O' N R
3
O
R'O \ Rt
where R, is H, N3, halo, CN, COOH or NH2, RZ is halo (particularly Cl, Br or
I, and
more particularly Br), R3 is alkoxy (particularly C1-3 alkoxy, and more
particularly
methoxy (-OCH3)) and R' is a group that facilitates the passage of the
compound
5 into a cell. As in the first formula, the NHS group can be converted, for
example to
NHCH3, NHCOC'H3, NHPh, NHCOPh, and NHCHZPh. The R' group can be, for
example, H, a phosphate, lipid or fatty acid group. Alternatively, sperm-
reactive
antibodies or cytokines could be used to derivatize these compounds (as well
as
those of the first formula) at the R' or R, positions for targeted delivery.
10 Pharmaceutically acceptable salt or ester forms, such as the sodium,
potassium or
ammonium salts, can be used as well.
The R' group forms a phosphate group. The H of an -OH member of the
phosphate can be replaced with C1-4 alkyl or aryl substituents (e.g. phenyl-,
naphthyl- or anthracinyl-substitution), which optionally may be substituted,
and SH
15 or NH~ groups can replace the OH of the phosphate, and in each of these
cases a H
of the NHZ or SH can be replaced in the same manner as the H of the OH group
discussed previously. The aryl phosphate group is surprisingly effective in
maintaining excellent anti-HIV activity. A general structure of an exemplary
aryl
phosphate OR' group is illustrated below:
Rs R5
0
R4 ~ 0_III_0_
R"
Rs Rs
where R4, R5, and R~ are the same or different and are selected from
hydrogen, methyl. ethyl, fluoro, chloro, bromo, iodo, dichloro, dibromo,
difluoro,
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trifluoromethyl, nitro, cyano, methoxy, trifluromethoxy and ethoxy,
particularly
hydrogen, fluoro, bromo and methoxy and R" is an amino acid residue that may
optionally be substituted and/or esterified, for example an alaninyl group (-
NHCH(Me)COOMe). In the case of the alaninyl group, the methyl group attached
to the CH group can be substituted, for example with a phenyl group, and the
methyl
esterification can be replaced with other C~ or C3 esterification.
Of these compounds, the following compound, hereinafter referred to as
WHI-07, is particularly preferred:
H3
Br ~NH
O Me0
i, H~ ..~~O
Br / \ O NHO
~ N3
Me"CO 2Me
Chemical name: (SR,6R)-and (SS,6S)-5-bromo-6-methoxy-5,6-dihydro-AZT-
5'-(para- bromophenyl methoxyalaninyl phosphate).
Examples of preferred anti-microbial, preferably anti-viral, compounds
disclosed in U.S. Patent Application No. 09/450,082, and corresponding
published
PCT Application No. PCT/LTS99/14774; and U.S. Patent Application No.
09/107,716, include aryl phosphate derivatives of nucleosides disclosed
therein,
particularly derivatives of d4T and AZT. Examples of suitable nucleoside
derivative disclosed therein include those of the formula:
Y
Rr-Y-P-R2
I
N-R3
R50
O
or a pharmaceutically acceptable salt thereof, in which Y is oxygen or sulfur,
preferably oxygen; R, is unsubstituted aryl or aryl substituted with an
electron-
withdrawing group; R, is a nucleoside of one of the following formulae:
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17
~o
H
R, K,
k..
in which R6 is purine or pyrimidine, preferably pyrimidine; and R,, R8, Rg,
R,o, R",
and R,, are independently hydrogen, hydroxy, halo, azido, -NO~, -NR,3R,4, or -
N(OR,S)R,6, in which R,3, R,4, R,S, and R,6 are independently hydrogen, acyl,
alkyl,
or cycloalkyl;
R3 is hydrogen, acyl, alkyl, or cycloalkyl; R4 is a side chain of an amino
acid; or R3
and R~ may be taken together to form the side chain of proline or
hydroxyproline;
and RS is hydrogen, alkyl, cycloalkyl, or aryl.
As used in the definitions of the aryl phosphate derivatives of nucleosides
disclosed above, and in U.S. Patent Application No. 09/450,082, the following
terms have the following meanings:
The term "aryl" includes aromatic hydrocarbyl, such as, for example, phenyl,
including fused aromatic rings, such as, for example, naphthyl. Such groups
may be
unsubstituted or substituted on the aromatic ring by an electron-withdrawing
group,
such as, for example, halo (bromo, chloro, fluoro, iodo), NO,, or aryl.
Preferably,
aryl substituted with an electron-withdrawing group is bromophenyl, more
preferably 4-bromophenyl.
The term "acyl" includes substituents of the formula R"C(O)-, in which R"
is hydrogen, alkyl, or cycloalkyl.
The term "alkyl" includes a straight or branched saturated aliphatic
hydrocarbon chain having from 1 to 6 carbon atoms, such as, for example,
methyl,
ethyl, propyl, isopropyl (1-methylethyl), butyl, tent-butyl (l,l-
dimethylethyl), and
the like. Such groups may be unsubstituted or substituted with hydroxy, halo,
azido,
-NO~, -NR,3R,4, or -N(OR,S)R,6, in which R,3, R,4, R,S, and R,6 are as defined
above.
The term "cycloalkyl" includes a saturated aliphatic hydrocarbon ring having
from 3 to 7 carbon atoms, such as, for example, cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, and the like. Such groups may be unsubstituted or
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18
substituted with hydroxy, halo, azido, -NO,, -NR,3R,4, or -N(OR,S)R,6, in
which R,3,
R,4, R,S, and R,6 are as defined above.
The term "purine" includes adenine and guanine.
The term "pyrimidine" includes uracil, thymine, and cytosine. Preferably,
the pyrimidine is thymine.
The term "side chain of an amino acid" is the variable group of an amino
acid and includes, for example, the side chain of glycine, alanine, arginine,
asparagine, aspartic acid, cysteine, cystine, glutamic acid, glutamine,
hydroxylysine,
isoleucine, leucine, lysine, methionine, phenylalanine, serine, threonine,
tryptophan,
tyrosine, valine, and the like. Preferably, the side chain of an amino acid is
the side
chain of alanine or tryptophan.
Generally, compounds substituted with an electron-withdrawing group, such
as an ortho-orpara-substituted halogen or NO, provide for more efficient
hydrolysis
to active inhibitory compounds. Preferred is halogen substitution, and most
preferred are para-bromo substitution and para-chloro substitution.
Preferred nucleoside derivative disclosed therein include those of the
formulas:
V
x -o-Q ~~ x
H ~ NH
HsCOzG"CH3 H3COz~CH3
Na
wherein X is an electron withdrawing group, for example halo or NO~, and
most preferably, X is bromo or chloro.
Gel-Microemulsiott Formulations with Additional Spermicidal Agents
Suitable examples of spermicidal agents, include any spermicidal agent
generally known that is compatible for formulation with the gel-microemulsion.
Such agents can be incorporated into the gel-microemulsion formulations to
provide
for additional spermicidal activity of the formulation. Many of the
spermicidal
agents discussed below also have anit-microbial activity, such as anti-viral
activity,
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19
and can be incorporated into the formulations as an anti-microbial agent as
well as a
spermicidal agent.
Preferred Spermicidal Agents For Incorporation Into tlZe Gel
Microemulsion Formulation: Preferred examples of spermicidal agents include
those disclosed in the following copending patent applications:
U.S. Patent Application No. 09/008.898, which is incorporated herein by
reference, and corresponding published PCT Application No. PCT/LTS99/01171
(International Publication Number WO 99/36063), which is incorporated herein
by
reference;
U.S. Patent Application No. 09/187,115, which is incorporated herein by
reference;
U.S. Patent Application No. 09/224,677, which is incorporated herein by
reference.
1 S Examples of preferred spermicidal compounds disclosed in copending U.S.
Patent Application No. 09/008,898, and corresponding published PCT Application
No. PCT/LJS99/Ol 171, include the vanadium (IV) compounds disclosed therein.
Examples of such vanadium (IV) compounds include organometallic
cyclopentadienyl vanadium IV complexes. Preferred such compounds include:
vanadocene dichloride, bis (methylcyclopentadienyl) vanadium dichloride,
vanadocene dibromide, vanadocene diiodide, vanadocene diazide, vanadocene
dicyanide, vanadocene dioxycyanate, vanadocene dithiocyanate, vanadocene
diselenocyanate, vanadocene ditriflate, vanadocene monochloro oxycyanate,
vanadocene monochloroacetonitrilo tetrachloro ferrate, vanadocene
acetylacetonato
monotriflate, vanadocene bipyridino ditriflate, vanadocene hexafluoro
acetylacetonato monotriflate, vanadocene acethydroxamato monotriflate, and
vanadocene N-phenyl benzohydroxamato monotriflate. Particularly preferred
compounds include vanadocene diselenocyanate, and vanadocene dichloride.
Examples of preferred spermicidal compounds disclosed in copending U.S.
Patent Application No. 09/187,115 include oxo-vanadium (IV) compounds
disclosed therein. Preferred the oxovanadium (IV) complexes include at least
one
bidentate ligand. Suitable bidentate ligands include N,N'; N,O; and O,O'
bidentate
ligands. Examples of suitable bidentate ligands include bipyridyl, bridged
bipyridyl,
SUBSTITUTE SHEET (R ULE 16)
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and acetophenone. Particularly, preferred oxovanadium compounds are those
having the formulas shown and described below.
Some suitable oxo-vanadium (IV) compounds include a bidentate ligand
wherein the bidentate ligand is a bipyridyl and the oxovanadium IV complex has
the
5 general formulae:
R~
~ 0
N\ % X
V
N/ \ X I
(OH2)n
R
where R and R, are the same or different and are independently selected
from: H, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano,
10 carboalkoxy (e.g. CZ-C6) and nitro; X and X' are the same or different and
are independently selected from: monodentate and bidentate ligands; and n
is0orl.
Other suitable oxo-vanadium (IV) compounds have a bidentate
ligand wherein the bidentate ligand is a bridged bipyridyl and the
15 oxovanadium IV complex has the general formulae:
Rz
2
U
N\ /X
Z N~ v ~X3
(OH2)n
R3
where Rz and R3 are the same or different and are selected from H, lower
alkyl,
halogen, lower alkoxy, halogenated alkyl, cyano, carboalkoxy (e.g. C~-C6) and
nitro;
20 X' and X3, are the same or different and are selected from monodentate and
bidentate
ligands; Z is selected from O, CHz, CHz-CH,, and CH=CH; and n is 0 or 1.
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21
Other suitable oxo-vanadium (IV) compounds have a bidentate
ligand wherein the bidentate ligand is a bridged bipyridyl, and the bridged
bipyridyl is phenanthroline, and the oxovanadium IV complex has the
general formulae:
0
Xa
V
R / \
s
X
I
(OH2)n
where R'~. R' and R6 are the same or different and are independently selected
from: H, lower alkyl, halogen, lower alkoxy, halogenated alkyl, cyano,
carboalkoxy (e.g. CZ-C6) and nitro; X4 and X5 are the same or different and
independently selected from: monodentate and bidentate ligands; and n is 0
or 1.
Other suitable oxo-vanadium (IV) compounds have a bidentate
ligand wherein the bidentate ligand is an O,O' bidentate ligand, the
oxovanadium IV complex has the general formulae:
R'
0
~Y
R8 v
o \YI
R9 (OH2)n
where R', and R9 are the same or different and are independently selected
from: H, lower alkyl, lower alkoxy, and halogenated alkyl; Rg is selected
from H, lower alkyl, halo, lower alkoxy, and halogenated alkyl; Y and Y' are
the same or different and independently selected from the group consisting
of: monodentate and bidentate ligands; and n is 0 or 1.
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Preferred monodentate ligands for the oxovanadium complex include H,O,
halides and carboxylates. Preferred bidentate ligands include N, N' bidentate
ligands, N, O bidentate ligands, and O, O' bidentate ligands. Examples of
suitable
N, N' bidentate ligands include diamines and other such known suitable N, N'
bidentate ligands. Examples of diamines include bipyridal, derivatives of
bipyridal,
bridged bipyridal, such as phenanthroline, derivatives of phenanthroline, and
other
such compounds. Examples of suitable N, O bidentate ligands include amino
acids
and Schiff base type groups. Examples of suitable O, O' bidentate ligands
include
dicarboxylate, 2-hydroxyacetophenone; acetylacetone type and catechol type
groups.
Particularly useful oxo-vanadium (IV) complexes are the following:
(diaqua)(2,2'-bipyridyl)oxovanadium(IV) sulfate;
(aqua)bis(2,2'-bipyridyl)oxovanadium(IV) sulfate;
(diaqua)(4,4'-dimethyl-2,2'-bipyridyl)oxovanadium(IV) sulfate;
(aqua)bis(4,4'-dimethyl-2,2'-bipyridyl)oxovanadium(IV) sulfate;
(diaqua)(1,10-phenanthroline)oxovanadium(IV) sulfate;
(aqua)bis(1,10-phenanthroline)oxovanadium(IV) sulfate;
(diaqua)(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate;
(aqua)bis(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate;
(diaqua)(5-chloro-1,10-phenanthroline)oxovanadium(IV) sulfate;
(aqua)bis(5-chloro-1,10-phenanthroline)oxovanadium(IV) sulfate; and
bis(5'-bromo-2'-hydroxyacetophenone) oxovanadium(IV).
Examples of preferred spermicidal compounds disclosed in copending U.S.
Patent Application No. 09/224,677 include phenethyl-5-bromopyridylthiourea
(PBT) and dihydroxalkoxybenzylopyrimidine (DABO) derivatives disclosed
therein.
Examples of such PBT derivatives include those having the following
chemical formula, or a pharmaceutically acceptable salt thereof:
SUBSTITUTE SHEET (R ULE 26)
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23
R~
R i
R~
S
~~N ~ R
H 3
~~ N
Br
where R, R,, R2, R3, and R4 are independently hydrogen, F, Cl, Br, or I, and
where at
least one of R, R,, R2, R3, and R4 is F, Cl, Br, or I. Preferably, one of R,
R,, R~, R3,
and R4 in structure above is F or Cl. Some, but not all of the suitable
halogen-
substituted PBT derivatives of the invention are listed below:
N-[2-(2- .fia~orophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea
N-[2-(2-chlorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea
N-[2-(3-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea
N-[2-(3-chlorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea
N-[2-(4-fluorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea
N-[2-(4-chlorophenethyl)]-N'-[2-(5-bromopyridyl)]-thiourea.
One of the more preferred PBT derivatives is N-[2-(2-fluorophenethyl)]-
N'-[2-(5-bromopyridyl)]-thiourea (F-PBT) which has the chemical structure
shown below:
F /
S
HN~N
H
\\ N
Br
F-PBT
PBT derivatives can be synthesized as described in Vig et al., BIOORG. MED.
CHEM., 6:1789-1797 (1998). In brief 2-amino-5-bromopyridine is condensed
with 1,1-thiocarbonyl diimidazole to furnish the precursor thiocarbonyl
derivative.
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24
Further reaction with appropriately halogen-substituted phenylethylamine gives
the
target halogenated PBT derivatives.
Examples of suitable DABO derivatives include those of the formula, or a
pharmaceutically acceptable salt thereof:
O
HN 3 5 R' RZ
S-~N O
R~ YO Rz
where R~ and RZ are alike or different, and are hydrogen, halo, alkyl,
alkenyl,
hydroxy, alkoxy, thioalkyl, thiol, phosphino, ROH, or RNH group, where R is
alkyl.
Preferably, one or more of Rl and RZ is an alkyl having 1 to 3 carbonatones,
(C1-
C3), such as methyl (Me), ethyl (Et), or isopropyl (i-Pr). Preferably, Ri is
alkyl,
alkenyl, ROH, or RNH2. Rz is preferably halo, alkyl, or Cl-C3 alkoxy;
Y is S or O, and is preferably S. R3 is alkyl, alkenyl, aryl, aralkyl, ROH, or
RNH group, where R is alkyl , and is preferably C,-C3 alkyl.
Preferred DABO derivatives include compounds having the chemical
structure shown below, or a pharmaceutically acceptable salt thereof:
O R~
R~
HN
S
N \ R2
Me-S
where R1 is Me, Et, or i-Pr and R~ is H or Me.
Some, but not all, of the suitable DABO derivative compounds include
compounds (a) through (d) listed below, or a pharmaceutically acceptable salt
thereof:
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(a) ~-methyl-2-[(methylthiornethyl)thio]-6-benzyl-pyrimidin-4-1H-
one,
(b) 5-ethyl-2-[(methylthiomethyl)thin]-6-benzyl-pyrimidin-4-1H-
one,
5 (c) 5-isopropyl-2-[(methylthiomethyl)thio]-6-benzyl-pyrimidin-4-
1 H-one, and
(d). ~-isopropyl-2-[(methylthiomethyl)thio]-6-(3,5-dimethylbenzyl)-
pyrimidin-4-1 H-one.
One of the more preferred DABO derivatives is the compound 5-isopropyl-
10 2-[(methylthiomethyl)thio]-6-(benzyl)-pyrimidin-4-(1H)-ones (S-DABO) , and
pharmaceutically acceptable salts thereof, which is exemplified by the
chemical
structure shown below:
O Me Me
S N
S-Me
S-DABO
DABO derivatives can be prepared as described in descrii~ed in Vig et al.,
BIOORC MED CHE,~LETTEx~', 8:1461-1466 (1998). The general synthesis scheme for
the
preparation of DABO derivatives (a) through (d) listed above is as follows:
SUBSTITUTE SHEET (R ULE 26)
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26
O
R I \ CN a' b R I \ OEt c H
/ ~O ~O
> \ R~
R~ R~ H
1 a-d
O R~
d H
S~~ \ R~
M~-
Sail
1- 3 I R~ R2
a H Me
b H Et
c H i-Pr
d Me i-Pr
Reagents and conditions a) f~CHBrCOOEt/Znm-iF, b) HG(aq), c) (HZN~CS/Na/EtOH,
d) DMF, f~CC~, Chioromethyl methyl sulfide, 15h.
Briefly, ethyl-2-alkyl-4-(phenyl)-3-oxobutyrates 1 a-d were obtained from
commercially available phenyl acetonitrile. The (3-ketoesters were condensed
with
thiourea in the presence of sodium ethoxide to furnish the corresponding
thiouracils
2a-d. Compounds ( 1 a-d and 2 a-d) were produced by a methods previously
described (Danel, K. et al., Acta Chemica Scandinavica, 1997, ~ 1, 426-430;
Mai, A.
et al., J. Med Chem., 1997, 40, 1447-1454; Danel, K. et al., J. Med. Chem.,
1998.
41, 191-198).
Subsequent reaction of thiouracil with methylchloromethyl sulfide in
N,N-dimethylformamide (DMF) in the presence of potassium carbonate afforded
compounds 3a-d in moderate yields. A mixture of thiouracil compound 2 (1
mmol),
SUBSTITUTE SHEET (R ULE 26)
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27
methylchloromethyl sulfide (lmmol), and potassium carbonate (lmmol) in
anhydrous DMF (Sml) was stirred overnight at room temperature. After treatment
with water (50 ml), the solution was extracted with ethyl acetate (3 x 50 ml).
The
combined extracts were washed with saturated NaCI (2 x 50 ml), dried (MgS04),
filtered and concentrated in vacuo to give the crude products 3a-d which were
purified by column chromatography (hexane : ethyl acetate eluent).
Other examples of therapeutic agents includes vanadium (IV) complexes
containing a substituted or un-substituted catacholate ligand. Examples of
such
compounds include complexes having the following structural formula, or
pharmaceutically acceptable salts thereof:
R1
O R2
RS~~~,O
R6~ i ~O O
'R3
(OH2)n
R
1 S wherein R', RZ, R3 and R'~ are the same or different and are independently
selected from H, halo, OH2, 03SCF3, N3, CN, OCN, SCN, SeCN, NO~, C1-C4 alkyl,
Ci-C4 alkoxy, and aryl; and n is 0 or l; and R' and R6 are the same or
different and
are either monodentate ligands or R5 and R6 together comprise a bidentate
ligand.
Suitable monodentate ligands include, for example, aryl, halo, H20, 03SCF3,
N3, COOH, CN, OCN, SCN, SeCN, NO2, C,-C4 alkyl, C1-C4 alkoxy. Preferred
monodentate ligands comprises one or more unsubstituted or substituted
aromatic
ring. More preferred monodentate ligands comprise substituted or un-
substituted
cyclopentadienyl ligands.
Suitable bidentate ligands include, for example, N,N'; N,O; and O,O'
bidentate ligands. Examples of suitable N, N' bidentate ligands include
diamines and
other such known suitable N, N' bidentate ligands. Examples of diamines
include
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bipyridal, derivatives of bipyridal, bridged bipyridal, such as
phenanthroline,
derivatives of phenanthroline, and other such compounds. Examples of suitable
N,
O bidentate ligands include amino acids and Schiff base type groups. Examples
of
suitable O, O' bidentate ligands include dicarboxylate, 2-hydroxyacetophenone.
acetylacetone type and catechol type groups. Preferred bidentate ligands
comprise
one or more aromatic ring. Preferred examples of suitable bidentate ligands
comprising aromatic rings include substituted or un-substituted bipyridyl,
bridged
bipyridyl, and acetophenone ligands. One example of a bridged bipyridyl
includes
phenanthroline.
Some preferred vanadium (IV) catacholate complexes include ''bent
sandwich" vanadocene monocatacholate complexes having the following structure
formula, or pharmaceutically acceptable salts thereof:
R1
R2
CPw V i0
Cps ~O R3
R4
wherein Cp is unsubstituted cyclopentadienyl, or cyclopentadieneyl
substituted with one or more substituents selected from substituted or
unsubstituted
aryl, C,-C4 alkyl, C,-C4 alkoxy, halo, OH2, 03SCF3, N3, CN, OCN, SCN, SeCN,
NO2. Preferably, Cp is unsubstituted cyclopentadienyl.
R1, RZ, R3 and R4 are the same or different and are independently selected
from H, halo, OH2, 03SCF3, N3, CN, OCN, SCN, SeCN, NOZ, C,-C4 alkyl, and C,-
C4 alkoxy Preferably, electron donating groups, for example electron donating
alkyl
SUBSTITUTE SHEET (R ULE 26)
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29
groups, are present as substituents of the catacholate ring in positions R1,
Rz, R3
and/or R4.
Particularly preferred such compounds include vanadocene catacholate,
vanadocene mono-tertbutyl catacholate, and vanadocene 1, 3-diisopropyl
catacholate.
Another example of vanadium (IV) catacholate complexes include
complexes having a bidentate ligand wherein the bidentate ligand is a
bipyridyl has
the general formula shown below, or pharmaceutically acceptable salts thereof:
R~
R1
O
UN \ I I /O R2
~ ~ ~O
'N (OH2)n \R3
R'1
R
where R' and R8 are the same or different and are independently
selected from: H, aryl, C~-C4 alkyl, halo, CI-C4 alkoxy, carboalkoxy (e.g.
Cz-C6), cyano, and nitro; n is 0 or 1; Rl, R2, R3 and R4 are the same or
different and are independently selected from H, halo, OHz, 03SCF3, N3, CN,
OCN, SCN, SeCN, NO2, C,-C4 alkyl, and C,-C4 alkoxy.
Another example of vanadium (IV) catacholate complexes include
complexes having a bidentate ligand wherein the bidentate ligand is a
bridged bipyridyl has the general formula shown below, or pharmaceutically
acceptable salts thereof:
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R9
Rl
On
UN \ I I /O R2
Z
R3
\N (OH2)n
R4
R1
where R9 and Rl° are the same or different and are selected from H,
aryl, C~-
C4 alkyl, halogen, CI-C4 alkoxy, halogenated alkyl, cyano, carboalkoxy (e.g.
CZ-C6) and nitro; Z is selected from O, CHz, CHZ-CH2, and CH=CH; n is 0
or l; and halo, OHM, 03SCF3, N3, CN, OCN, SCN, SeCN, NO2, CI-C4
alkyl, and C~-C4 alkoxy.
Another example of vanadium (IV) catacholate complexes include
complexes having a bidentate ligand wherein the bidentate ligand is a
10 bridged bipyridyl, and the bridged bipyridyl is phenanthroline, has the
general formula shown below, or pharmaceutically acceptable salts thereof:
Rl
O R2
R1 ~~V~O
~ ~O
R3
(OH2)n
R4
15 where R1', R12 and R13 are the same or different and are independently
selected from: H, aryl, C,-C4 alkyl, halogen, lower alkoxy, halogenated
alkyl, cyano, carboalkoxy (e.g. CZ-C6) and nitro; n is 0 or 1; and R', R2, R3
and R4 are the same or different and are independently selected from H, halo,
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OH2, 03SCF3, N3, CN, OCN, SCN, SeCN, NO~, C~-Ca alkyl, and C,-C~
alkoxy.
Another example of vanadium (IV) catacholate complexes include
complexes having a bidentate ligand wherein the bidentate ligand is an O,O'
bidentate ligand, and the complex has the general formula VI, is shown
below, or pharmaceutically acceptable salts thereof:
R14 Rl
O R2
R 1 i~ ~V i0
~ ~ ~O
itl (OH~)n \R3
R4
where R14 and RI6 are the same or different and are independently selected
from: H, aryl, C,-C4 alkyl, and C,-C4 alkoxy, and halogenated alkyl; R15 is
selected
from H, C,-Ca alkyl, halo, C~-C4 alkoxy, and halogenated alkyl; n is 0 or l;
and Rl,
R2, R3 and R4 are the same or different and are independently selected from H,
halo,
OH2, 03SCF3, N3, CN, OCN, SCN, SeCN, NOZ, C,-C4 alkyl, and C1-C4 alkoxy.
IS
Formulation of the gel-microemulsions formulations including additional
tlzerapeutic agents
Gel-microemulsions formulations including additional therapeutic agents, for
example the anti-microbial agents or spermicidal agents discussed above, are
formulated and prepared in substantially the same way as the primary gel-
microemulsions discussed above, with the only difference being the addition of
the
additional therapeutic agent. The amount of additional ingredient added is
dependent upon the desired effective amount of the ingredient in the final gel-
microemulsion formulation. It is still desirable to provide a dispersion with
a
viscosity in the range of 200-1000 centipoise, and a submicron particle size,
preferably in the range of 30-80 nm.
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Representative examples of constituent concentration ranges for base
components of some gel-microemulaion formulations embodying the invention can
be found in Table 3, wherein the values are given in wt. % of the ingredients
in
reference to the total weight of the formulation.
Table 3
Constituent Ranges Preferred RangesMore Preferred
Ranges
Therapeutic up to up to S up to 2
Ingredient 10
Lipid 2 to 6 to 23 8 to 15
25
Surfactant 3 to 4 to 17 8 to 15
30
Humectant 2 to 3 to 12 S to 10
24
Polymer Gel 0.5 to 1 to 2 1.2 to 1.8
4
Additives (e.g.0 to 0.1 to 0.3 0.15 to 0.2
Preservatives)0.5
Water Balance Balance Balance
In some preferred embodiments, the formulation includes the specific
constituent concentrations for base components as found in Table 4, wherein
the
values are given in wt. % of the ingredients in reference to the formulation
weight.
Table 4
Ingredients Preferred RangesMore Preferred
Ranges
Anti-microbial agent up to 2 1 to 2
or
Spermicidal agent,
or
mixtures thereof
Medium Chain Tryglyceride6 to 23 8 to 15
Ethoxylated Castor 3 to 11 5 to 9
Oil
Phospholipid 1.5 to 6 3 to 6
Propylene Glycol 1.5 to 6 3 to 6
PEG-200 1.5 to 6 3 to 6
Natural Hydrogels 1 to 2 1.2 to 1.8
Preservative 0.1 to 0.2 0.15 to 0.2
Water Balance Balance
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The following generally describes a simple formulation procedure for
producing formulations with additional therapeutic agents, such as an anti-
microbial
agent or a spermicidal agent: Combine surfactants, hydrophilic components, and
the
lipids (preferably medium chain tryglycerides) in an appropriate container.
Mix the
components using a stir bar with mild heat until a clear and homogeneous
microemulsion is formed. Add the appropriate amount of therapeutic agents with
continued stiffing for approximately 10 minutesto assure complete
solubilization of
the drug. Remove the composition from the heat, and wait until it reaches room
temperature. Add two parts of a pre-prepared polymer dispersion to each part
of
microemulsion with continued mixing. The resulting gel-microemulsion is a
dispersion with a viscosity in the range of 200-1000 centipoise, and a
submicron
particle size, preferably in the range of 30-80 nm.
Use Theraputic Agent Containing Gel-Microemulsion Formulations
When used as a spermicide, or a dual function spermicide/anti-microbial
composition, the gel-microemulsion formulations resulting from the addition of
additional theraputic agents are contemplated for used in generally the same
manner
as the gel-microemulsion spermicide use discussed above. However, it is
contemplated that formulations including other theraputic agents, for example
anti-
viral agents, can be used in non-spermicidal applications.
In such applications, the formulation is preferably administered to a site
appropriate for the theraputic activity desired in a dosage which is effective
to
effectuate the desired theraputic effect. For example, in anti-microbial
applications,
the formulation is preferably administered to a site appropriate for desired
anti-
microbial activity in a dosage which is effective to effectuate the desired
anti-
microbial effect. Appropriate amounts can be determined by those skilled in
the art.
Such theraputic compositions are intended particularly for use in mammals, but
use
outside of mammals is contemplated. It is expected that the formulations will
be
used by humans in most practical applications.
The invention may be further clarified by reference to the following
Examples, which serve to exemplify some of the preferred embodiments, and not
to
limit the invention.
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EXAMPLES
Example 1: Synthesis of Gel-Microemulsion Formulations
Materials: Captex 300 was obtained from ABITEC Corp., Janeswille,
WI. Cremophor EL was from BASF Corp., Mount Olive, NJ. Phospholipon 90G
was purchased from American Lecithin Co., Danbury, CT. PEG-200 was from
Union Carbide Corp., Danbury, CT while propylene glycol was obtained from
Spectrum Quality Products Inc., New Brunswick, NJ. Seaspan and viscarin
carrageenan were obtained from FMC Corp., Newark, DE. N-9 (IGEPAL CO-630)
was a generous gift from Rhone Poulenc, Cranbury, NJ.
Gel-Microemulsion Formulation: A lipophilic sub-micron (30-80 nm)
particle size microemulsion was developed using commonly used pharmaceutical
excipients through systemic mapping of ternary phase diagrams (Eccleston GM,
In:
Swarbrick J, Boylan JC, eds. Encyclopedia of Pharmaceutical Technology, New
York:Marcel Dekker, 1992:375-421.; Ritschel WA, Meth Find Exp Clin
Pharmacol, 1993; 13: 205-20.). Several microemulsion compositions were
screened for particle size, stability, and responses to in vitro spermicidal
activity.
The ingredients tested included: medium chain triglycerides, purified soya
phospholipid, Pluronic F-68, ethoxylated castor oil, propylene glycol, and
polyethylene glycol. The ingredients selected included, drug solubilizers and
stabilizers (Captex 300, Cremophor EL, phospholipon 90 G, propylene glycol,
and
PEG 200) and a preservative (sodium benzoate). Various polymeric gels were
screened to produce a gel with desirable viscosity. Polymer suspensions of
seaspan
and viscarin carrageenan were selected as additives to the microemulsion-based
system to obtain a gel with desirable viscosity with high thickening
capability and
compatibility with vaginal mucosa.
A submicron (30-80 nm) particle size microemulsion-based system
containing the pharmacological excipients, Captex 300, Cremophor EL,
phospholipon 90G, propylene glycol, PEG 200, and sodium benzoate, with high
solubilizing capacity for lipophilic drugs was identified through systematic
mapping
of ternary phase diagrams, and lipophilic drug solubilization studies. The
ternary
phase diagram of the microemulsion components used for the preparation of GM-4
is shown in Figure 1. The non-grid area represents the single phase
microemulsion
region. The asterisk represents the microemulsion which was used for GM-4
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formulation listed in Table 5. Polymer suspensions of seaspan and viscarin
carrageenan were selected as additives to the microemulsion-based system to
obtain
a gel of desirable viscosity with high thickening capability and compatibility
with
the microemulsion. These polymers did not cause precipitation or alter the
5 microemulsion particle size. The GM-4 was found to be very stable at ambient
temperature.
Particle size determination was made using Nicomp Model 380 laser diode
source (Particle Sizing Systems, Santa Barbara, CA). Viscosity measurements
were
made using the Brookfield digital viscometer (Model DV-II+; Brookfield
10 Engineering Laboratories, Spoughton, MA).
Table 5: Components of GM-4 Formulation
xcipient Type Final concentration
%, b wt
Ca tex 300 Li id 10.8
Cremo hor EL Surfactant '7,6
hos holi on 90G hos holi id 5.1
ro lene GI col Humectant 4.2
PEG-200 Humectant 4.2
Seas an carra eenanatural of mer 0.9
iscarin carra~eenanatural olvmer 0.5
Sodium benzoate Preservative 0.2
Water Diluent 66.5
15 EXAMPLE 2: Screening of Pharmaceutical Excipients of GM-4 for
Spermicidal Activity Against Human Sperm
Methods and Materials
Computer-Assisted Spermicidal Assay: To evaluate the spermicidal
20 activity of the pharmaceutical excipients used in the GM-4 formulation, a
highly
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motile fraction of pooled donor sperm (n = 9) was prepared by discontinuous
(90-
45%) gradient centrifugation using Enhance-S-Plus cell isolation medium
(Conception technologies, San Diego, CA) and the "swim-up" method as
previously
described (D'Cruz OJ, et al., Biol Reprod 1995;53:118-30.; D'Cruz OJ, et al.,
Biol
Reprod, 1998; 59:503-1 ~.). All donor specimens were obtained after informed
consent and in compliance with the guidelines of the Parker Hughes Institute
Institutional Review Board. Highly motile fraction of sperm L 1Ox106/ml) were
suspended in 1 ml of Biggers, Whitten, and Whittingam's medium (BWW)
containing 25 mM HEPES (Irvine Scientific, Santa Ana, CA) and 0.3% BSA
(fraction V; Sigma Chemical Co., St. Louis, MO) in the presence and absence of
serial 2-fold dilutions of test substance. The ingredients evaluated were
Captex 300
(1.35% - 10.8%), Cremophor EL (0.95% - 7.6%), phospholipon 90G (0.637% -
5.1 %), propylene glycol (0.52% - 4.2%), PEG 200 (0.52% - 4.2%), seaspan
carrageenan (0.11 % - 0.9%), viscarin carrageenan (0.06% - 0.5%), and sodium
benzoate (0.025% - 0.2%). After 3 h incubation at 37°C, the sperm head
centroid-
derived sperm motility parameters were determined using a Hamilton Thorne
Research (Danvers, MA) Integrated Visual Optical System (IVOS), version 10
instrument, as previously described (D'Cruz OJ, et al., Biol Reprod,
1998;58:1515-
26.; D'Cruz OJ, et al., Mol Hum Reprod, 1998;4:683-93.; D'Cruz OJ, et al.,
Biol
Reprod, 1999;60:435-44.; D'Cruz OJ, et al. Biol Reprod, 1999;60:1419-28.). The
attributes of sperm kinematic parameters evaluated included numbers of motile
(MOT) and progressively (PRG) motile sperm; curvilinear velocity (VCL);
average
path velocity (VAP); straight-line velocity (VSL); beat-cross frequency (BCF);
and
the amplitude of lateral head displacement (ALH) and the derivatives,
straightness
(STR) and linearity (LIN). Data from each individual cell track were recorded
and
analyzed. For each aliquot sampled, >200 sperm were analyzed. The percentage
motilities were compared with those of sham-treated control suspensions of
motile
sperm. The spermicidal activity of the test compound was expressed as EC50
(the
final concentration of the compound in the medium that decreased the
proportion of
motile sperm by 50%).
Results
The effects of individual components of GM-4 on the motility of washed
and enriched motile fraction of sperm evaluated by CASA are summarized in
Figure
2. At the final concentrations used for GM-4 formulation, Captex 300, PEG 200,
seaspan carrageenan, viscarin carrageenan, and sodium benzoate, demonstrated
little
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or no inhibitory effects on human sperm motility. Further, sperm motion
kinematics
using CASA confirmed that these excipients did not significantly alter the
sperm
motion parameters, such as the progressive velocity, straightness of the
swimming
pattern, linearity of the sperm tracks, beat-cross frequency, and the
amplitude of
lateral sperm head displacement. In contrast, Cremophor EL and phospholipon
90G
were spermicidal over the entire range of concentrations tested whereas
propylene
glycol was partially spermicidal at the highest concentration tested (EC50 =
>4.2%).
The concentration-dependent spermicidal activity by Cremophor EL and
phospholipon 90G was associated with a parallel decline in sperm kinematics,
particularly with respect to track speed (VCL), path velocity (VAP), and
straight line
velocity (VSL).
Example 3: Spermicidal Activity of Pharmaceutical Excipients of GM-4 and
of the GM-4 Formulation in Human Semen
Methods and Materials
The effect of duration of incubation on spermicidal activity in the presence
of
each of the eight pharmaceutical excipients was tested by mixing an aliquot of
semen with equal volume of test compounds in BWW-0:3% BSA to yield the final
concentrations contained in GM-4. At timed intervals of 15, 30, 45 and 60 min,
5-
~.1 samples were transferred to two 20-~m Microcell (Conception Technologies)
chambers, and sperm motility was assessed by CASA. Sperm motility in samples
too viscous for CASA analysis (seaspan carrageenan, viscarin carrageenan,
Cremophor EL and phospholipon 90G) were determined by phase contrast
microscopy, and the number of motile sperm per treatment were enumerated for a
total of 200 sperm. The time course test was performed in 3 separate trials,
with
semen obtained from three different donors.
Modified Sander-Cramer Assay: The spermicidal activity of GM-4
formulation, as produced in Example 1, was tested by a modified Sander-Cramer
assay (Sander FV, et al., Hum Fey°til, 1941;6:134-7.; D'Cruz OJ, et
al.,
Contraception, 1999;59:319-31.). Briefly, aliquots (0.1 ml) of freshly
liquefied
semen were rapidly mixed with an equal volume of freshly prepared GM-4
formulation. A 5-~l sample was transferred to a 20 ~m Microcell chamber
(Conception Technologies) and examined immediately under a phase contrast
microscope (Olympus BX-20; Olympus Corporation, Lake Success, NY) attached to
a CCD camera (Hitachi Deneshi Ltd., Tokyo, Japan) and a videomonitor. The time
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required for sperm immobilization was recorded in seconds. This test was
performed
in six separate trials, with semen obtained from six different donors.
Results:
The results of spermicidal activity of individual components of GM-4 tested
in human semen rather than using washed motile fraction of sperm are shown in
Figure 3. A time course study of sperm motility impairment by each of the
individual components of GM-4 formulation revealed that none of the eight
components tested including Captex 300, phospholipon 90G, Cremophor EL,
propylene glycol, PEG 200, viscarin carrageenan, seaspan carrageenan and
sodium
benzoate was spermicidal in human semen (t 1 /2 = > 60 min).
By contrast, the submicron particle size GM-4 formulation completely
immobilized sperm in human semen in less than 2 min (1.2 ~ 0.3 min). Thus, the
combination of these pharmaceutical excipients as a gel-microemulsion
formulation
was a potent spermicide in semen.
EXAMPLE 5 Preparation and Characterization of GM-4 Formulation
Containing 2% WHI-07.
WHI-07 is a phenyl phosphate derivative of bromo-methoxy zidovudine
(WHI-07) with potent anti-HIV and spermicidal activities. WHI-07 is a
lipophilic
zidovudine (AZT) derivative which has extremely low solubility in water. WHI-
07
has the following chemical structure:
H3
Br ~NH
O Me0
Br ~ ~ O-~P~~-O HO
NH
~ N
M~C02Me
Chemical name: (SR,6R)-and (SS,6S)-5-bromo-6-methoxy-5,6-dihydro-
AZT-5'-(para- bromophenyl methoxyalaninyl phosphate). Molecular weight:
698
WHI-07 was synthesized using the following synthetic scheme.
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Synthetic Scheme for WHI-07
39
/ \ ~ E3N/Eb_O /
Br~OH + CI-P-CI --> B~O P-CI
CI CI
Me
NHzCHMeCOzMe R ~ AZT/THF
> B / \ O-P-N C02Me >
Et3N/CHzCIz y
CI H N Meth limidazole
V O
H3~NH
H N'~O Br'-/MeOH O M ~ ~H
B ~ ~ o- Ho o > B / \ o_'~;-o Ho 0
NH
~ 3
M~COZMe M~C02Me
Chemical Characterization:
Melting Point : 59-600C; Rf : 0.56 ( 10%MeOH/ 90% CHC13 ); UV (MeOH) 209,
218, 221, & 261 nm; IR (Neat): 3218, 3093, 2925, 2850, 2105, 1712, 1484, 1378,
1241, 1153, 1010, 929 cm 1.
1H NMR (CDC13) d 8.66 (1H, br, 3-NH), 7.43 (2H, d, J = 9.OHz, Aryl H), 7.14
(2H, d, J = 9.0 Hz, Aryl H), 6.01 (0.68H, t, J = 6.3 Hz, -CH at C-1'), 5.37
(0.32H,
m, -CH at C-1'), 4.87 (0.68H, s, -CH at C-6), 4.61 ( 0.33H, s, -CH at C-6 ),
4.35-
3.96 (6H, m, -CH at C-3', 4', 5' and Ala-NH, a-CH), 3.74 (3H, s, -COOCH3),
3.44 (3H, s, -OCH3 at C-6), 2.56-2.30 (2H, m, -CH2 at C-2'), 1.93 (3H, s, -CH3
at C-5), 1.38 (3H, m, a-CH3 of Ala).; 13C NMR (CDCl3) d 173.6, 166.7, 150.1,
148.9, 132.7, 132.5, 121.9, 121.7, 118.1, 87.7, 85.1, 81.7, 81.5, 81.4, 65.7,
65.6,
60.0, 57.9, 57.8, 53.6, 52.7, 50.3, 50.2, 36.9, 36.7, 22.8, 22.7, 21.2, 21.1.;
31P NMR
(CDC13) d 2.70, 2.60, 2.54, 2.32.; MS (CI, m/e) 700.6 (M+, 8lBr+8lBr), 698.6
(M+, 8lBr+79Br), 696.6 (M+, 79Br+79Br), 588.8 (M+-Br-OCH3, 8lBr), 586.9
(M+ - Br-OCH3, 79Br).; HPLC: 39.06, 40.28, 45.33, & 49.25 min (Column:
LiChrospher 100 RP-18e (5 Vim); Flow rate: 1 mL/min; Solvent: H20 (0.1 % TEA +
0.1% TFA) : CH3CN = 62:38).
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Appearance: White solid at room temperature: Solubility: < 0.003% solubility
in
water, soluble in oil (medium chain triglyceride, 4.1 %), and quite soluble in
some
hydrophilic cosolvents such as polyethylene glycol 300 ( 13.1 %). It is also
soluble in
several organic solvents including chloroform, ethanol, methanol, and DMSO.;
5 Octanol/Water partition coefficient: Log KD = 2.05 (see below)
Partition coefficient of WHI-07: Four samples of WHI-07, 51.8 mg, 64.6 mg,
56.5 mg, and 74.5 mg were weighed and dissolved in 5 ml of octanol in four
test
tubes. After the drug was completely dissolved in octanol, 5 ml of water was
added
10 to each octanol solution. The mixtures were handshaken vigorously for 10
min, and
afterward let to stand overnight until a complete phase separation occurred.
Samples of water and octanol were taken from each mixture, and directly
injected into the HPLC for analysis. Peak areas of WHI-07 in the water layer
and in
corresponding octanol layer are shown in Table 6. The peak area ratios
represent the
15 partition coefficient.
Table 6
Sample Octanol layer Water layer Peak area ratio
Peak area Peak area Octanol/water
mAU*s) mAU*s
1 5324.3 46.74 113.9
2 6159.2 55.32 111.3
3 5712.4 49.32 115.8
4 7347.4 67.25 109.2
1 m a~cra~c uc~anouwater partmon coetticient is: Log KD = 2.05
20 The suitable microemulsion compositions were identified by first
constructing a series of ternary phase diagrams. The solubilizations of WHI-07
in
several microemulsions selected from within the single phase microemulsion
region
in the phase diagrams were determined to identify microemulsions with high
solubilizations of WHI-07.
Solubility of WHI-07
The solubility of WHI-07 in Captex 300 and polyethylene glycol 300 was
carried out using UV-Vis spectrophotometer at 272 nm.
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Calibration curve: Two stock solutions of WHI-07 were prepared in
ethanol at concentrations of 4.2 mg/ml and 3.85 mg/ml respectively. These
stock
solutions were diluted in ethanol to prepare the standard solutions with
concentrations and A272 shown in Table 7. The plot of the calibration curve is
shown in Curve 1.
Table 7
Calibration sampleConc. in mg/ml A272
1 0.06 0.07
2 0.12 0.128
3 0.24 0.240
4 0.48 0.467
5 0.96 0.927
6 1.93 1.802
7 0.11 0.084
8 0.21 0.182
0.42 0.414
0.53 0.479
11 0.7 0.652
12 1.05 0.969
13 2.10 1.886
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Curve 1
s
WHI-07 Conc mg/ml = 0.0134 + 0.912 x
Correlation coefficient R"2 = 0.999
0
2 3
Abs (272 nm)
Sample preparation and assay
S Appropriate amounts of WHI-07 and Captex 300 or PEG300 were weighed
and placed in glass containers. Each sample was stirred with a stir bar at
ambient
temperature for 2 hours. Each sample was then filtered through 0.45 ~m filter,
and
diluted with ethanol for UV-Vis measurement. The results are shown in Table 8.
Table 8
WHI-07 Solubility (mg/ml)
Captex 300 40.6 +/- 4.1 (n = 3)
PEG300 131.5 +/- 4.9 (n = 3)
Polymer suspensions were added to the microemulsions to increase their
viscosity. Two types of polymers, xanthan gum and Carrageenan, were found to
be
particularly suitable. but other types of polymers can also be used to produce
gel-
microemulsions. In general, a total polymer concentration of about between
0.5%
and 3% is needed to provide a gel-microemulsion with adequate viscosity.
Based on these results, we postulated that WHI-07 can be delivered using
the GM-4 formulation. To this end, 18.7 mg of WHI-07 was first dissolved in
305
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mg of a microemulsion consisting 32.4% of Captex 300 (medium chain
triglyceride),
4.3% of purified water, and 63.3% of surfactant-cosolvent mixture containing
36%
of Cremophor EL, 24% of Phospholipon 90G (purified Soya lecithin), 20% of
polyethylene glycol 200 and 20% of propylene glycol all by weight. The mixture
was heated to 700C. The drug was completely dissolved in the mixture after
about 5
minutes of mixing with a stir bar. The drug solution was then removed from the
heat. The resulting composition was a clear microemulsion.
This WHI-07-containing microemulsion concentrate was diluted with water
to bring the intensity to between 300 and 500 KHZ for optimal particle size
measurement. The particle diameter of the microemulsion in the absence of
polymers was determined by laser light scattering using Nicomp 380 Submicron
Particle Sizer (Particle Sizing Systems, Inc., Santa Barbara, CA). The size
distribution and mean particle diameter value became stabilized quickly after
a few
minutes of run time. A average (meantSD) particle diameter of 18.58.1 nm was
determined by Nicomp 380 photon correlation light scattering particle sizer.
WHI-07 concentration ana~sis in WHI-07 Formulations
Routine analysis of WHI-07 were performed by dissolving the samples in
ethanol or acetonitrile and analyzed with HPLC after appropriate dilution. WHI-
07
gel formulations, however, are not totally soluble in either ethanol or
acetonitrile
because of the polymers in the formulation. The following WHI-07 analytical
methods were developed for the gel formulations. The accuracy and precision of
the
methods meet pharmaceutical requirements.
WHI-07 calibration curve
101.3 mg of WHI-07 (96% purity) was dissolved in a 10 ml glass vial with
1.5 ml of a microemulsion with the following composition: 36% Cremophor EL,
24% Phospholipon 90G, 20% PEG 200, and 20% propylene glycol. The mixture
was stirred for 10 min at SOOC until the drug was completely dissolved and the
mixture was clear. The vial was removed from the hot plate and 3.5 ml of the
polymer suspension (1.3% Seaspen Carrageenan, 0.7% Xantural, and 0.3% sodium
benzoate in DI water) was added to the vial. The vial was hand shaken for 1
min
and vortexed for 1 min or until the mixture was homogeneous.
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The control (drug-free) gel microemulsion was prepared by mixing 1.2 ml of the
microemulsion with 2.8 ml of the polymer suspension. The calibration curve was
prepared as following:
Five standard solutions with varying WHI-07 contents were prepared as
shown in Table 9. Each vial was hand shaken and the polymers in the gel was
precipitated out. The mixture was then centrifuged at room temperature for 10
min
at 2000 rpm. The vials were carefully removed from the centrifuge and 0.5 ml
of the
supernatant was pipetted out from each vial for HPLC analysis.
The condition for HPLC analysis (WHI-07) was as followed:
Column: RPl8e (Spm) Lot # L228433
Eluent: Acetonitrile / (0.1 % TFA and 0.1 % TEF) = 45 / 55
Eluent flowrate: 1 ml/min
Sample injection volume: 20 pl
Method run time: 30 min
Under these conditions, the retention times for the three isomeric peaks of
WHI-07 were 20.5 min, 23.2 min, and 24.9 min. The area ratio of these peaks
were
31.9 : 5.7 : 3.3. The peak with retention time of 20.5 min was used to
construct the
calibration curve by plotting WHI-07 concentration vs. peak area. The
calibration
curve was linear in the WHI-07 concentration range of 0 to 1.3 mg/ml with a
correlation coefficient of 1.000 (Curve 2).
Table
9
Vial WHI-07 Control ACN added WHI-07 concentrationPeak(20
# gel gel min)
added (ml)added (ml) (mg/ml) (mAU*s)
(ml)
(20.26
mg/ml)
1 0 0.50 4.0 0 0
2 0.05 0.45 4.0 0.225 1121.2
3 0.10 0.40 4.0 0.450 2348.4
4 0.20 0.30 4.0 0.900 4682.6
5 0.30 0.20 4.0 1.351 6998.1
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4~
Curve 2: Calibration curve for WHI-07 gel formulation
WHI-07 Conc mg/ml = 0.0022 + 7.g2p x Peak area /
Correlation coefficient R"2 =
0
sooo sooo
HPLC Peak areas
Precision of the analytical method
To test the precision of the method, ten WHI-07 gel samples were prepared
as following, and HPLC analysis was performed. 101.3 mg of WHI-07 (99.4%
purity) was dissolved in a 10 ml glass vial with 1.5 of the microemulsion with
composition of 36% Cremophor EL, 24% Phospholipon 90G, 20% PEG 200 and
20% propylene glycol. The mixture was stirred for 10 min at SOOC until the
drug
was dissolved and the mixture was clear. The vial was removed from the hot
plate
and 3.5 ml of the polymer suspension (1.3% Seaspen Carrageenan, 0.7% Xantrual
and 0.3% sodium benzoate in DI water) was added to the vial. The vial was hand
shaken for 1 min and vortexed for 1 min or until the mixture was homogeneous.
The control gel was prepared by mixing 1.2 ml of the microemulsion with
2.8 ml of the polymer suspension. To each of the 10 vials, 0.2 ml of the WHI-
07
gel, 0.3 ml of the control gel and 4 ml of acetonitrile were added. The vials
were
then handshaken for 1 min and vortexed for 1 min. The mixture was then
centrifuged at room temperature for 10 min at 2000 rpm. The vials were
carefully
removed from the centrifuge and 0.5 ml of the supernatant was pipetted out
from
each vial for HPLC analysis. The data was analyzed to determine the precision
of
the method. The results are shown in Table 10. The relative standard deviation
of
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the method was 0.61 %. These data indicate that the developed method has a
good
precision for the analysis of WHI-07 content in the gel formulation.
Table 10
Vial # Peak area(mAU*s)deviation relative standard
deviation
(RSD)
1 2312.8 18.04
2 2285.2 -7.56
3 2272.0 -22.76
4 2279.4 -15.36
5 2296.0 1.24
6 2275.6 -19.16
7 2299.4 4.64
8 2291.2 -3.56
9 2332.8 38.04
10 2303.2 8.44
Average 2294.76 14.08 0.61
Procedures for WHI-07 formulation sample preparation:
240 mg of WHI-07 was weighed in a 10 ml glass shell vial. 4.0 ml of the
microemulsion was transferred to the vial with a 1.0 ml Drummond pipette. A 12
X
4 mm magnetic stir bar was placed in the vial and the vial was placed on the
Corning
stir/hot plate for mixing. The dials for both stir and heat were set at #3.
The mixture
was heated for 5 min until the drug crystals were disappeared and the mixture
was
clear. WHI-07 concentration in this microemulsion was 60 mg/ml.
2% WHI-07 gel was prepared by mixing 2 ml of the concentrated WHI-07
microemulsion with 4 ml of the polymer suspension in a 10 ml glass shell vial.
1
WHI-07 gel was prepared by mixing 1 ml of the concentrated WHI-07
microemulsion with 1 ml of the microemulsion and 4 ml of the polymer
suspension.
0.5% WHI-07 gel was prepared by mixing 0.5 ml of the concentrated WHI-07
microemulsion with 1.5 ml of the microemulsion and 4 ml of the polymer
suspension.
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The control gel was prepared by mixing 2 ml of the microemulsion and 4 ml of
the
polymer suspension.
WHI-07formulatiou couceutratiou analysis: Two samples, 100 ~1 each,
were taken from each preparation for concentration analysis. The samples were
placed in 5 ml glass shell vials. 4.2 ml of acetonitrile was added to each of
the
sample vials. The vials were hand shaken for 10 seconds and were placed in a
shaker at 200 rpm for 30 min. The vials were hand shaken again for 10 seconds
and
span for 10 min at 2000 rpm in the Beckman GS-6 Centrifuge. The vials were
carefully removed from the centrifuge and 1 ml each of the supernatant was
pipette
out for HPLC analysis. The results were shown in Table 11. All of the samples
meet pharmaceutical requirement (RSD less than 5.0%).
Table 11
Vial # Sample ID WHI-07 Average RSD
concentration
1 0.5% WHI-07 0.52%
2 0.5% WHI-07 0.49% 0.5% 0
3 1.0% WHI-07 1.02%
4 1.0% WHI-07 0.92% 0.97% 3.0%
5 2.0% WHI-07 1.88%
2.0% WHI-07 1.99% 1.94 3.0%
We next examined the shelf life/stability of the WHI-07. A gel-
microemulsion with WHI-07 concentration of 0.5% and 2.0% were prepared and
were analyzed for WHI-07 concentrations at day l, day 4 and day 8. The result
is
shown in the following Table 12:
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Table 12
Time (day) 0.5% WHI-07 2.0% WHI-07
gel gel microemulsion
microemulsion
1 0.5 2.0
4 0.49 2.0
8 0.51 1.97
Percent change 2% 1.5%
at day
8
The result indicates that WHI-07 was stable in the gel microemulsion
formulations were stable at room temperature in the 8 day observations.
Formation ofgel-microemulsion: This drug-containing microemulsion
was then mixed with 600 mg of a polymer suspension containing 1.3% of Seaspan
Carrageenan, 1.3% of Viscarin Carrageenan, and 0.3% of sodium benzoate by
weight with gentle mixing. The resulting gel microemulsion had a pH of 7.2 and
was a translucent gel with the following composition:
(by weight)
WHI-07 2.0
Captex 300 10.7
Phospholipon 90G 5.0
Cremophor EL 7.5
Propylene glycol 4.2
PEG 200 4.2
SeaSpan Carrageenan 0.9
Viscarin Carrageenan0.9
Sodium benzoate 0.2
Water 64.4
The viscosity of the GM-4 formulation with and without WHI-07 was
determined using a Brookfield DV-E Viscometer with spindle #3 (speed: 10 rpm).
Drug-free GM-4 formulation had a viscosity of 301.9 centripoises. The GM-4
formulation containing 2% WHI-07 had a viscosity of 64.7.
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Although a viscosity of 64.7 appeared to be sufficient for the utility of the
2% WHI-07 containing GM-4 formulation to prevent transvaginal or transrectal
transmission of FIV, the viscosity could easily be increased by using a
different
polymer suspension. For example, a thicker formulation was prepared as
follows:
This drug-containing microemulsion in Example was mixed with 600 mg of a
polymer suspension containing 1.3% of Seaspan Carrageenan, 0.7% of Xantural,
and
0.3% of sodium benzoate by weight with gentle mixing. The resulting gel
microemulsion was a translucent gel-like liquid with the following
composition:
% (by weight)
WHI-07 2.0
Captex 300 10.7
Phospholipon 90G 5.0
Cremophor EL 7.5
Propylene glycol 4.2
PEG 200 4.2
SeaSpan Carrageenan 0.9
Xantural 0.5
Sodium benzoate 0.2
Water 64.8
The viscosity of this formulation was 380 centripoises. This formulation
was also found to have good colloidal stability when stored at room
temperature for
several months.
EXAMPLE 4: Preclinical Studies:
Methods and Materials
Rabbits: Fifty nine female and 12 male, sexually mature (> 6 months old; >
7 lbs), specific-pathogen-free. New Zealand White rabbits were obtained from
Charles River Laboratories (Wilmington, DE). For each fertility trial, 24 does
and
12 bucks were used. All rabbits were uniquely identified with metal ear tags.
Tap
water and rabbit food pellets (Teklad LM-485; Harlan Teklad) were available ad
libitum. The does and bucks were maintained in separate rooms that were kept
at 22
~ 2°C with relative humidity of 50 ~ 20% and a 12-h fluorescence light
cycle. The
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rabbits were isolated for a minimum of 4 weeks before the fertility trials.
All
procedures were approved by the Parker Hughes Institute Animal Use and Care
Committee. All animal husbandry operations were conducted under current USDA
Guidelines.
5 Mice: Twenty, female B6C;F1 mice of approximately 6 weeks of age were
obtained from Charles River Laboratories (Wilmington, DE) and were uniquely
identified with metal ear tags and ear notches. Tap water and laboratory diet
(Teklad
LM-485; Harlan Teklad) were available ad libitum. The animals were maintained
in a room that was kept at 22 ~ 2°C with relative humidity of 50 ~ 10%
and a 12-h
10 fluorescence light cycle. All animal husbandry operations were conducted
under
NIH 1996 Guidelines.
In T~ivo Contraceptive Effccacy in the Rabbit Model: For each fertility
trial, 24 does and 12 bucks were used. For each contraceptive test, the does
were
divided into 3 subgroups of 8; 1) control does; 2) GM-4 group and; 3) N-9
group.
1 S Semen was obtained from bucks (n = 12) of proven fertility via a prewarmed
(45°C)
artificial vagina immediately before use. Sperm count and motility was
assessed to
ensure that the males were ejaculating good quality semen. Prior to artificial
insemination, semen samples without the contamination of urine or gel were
pooled
and 0.5 ml (>30 x 10~ sperm/ml) aliquots were transferred to 1 ml tuberculin
20 syringes. Two ml of a GM-4 formulation or a commercial 2% N-9 formulation
(Gynol II; Ortho Pharmaceutical Corp., Raritan, NJ) was applied intravaginally
by
means of a 3 ml disposable plastic syringe. The doe was held in a supine
position
during the application of 2 ml of the test agent followed by the application
of semen
dose (0.5 ml) which was deposited within 1-2 min by inserting approximately 8
cm
25 of the syringe into the vagina for the delivery of the test agent. At the
time of
artificial insemination, ovulation was induced by an intravenous injection of
100 IU
of human chorionic gonadotropin (Sigma Chemical Co., St. Louis, MO) into the
marginal ear vein. After ovulation and artificial insemination, the does were
allowed
to complete their pregnancy (31 ~ 2 days). Pregnant does were transferred to
cages
30 containing nest boxes ( 16 x 12 x 6 in). The litter size, weight, fetal
length, and the
condition of each offspring at birth were recorded. The in vivo spermicidal
effect of
GM-4 formulation versus 2% N-9 formulation was assessed based on the level of
pregnancy reduction achieved in comparison to controls and the consistency of
this
response. The vaginal delivery/artificial insemination and pregnancy cycle was
35 repeated a second time.
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Rabbit Vaginal Irritation Test: For the vaginal irritation study, eleven
female rabbits were treated intravaginally with 1 ml of GM-4 (seven rabbits)
or 1
ml of GM-4-containing 4% N-9 (four rabbits), for 10 consecutive days. Animals
were sacrificed on day 1 l and the reproductive tract was examined grossly and
microscopically after completion of the study (Eckstein P, et al., J Reprod
Fertil,
1969;20:85-93.). The vaginal tissues were rapidly removed and parts of the
caudal,
mid, and distal regions of each vagina were fixed in 10% buffered formalin.
Tissues
were embedded in paraffin, sectioned at 4-6 pm and stained with hematoxylin
and
eosin and examined under x200 and x400 magnification using a Leica light
microscope (Milton Keynes, Buckinghamshire, UK) interfaced with an image
analysis system. The images were captured using the ImagePro Plus program
(Media Cybernetics, Silver Spring, MD) in conjunction with a 3CCD camera
(DAGE-MTI Inc., Michigan City, KS), and images were transferred to Adobe
Photoshop ~.5 software (Adobe Systems Inc., San Jose, CA) for observation and
analysis. Each of the three regions of vagina were examined for epithelial
ulceration, edema, leukocyte infiltration, and vascular congestion. The scores
were
assigned based on the scoring system of Eckstein et al., (Eckstein P, et al.,
JReprod
Fertil, 1969;20:85-93.) which was as follows: Individual score: 0 = none, 1 =
minimal, 2 = mild, 3 = moderate, 4 = intense irritation; Total score: <8
acceptable,
9-10 marginal, and >_11 unacceptable. Results were expressed as the mean ~ SD
values.
Thirteen-Week Toxicity Study in Mice: Twenty, female B6C3F1 mice
were allocated to two groups. The test group of 10 mice received 50 pl of the
GM-4
formulation intravaginally for 5 days per week for 13 consecutive weeks. Ten
mice
without intravaginal treatment served as the control group. The GM-4
formulation
was prepared weekly and the intravaginal treatment was performed inside a
microisolator. All animals were individually observed daily for signs of toxic
effects. Body weights were obtained before exposure (day 0), weekly during
exposure, and preceding sacrifice. At the end of the study, animals were
sacrificed
for pathologic and histopathologic examinations and determination of blood
chemistry. Hematological analyses were performed from blood obtained from ~
control and 5 test mice.
Hematology Parameters: Complete blood counts and differentials were
obtained using an Abbot CELL-DYN 3200 multiparameter, automated hematology
3~ analyzer (Abbot Laboratories, Abbot Park, IL) which was standardized for
mouse
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blood. This instrument uses flow cytometric techniques to provide the
hemograms
for anticoagulated whole blood samples: red blood cell count (RBC; 106/pl),
total
and differential leukocyte count (lymphocytes [LYM], neutrophils [NEU],
monocytes [MONO], eosinophils [EOS], and basophils [BASO] as 103/X1 or %),
hemoglobin concentration (HGB; g/dl), hematocrit (HCT; %), mean corpuscular
volume (MCV; fl), mean cell hemoglobin content (MCH; pg), mean cell hemoglobin
concentration (MCHC; g/dl), red cell distribution width (RDW; %), platelet
count
(PLT; 103/X1), and mean platelet volume (MPV; fl).
Clinical Chemistry Profiles: Biochemical analyses were performed using a
Beckman SYNCHRON CXSCE random access analyzer (Beckman Coulter Inc.,
Fullerton, CA). After 13 weeks of intravaginal application of GM-4, blood was
obtained from GM-treated and control mice in lithium heparin tubes and the
clarified plasma was used for the determination of serum/plasma levels of
total
protein (TP), albumin/globulin (ALB/G), blood urea nitrogen (BUN), creatinine
1 S (CRE), total cholesterol (CHO), triglycerides (TG), aspartate
aminotransferase
(AST), alanine aminotransferase (ALT), amylase (AMY), total bilirubin (TBIL),
glucose (GLU), and calcium (Ca), phosphorous (P), sodium (Na), potassium (K),
and chloride (Cl) using reagents and methods provided by the manufacturer.
Necropsy and Histopathology: Mice were killed after 13 weeks of
intravaginal exposure of GM-4 for complete necropsy evaluations. The thymus,
lungs, heart, liver, pancreas, spleen, kidneys, reproductive organs (ovaries,
uteri and
vaginal tissue) and brain from each of 10 mice were weighed at necropsy. Organ
weights were recorded as absolute weights and as a percentage of body weight.
The
above mentioned organs and the bone and bone marrow, large and small
intestine,
skeletal muscle, skin, spinal cord. and urinary bladder were fixed in 10%
buffered
formalin solution, trimmed, embedded in paraffin, sectioned at 4-6 Vim, and
stained
with hematoxylin and eosin. Complete histopathological examination of all
tissues
was performed on mice from the control and GM-4 group.
Statistical Analysis: Group means and standard deviations were calculated
from initial and terminal body weights, organ weights, hematology and clinical
chemistry parameters. Statistical significance of the differences between the
treated
group mean versus the control group was analyzed by a one-way analysis of
variance, followed by Dunnett's multiple comparison test using GraphPad Prism
software (San Diego, CA). The significance of differences in fertility between
the
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groups was analyzed by Fisher's exact test. Differences were considered
statistically
significant ifp < 0.05.
Cat Studies: FeT-J cell line is a feline T-cell line chronically infected with
the FIV strain Bangston (FIVB~g). Cats were challenged with FIV by inoculating
7x106 infected FeT-J cells mixed in 0.2 mL of infected culture fluid in the
vagina or
in the rectum 1 min after insertion of 0.4 mL of the WHI-07 gel formulation.
The
FIV load in the peripheral blood mononuclear cells (PBMC), lymph node (LN)
cells,
and bone marrow (BM) cells was measured by quantitative virus isolation and
PCR
analysis, as previously described (Rey MA et al., Biochem Biophys. Res.
Commun.
1984, 121:126-33; Diehl LJ et al., J Virol 1995, 69:2328-32; Greene WK et al.,
Arch Virol 1993, 133:51-62; Okada S et al., AIDS Res Hum Retroviruses 1994,
10:1739-46; Tellier MC et al., Vet Microbiol 1997, 57:1-11). The cells from
treated
and untreated cats ( 5x106 cells/culture) were cocultured for 3 weeks with
5x106 T-
cell enriched FBMCs from specific pathogen-free (SPF) cats in a total volume
of 5
ml in a 25 cm2 flask. Culture supernatants were harvested and cells were
resuspended in fresh culture media every 3 days. Viral production was
determined
by measuring the levels of RT activities in the culture supernatants and
examining
the cells for proviral DNA by FIV gag-specific and env-specific PCR at the
termination of cultures (Diehl LJ et al., J Virol 1995, 69:2328-32; Greene WK
et
al., Arch Virol 1993, 133:51-62; Okada S et al., AIDS Res Hum Retroviruses
1994,
10:1739-46). Serum samples were also examined by standard Western blot
analysis
for the presence of FIV core antigen p25. Cats were considered FIV positive if
one
of the following criteria was met during the 18-week observation period: (1)
Serum
samples from two different bleeding dates were positive by Western blot
analysis;
(2) A single Western blot result and a single virus isolation test from a
different
bleeding date were positive, with or without a positive PCR test; (3) PBMC,
lymph
node or bone marrow cells from two different bleeding dates were positive by
virus
isolation test; or (4) mononuclear cells from two different bleeding dates
were
positive by PCR with the same tissue source.
Results
In Vivo Contraceptive Activity of GM-4 versus N-9 Formulation iu the
Rabbit Model: Because of the rapid spermicidal activity of GM-4 formulation,
we
performed in vivo contraceptive efficacy studies of GM-4 formulation in the
standard rabbit model. Gynol II, a commercial contraceptive containing 2% N-9,
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was tested in the same way for comparison. Ovulated NZW rabbits were given
intravaginal application of GM-4 formulation or N-9 formulation immediately
prior
(< 2 min) to artificial insemination with fresh pooled semen and the females
were
allowed to complete their pregnancy. The efficacy of GM-4 formulation versus
Gynol II for preventing pregnancy in the rabbit model are summarized in Table
13.
In the control group, 15 out of 16 (93.7%) rabbits artificially inseminated
became
pregnant and delivered a total of 123 newborn rabbits. By contrast, none of
the 16
rabbits given GM-4 formulation prior to artificial insemination became
pregnant (p
< 0.0001, Fisher's exact test). Whereas rabbits given Gynol II, 5 out of 16
(31.2%)
rabbits became pregnant (p = 0.0006) and delivered a total of 34 newborn
rabbits.
Thus, the GM-4 formulation was far more effective than Gynol II as a vaginal
spermicidal contraceptive ( 100% vs 68.7%, p < 0.05, Fisher's exact test).
Table 13
Fertility of female rabbits after artificial insemination/ovulation induction
with
and without intravaginal application of GM-4 formulation or Gynol II
containing 2% N-9
1 reatment No. of does o. of does fertileLitter size
inseminated (%)
one 16 15 93.7 123
GM-4 16 0 0 0
~
G nol II 2%-N-916 5 31.2 34
Aliquots (0.5 ml) of fresh, pooled semen obtained from fertile bucks (n = 12)
were used to artificially inseminate the does within 1-2 min following
intravaginal application of 2 ml of GM-4 or N-9 formulation. Does were
induced to ovulate by an intravenous injection of 100 IU of hCG and allowed to
complete term pregnancy.
Significantly different from control by Fisher's exact test, (p < 0.0001 )
Significantly different from control by Fisher's exact test, (p = 0.0006)
Lack of Vaginal Irritation from GM-4 in the Rabbit Model: Histological
evaluation of three different regions of the vaginal tissue after daily
intravaginal
application of GM-4 for 10 consecutive days showed lack of significant vaginal
irritation in all seven rabbits examined (mean individual scores 0-l; total
score 2,
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range 0-3) [Table 14J. In contrast, all rabbits treated with 4% N-9 had
epithelial
ulceration, edema, leukocyte influx, and vascular congestion characteristic of
inflammation (mean individual scores 1-3; total score 9, range 7 to 11) as
quantitated by histological scoring according to the method of Eckstein et al
(Eckstein P, et al., JReprod Fertil, 1969;20:85-93.). Figure 4 shows the
representative vaginal section from a GM-4-treated rabbit which showed intact
vaginal epithelium when compared with the vaginal section of a N-9-treated
rabbit
which revealed disruption of the epithelial lining, and an inflammatory
response
with influx of leukocytes, consistent with previously published observations
in rats
10 (Tryphonas L, et al., Toxicol Lett, 1984;20:289-95.).
Table 14 Scoring of histological changes in the rabbit vaginal tissue after
10 days of intravaginal application of GM-4 formulation with and without 4%
N-9
GM-4 GM-4 + 4% N-9
(n W) (n - 4)
Epithelial ulceration 0* 3 + 2*~'~
Lamina propria thickness 1 ~ 1 2 ~ 1
Leukocyte Infiltration 1 ~ 1 3 ~ 2
Vascular congestion 0 1 ~ 1
Total score 2 ~ 1~ 9 ~ 2
Seven rabbits were administered intravaginally with 1 ml of GM-4 and 4 rabbits
were exposed to GM-4 containing 4% N-9.
Mean ~ SD values representing the caudal, middle, and distal sections of
vagina
from each rabbit.
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~Semiquantitative scoring based on Eckstein et al [35]. Individual score: 0 =
none,
1 = minimal. 2 = mild, 3 = moderate, 4 = intense. Total score: <8 acceptable,
9-10 marginal, and >-11 unacceptable.
Lack of Systemic Toxicity from GM-4 in Mice: Female B6C3F 1 mice (n
= 10) were treated with intravaginal application of GM-4, 5 days per week, for
13
consecutive weeks. Mortality did not occur and there were no clinical signs
attributed to intravaginal exposure of GM-4 throughout these studies. All
animals
were clinically healthy at the end of the study. Mean body weight gain and
final
mean body weight of control mice (27.4 ~ 1.6 g; n = I 0) and test mice (28.0 ~
3.0 g;
n = 10) exposed to GM-4 formulation were similar (Figure 5).
Complete blood counts of mice revealed no biologically significant
differences between GM-4 -treated and control mice. The values of hematologic
parameters for red cell, leukocyte, lymphocyte, platelet counts, and
hemoglobin were
within normal limits (Table 15). Analysis of blood chemistry parameters for
female
mice revealed no significant treatment-related differences between GM-4-
treated
and control groups (Table 16). The kidney function (BUN and CRE), liver
function
(TBIL, AST, ALT, ALB/G, GLU, and TG), pancreas function (AMY and GLU), and
nutritional status (TP), were not affected adversely by repeated intravaginal
exposure
to GM-4 formulation.
Table 15 Hematological Findings for B6C3F1 Mice Given GM-4
Intravaginally for 13 Weeks
Parameter Control GM-4
RBC (x 104/p.l) 949 ~ 62* 905 ~ 61
WBC (x 104/~l) 2.6 ~ 1.2 2.0 ~ 0.7
LYM (%) 76.3 ~ 17.7 85.4 ~ 5.6
NEU (%) 3.0 ~ 1.2 2.5 ~ 0.8
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Others (%) ~ 20.6 ~ 17.9 19.8 ~ 14.9
HGB (g/dl) 14.3 ~ 0.8 13.5 ~ 0.8
HCT (%) 54.3 ~ 3.5 51.4 ~ 3.0
MCV (fl) 57.2 ~ 1.6 56.4 ~ 0.8
MCH (pg) 15.1 ~ 0.2 15.0 ~ 0.1
MCHC (g/dl) 26.4 ~ 0.9 26.4 ~ 0.4
W' 9.2 ~ 3.5 8.8 ~ 1.9
PLT (x 104/~.~1) 358 ~ 226 413 ~ 327
MPV (fl) 7.4 ~ 2.7 6.2 ~ 0.7
Mean ~ SD for groups of 5 mice.
Others = MONO, EOS, AND BASO.
RBC, red blood cells; WBC, white blood cells; LYM, lymphocytes;
NEU, neutrophils; MONO, monocytes; EOS, eosinophils; BASO,
basophils; HGB, hemoglobin concentration; HCT, hematocrit; MCV,
mean corpuscular volume; MCH, mean cell hemoglobin; MCHC,
mean cell hemoglobin concentration; RDW, red cell distribution
width; PLT, platelets; MPV, mean platelet volume.
Table 16 Blood Chemistry Profiles for B(,C3F1 Mice Given GM-4
Intravaginally for 13 Weeks
Parameter Control GM-4
TP g/dl 4.8 ~ 0.1 * 4.8 ~ 0.1
ALB/G g/dl 3.6 ~ 0.2 3.8 ~ 0.3
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BUN mg/dl 24 2 26 4
CRE mg/dl 0.25 0.05 0.27 0.04
CHO mg/dl 109 6 104 6
TG mg/dl 174 38 166 33
AST IU/1 210 138 315 206
ALT IU/1 125 87 103 89
AMY IU/1 1783 134 1667 377
TBIL mg/dl 0.16 0.05 0.18 0.06
GLU mg/dl 325 23 310 50
Ca mg/dl 9.2 0.2 8.6 0.7
P mg/dl 6.9 1.4 6.4 1.2
Na mg/dl 152 2 150 3
K mg/dl 3.9 0.4 5.2 1.4~
Cl mg/dl 109 1 110 2
Mean SD for
groups of
8 mice.
Significantly
different
from control
group (p
< 0.05).
13-Week NecropsylOrgan
Weights and
Histopathology:
Table 17
summarizes the terminal bodyt, terminal
weigh absolute and
relative organ
weights
observed at week study. tically significant
the conclusion No statis
of the 13-
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differences were observed between the absolute and relative organ weights of
test
versus control mice. Microscopic examination of bone and bone marrow, brain,
gut,
heart, kidney, liver, lung, ovaries, pancreas, skeletal muscle. skin, spinal
cord,
spleen, urinary bladder, uterus, and vaginal specimens taken from the study
animals
did not reveal any treatment-related lesions (data not shown). No
histopathological
lesions were observed in the ovarian, uterine and vaginal tissues of GM-4-
treated
mice which suggests lack of toxicity to repeated intravaginal exposure of the
lipophilic and spermicidal GM-4 formulation.
Table 17: Absolute and Relative Organ Weights of B6C3F1 Mice Given
GM-4 Intravaginally for 13 Weeks
Control GM-4
Organ Absolute Relative Absolute Relative
(g) (g%) (g) (g%)
Terminal 27.4 ~ 1.6* 28.0 ~ 3.0*
Body weight
Thymus 0.09~0.01* 0.32~0.03 0.10~0.03* 0.35~0.10
Lung 0.28 ~ 0.02 1.02 ~ 0.07 0.29 ~ 0.03 1.03 ~ 0.10
Heart 0.14 ~ 0.01 0.51 ~ 0.03 0.13 ~ 0.01 0.46 ~ 0.03
Liver 1.62 ~ 0.13 5.91 ~ 0.47 1.73 ~ 0.22 6.17 ~ 0.78
Pancreas 0.15 ~ 0.02 0.54 ~ 0.07 0.14 ~ 0.03 0.49 ~ 0.10
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Spleen 0.12 ~ 0.02 0.43 ~ 0.07 0.10 ~ 0.01 0.35 ~ 0.03
Rep Orgy 0.17 ~ 0.02 0.62 ~ 0.07 0.17 ~ 0.04 0.60 ~ 0.14
Kidney 0.18 ~ 0.01 0.65 ~ 0.03 0.17 ~ 0.01 0.60 ~ 0.03
Brain 0.55 ~ 0.02 2.00 ~ 0.07 0.53 ~ 0.04 1.89 ~ 0.14
Mean ~ SD for groups of 10 mice.
Reproductive organs (ovaries, uteri, and vagina).
5 WHI-07/GM-4 Gel Formulation Prevents Vaginal and Rectal FIV
Transmission in Cats.
A total of 10 cats, including 5 control cats and 5 cats treated with WHI-
07(2%)/GM-4 vaginal gel formulation were challenged with an intravaginal
inoculum of 7x106 FIVB~g infected FeT-J cells mixed in 0.2 mL of infected
10 culture fluid. As shown in Table 18, WHI-07/GM-4 formulation provided 60%
protection against FIV. Similarly, WHI-07/GM-4 was also able to prevent the
transrectal transmission of FIV (Table 19).
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Table 18 WHI-07(2%)/GM-4
Gel Formulation
Prevents
Vaginal
Transmission
of FIV
in Cats
Cat No. Treatment WB Virus IsolationPCR Overall Protection
SerumPBMC PBMC Conclusion Rate
1 None 3X 3X 3X +
2 None SX 4X 2X +
3 None 1 2X 2X + Not applicable
X
4 None SX SX 6X +
S None 4X 2X 2X +
6 WHI-07/GM-40 0 0 -
7 WHI-07/GM-40 0 0 -
8 WHI-07!GM-4SX SX 4X +
9 WHI--07/GM-40 0 0 -
10 WHI-07/GM-4SX 6X SX +
The FIV status of cats was examined at 0, 3, 6, 9, 12, 15, and 18 weeks post
intravaginal FIV challenge. The number of different assessment times with
positive
test results is indicated for each assay. WB: Western blot; PCR: polymerase
chain
reaction; PBMC: peripheral blood mononuclear cells.
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Table 19 WHI-07(2%)/GM-4 Gel Formulation Prevents Rectal
Transmission of FIV in Cats
Cat No. Treatment WB Virus Isolation PCR Overall Infection
Serum PBMC PBMC Conclusion Rate
1 None 6X 6X 6X +
2 None 0 0 0 - 67%
3 None 2X 3X 4X +
4 WHI-07/GM-4 0 0 0 -
5 WHI-07/GM-4 0 0 0 - 0%
6 WHI-07/GM-4 0 0 0 -
The FIV status of cats was examined at 0, 3, 6, 9, 12, 15, and 18 weeks post
intrarectal FIV challenge. The number of different assessment times with
positive
test results is indicated for each assay. WB: Western blot; PCR: polymerase
chain
reaction; PBMC: peripheral blood mononuclear cells.
Discussion:
The first objective of our studies was to determine the in vivo contraceptive
efficacy of spermicidal GM-4. Since the rabbit provides a standard animal
model
for testing vaginal agents for antifertility activity (Castle PE, et ai.,
l3iol Reprod,
1997;56:153-9.; Castle PE, et al., Contraception, 1998;58:51-60.), we tested
the
ability of intravaginally applied GM-4 to prevent pregnancy in ovulated
rabbits. We
confirmed that vaginal delivery of GM-4 formulation prior to artificial
insemination
can prevent pregnancy in the rabbit. Our in vivo contraceptive efficacy
studies
included term pregnancy as well as the analysis of normalcy of the resulting
pregnancies. The GM-4 formulation showed remarkable contraceptive activity in
the rigorous rabbit model. In two separate fertility trials, a 100%
contraceptive
effect was obtained despite the fact that the rabbit ejaculate used contained
>1000-
fold larger inseminating doses than in humans (Castle PE, et al., Biol Reprod,
1997;56:153-9.). To our knowledge, these experiments are the first to
demonstrate
the in vivo contraceptive efficacy of a GM formulation prepared from commonly
used pharmaceutical excipients. The 100% contraceptive efficacy obtained with
GM-4 is most likely due to rapid spreadability of GM-4 across the vaginal
mucosa
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as well as to its rapid spermicidal activity. In contrast, the contraceptive
effect of N
9 has been shown to be highly dependent on the time interval between
delivering the
agent to the vagina and coitus or artificial insemination. It takes several
minutes for
N-9 gel to distribute in the rabbit vagina (Castle PE, et al., Contraception,
1998;58:51-60.). Thus, gels may be slower to mix with vaginal secretions than
the
GM-4 formulation. Therefore, a large excess of N-9 (400-fold greater dose) is
required to achieve in vivo contraceptive activity (Castle PE, et al.,
Contraception,
1998;58:51-60.). In fact, in over-the-counter formulations, N-9 is being used
at
concentrations of 2 to 6% in creams and gels, 12% in foams and as high as 18%
in
condom lubricants. The partial (68.7%) contraceptive effect of a commercial 2%
N-
9 gel observed in our study when compared with 100% efficacy of GM-4 is in
agreement with the high contraceptive failure rates reported for N-9 (Trussell
J, et
al. Stud Fam Plann, 1987;18:237-83.; Kulig JW, Ped Clinic North Am.
1989;36:717-30.; Raymond E, et al., Obstet Gynecol, 1999;93:896-903.). Our
studies suggest that this is most likely due to incomplete mixing of semen
with N-9
gel or inadequate distribution of the agent throughout the vagina.
The second objective of these studies was to determine the toxic effects, if
any, resulting from repeated intravaginal application of spermicidal GM-4.
Because
of the potent in vitro and in vivo spermicidal activity of GM-4 formulation,
it was
necessary to evaluate the toxicity to vaginal mucosa particularly in the
rabbit vaginal
irritation test. In the rabbit vaginal tolerance test, the GM-4 formulation
lacked
mucosal toxicity in contrast to 4%-N-9-containing GM-4 formulation after daily
application for 10 days. Our results clearly demonstrated that the GM-4 is not
damaging to vaginal mucosa of the rabbit despite the fact that it was a potent
spermicidal agent when added to human or rabbit semen.
The spermicidal components used for GM-4 formulation are non-toxic
solubilizers for lipophilic drugs used in the preparation of a variety of
topical, oral,
and injectable medications. Cremophor EL (polyethoxylated castor oil),
phospholipon 90G (purified soya lecithin), PEG 200, propylene glycol and
Captex
300 (medium chain triglyceride), are widely used parenteral vehicles as non-
toxic
solubilizers for lipophilic drugs and vitamins (Castle PE, et al.,
Contraception,
1998;58:51-60.; Lundberg BB, JPharm Pharmacol, 1997;49:16-21.) Cremophor
EL when used up to 10% w/v did not cause any apparent membrane damage to cell
monolayers and did not cause lysis of human leukemic cells (Woodcock DM, et
al.,
Cancer Res, 1990;50:4199-203.; Nerurkar MM, et al., Pharmaceutical Research,
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1996;13:528-34.). These components by themselves were not spermicidal in human
semen. Therefore, unlike the currently used non-ionic and cationic detergent
spermicides, the submicron particle-based GM-4 formulation is not likely to
cause
harmful side effects following repetitive intravaginal application.
In short-term toxicity studies, intravaginal administration of the lipophilic
and spermicidal GM-4, to female B6C3F1 mice for 13 weeks displayed no adverse
effects on survival, growth, hematological, clinical chemistries, absolute or
relative
organ weights and histopathology. The kidney, liver, and pancreas function as
well
as the nutritional status were not affected adversely by GM-4 formulation
exposure.
Based on the preclinical results reported here, we are hopeful that repetitive
intravaginal application of GM-4 will have no significant adverse systemic
side
effects in clinical settings. Experiments to formally test the safety of the
intravaginally applied GM-4 on the long-term health and reproductive
performance
of test animal species are currently in progress.
Conclusion
Within the above examples we described the in vitro and in vivo spermicidal
activity and safety of a novel pharmaceutical formulation, in the form of a
gel-
microemulsion GM, which contains common pharmaceutical excipients as the
active
ingredients. In some embodiments of the Examples, drug solubilizing agents,
for
example Cremophor EL and Phospholipon 90G, are contemplated as the active
ingredients since these agents were spermicidal against highly motile fraction
of
sperm. Although, the individual components of GM-4 formulation alone lacked
spermicidal activity in semen, the GM-4 formulation containing all eight
pharmacological excipients rapidly inactivated sperm in human semen. The lack
of
cytotoxicity of individual components of GM-4 in human semen and their
synergestic spermicidal property in a GM formulation shows uniqe clinical
potential
to formulate them as the active ingredients for a novel and effective vaginal
contraceptive.
The microemulsion-based lipophilic and vaginal spermicide, GM-4, appears
to offer several benefits for vaginal delivery including increased absorption,
improved contraceptive efficacy, and decreased toxicity. Under the described
conditions of its intended use, a 13-week intravaginal application of GM-4
formulation in B6C3F1 mice did not result in systemic toxicity and no other
specific
target organs were identified. Therefore, the spermicidal GM-4 formulation
shows
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unique clinical potential to become a clinically useful vaginal contraceptive
for
preventing the sexual transmission of STDs while preventing unwanted
pregnancies.
As a potent contraceptive agent which is inexpensive and devoid of mucosal
toxicity, the lipophilic GM-4 formulation meets the criteria for a vaginal
spermicide
and warrant further evaluation in vivo in humans.
In addition. this non-toxic lipophilic gel-microemulsion formulation may
also be useful for intravaginal application of anti-microbial agents to
prevent the
sexual transmission of diseases such as AIDS, genital herpes, gonorrhea and
chlamydia.
All publications, patents, and patent documents described herein are
incorporated by reference as if fully set forth. The invention described
herein may
be modified to include alternative embodiments. All such obvious alternatives
are
within the spirit and scope of the invention, as claimed below.
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