Note: Descriptions are shown in the official language in which they were submitted.
CA 02321406 2000-11-21
P A TFTvIT
Attornev_ Docket No.: 23072-l 14100US
UC Case No. 2000-294-1
RESTORATION OF PERTURBED BARRIER
FUNCTION BY APPLICATION OF
ANTIANDROGENS
STATEMENT AS TO R(GHTS TO INVENTIONS MADE UNDER
FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT
This invention was made with Government support by the Veterans
Administration Gram: Nos. HD 29706 and AR 19098. awarded by the National
Institutes of
Health. The Government has certain rights in this invention.
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention resides in the technical field of formulations for the
treatment
of skin conditions, and relates in particular to the treatment of subjects
suffering from skin or
mucous membrane diseases or disorders that display disruptions of the barrier
function.
2. Description of the Prior Art
The cutaneous permeability barrier resides in the outermost layer of the
epidermis, the stratum corneum (SC). The SC is a highly resilient tissue
organized into a
unique two-compartment system of nucleated corneoc~~tes embedded in a lipid-
enriched,
intercellular matrix. ~,~~hich is enriched in ceramides, cholesterol. and free
fatty acids. This
hydrophobic mixture is organized into stacks of broad lamellar bilayers, which
mediate both
2(1 transcutaneous water loss and the l7ercutaneous absorption of xenobiotics.
Acute and chronic
perturbations in the permeability ban-ier stimulate a coordinated lipid
synthetic and secretory
response in the underlying epidernais that leads to rapid restoration of
barrier homeostasis.
Yet. little is known about the reguiation of epidermal lipid metabolism by
circulating
hormones or other biological response modifiers. Both male primates and
rodents exhibit
more robust cutaneous lipid synthesis than age-matched females. Moreover,
epidermis/keratinoc~~tes express androgen receptors and possess the enzymatic
apparatus to
convert testosterone to dihydrotestosterone. Yet, priior studies failed to
discern gender-related
differences in human barrier function (Reed, .1.T., et al., Archiv. Dermatol.
131:1134-1 I38
(1995)).
CA 02321406 2000-11-21
In contrast, sex hormones exert important influences on the latest stages of
development of the permeability barrier in utero (Williams, M.L., et al., J.
Invest Dermatol.
Symposium Proceedings 3:75-79 (19!8)). Whereas exogenous estrogens accelerate
barrier
ontogenesis both in utero and in grovr~th-factor-free explant cultures,
androgens retard the
kinetics of barrier ont:ogenesis (Hanle;y, K., et al., J. Clin. Invest.
97:2576-2584 (1996)).
Moreover, male fetuses display a delay in barrier development vs. female
littermates, an
effect that is reversible when pregnant mothers are treated with the
testosterone receptor
antagonist, flutamide (Hanley, K., et .al., J. Clin. Invest. 97:2576-2584
(1996)). Although
decreased survival rates in male premature infants have been ascribed to
increased respiratory
distress rates (Khoury, M.J., et al., ,Arn. J. Obstet. Gynecol. 151:772-782
(1985)), a negative
influence of androgens on skin development could explain ongoing differences
in premature
human male vs. female infants, even when treated with surfactant replacement
therapy
(Allen, M.C., et al., l~~ Engl. J. Mee:f. :329:1597-1601 (1993); La Pine,
T.R., et al., Pediatrics
96:479-483 (1995)).
SUMMARY OF THE INVENTION
It has now been discovered that the formation of a functional epidermal
barrier
is accelerated by the administration of antiandrogens. While the prior art has
addressed the
administration of antiandrogens to pregnant female rats to observe its effect
on the skin of the
fetal rat, this invention resides in the discovery that antiandrogens are
effective on
mammalian subjects i.n general, and also that they arcs effective as topical
treatments.
Included among the definition of anti~androgens are (i) antagonists of the
androgen receptor,
(ii) agents that suppress androgen production at the level of the hypothalamic-
pituitary axis,
and (iii) inhibitors of the conversion of testosterone to dihydrotestosterone.
The invention
thus resides in the use of antiandrogens the treatment of mammalian skin
suffering from a
deficient or perturbed barrier function. The invention is particularly useful
in the treatment of
premature infants, as well as various forms of dermatitis, inflammation to
mucous
membranes, ulcers and erosions, and any physiological condition or disease in
which a
perturbed barrier function is present.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graphical representation of a study showing the improvement of
barrier recovery after surgical castration of male mice.
2
CA 02321406 2000-11-21
FIG. 2 is a graphical representation of a study showing the worsening of
barrier recovery in castrated male rnic.e after systemic testosterone
replacement.
FIG. 3 is a graphical representation of a study showing the improvement of
barrier recovery in male mice after systemic flutamide treatment.
FIG. 4 is a graphical representation of a study showing the improvement of
barrier recovery in male mice after topical flutamide treatment.
FIG. 5 is a graphical representation of a study showing that barrier recovery
is
faster in pre-pubertal mice than it is in young adult male mice.
FIG. 6 is a graphical representation of a study showing the changes in barrier
recovery in relation to testosterone therapy in a hypogonadal human male.
DETAILED DESCRIPTION OF T'HE INVENTION AND
PREFERRED EMBODIMENTS
Antagonists of the androgen receptor are well known in the pharmaceutical
industry for uses other that those provided by the present invention. Included
among these
antagonists are those of the formula
R2
O
R~-_ ~ ~ N , _R3
in which:
R' is a. CZ-Cb alkyl, CS-C~ cycloalkyl, CS-C~ cycloalkyl substituted with C1-
C6
alkyl, or C2-C6 alkyl substituted with one or more of the following:
halogen, hydroxyl, phenylsulfonyl, and halogen-substituted
phenylsulfonyl, and
RZ and R3 are either the same or different, and being either nitro,
trifluoromethyll, halogen, cyano, C1-C3 alkyl, or C1-C3 alkoxy.
Certain subgenera within the scope of this formula are preferred. For example,
R' is
preferably CZ-C6 alkyl or CZ-C6 alkyl substituted with one or more of halogen,
hydroxyl,
phenylsulfonyl, and halogen-substituted phenylsulfo:nyl. More preferably, R'
is CZ-C6 alkyl
or CZ-C6 alkyl substituted with hydroxyl, phenylsulfonyl, halogen-substituted
phenylsulfonyl,
or a combination of hydroxyl and halogen-substituted phenylsulfonyl.
Antiandrogens of
these structures are disclosed in United States Patent No. 3,847,988 and
United States Patent
CA 02321406 2000-11-21
No. 4,636,505. The disclosures of both of these patents are incorporated by
reference.
Examples of specific antiandrogens within this formula are flutamide, whose
scientific name
is 2-methyl-N-[4-nitro-3-(trifluorome;thyl)phenyl]propanamide and whose
molecular formula
is
CF,
O
CH3
CH-~~ N O
CH3
and bicalutamide, whose scientific name is (~)-N-[4-cyano-3-
(trifluoromethyl)phenyl]-3-[(4-
fluorophenyl)sulfonyl]-2-hydroxy-2-methyl propanamide and whose structural
formula is
O O HO CH3
H
S. , N \ CF3
O
F CN
Antiandrogens that are inhibitors of the conversion of testosterone to
dihydrotestosterone are exemplified by certain substituted androstenones and
certain
pregnadienediones. One example is megestrol acetate, whose scientific name is
17a-
hydroxy-6-methylpregna-4,6-dime-3,20-dione acetate and whose structural
formula is
CH3
C O O
C CH3
O
Another example is finasteride, whose scientific name is (Sa,, 17(3)-N-(1,1-
dimethylethyl)-3-
oxo-4-azaandrost-1-ene-17-carboxarr~ide, and whose structural formula is
4
CA 02321406 2000-11-21
H
C/N\C CH
( z)z
Disclosures of compounds of this general structure are found in United States
Patents Nos.
3,356,573 and 3,400,137, the disclosures of both of which are incorporated
herein by
reference for all legal purposes capable of being served thereby.
Antiandrogens that are, agents that suppress androgen production at the level
of the hypothalamic-pituitary axis are exemplified by LHRH (leutinizing
hormone releasing
hormone) and its analogues, whose structures are known to those skilled in the
art.
In the practice of this invention, the antiandrogens will be administered as
active ingredients in a formulation thief is pharmaceutically acceptable for
the particular
means of administration desired. In certain embodiments of this invention, the
formulation is
intended for topical administration, while in others the formulation is
intended for systemic
administration or for intralesional administration. These formulations may or
may not
contain a vehicle, although the use of a vehicle is preferred. Preferred
vehicles are non-lipid
vehicles, particular water-miscible liquids or mixtures of water-miscible
liquids. Examples
of such liquids are methanol, ethanol., isopropanol, ethylene glycol,
propylene glycol, and
butylene glycol. The most appropriate vehicle will depend on the particular
type of
administration that the formulation is designed for.
The concentration of active ingredient in the formulation is not critical to
the
invention and can vary widely, depending on the vehicle, the type and stage of
condition
being treated, the site where treatment is being administered, and similar
considerations. In
most applications, the optimum concentration will range from about 10 p.g/mL
to about
100 pg/mL. In general, however, the optimum amounts for any given application
or active
agent will be readily determinable by routine experimentation.
The formulation can be a lotion, a solution, a gel, a cream, an emollient
cream,
an unguent, a spray, or any other .form that will permit topical application.
The formulation
O 1V
H
H
CA 02321406 2000-11-21
may also contain one or more agents that promote the spreading of the
formulation over the
affected area, but are otherwise biologically inactive. Examples of these
agents are
surfactants, humectants, wetting agents, emulsifiers and propellants.
Examples of skin conditions that are susceptible to treatment by the practice
of
this invention are:
Barrier Abnormality Representing Primary Process
Chronological aging (epidermis)
Photoaging (epidermis)
Atopic; dermatitis
The skin of premature infants of gestational age less than 33 weeks
Cheilitis
RXLI, Gaucher's (I:f); Neimann-Pick (I)
Burns
Ulcers (ischemic, vascular, diabetic)
Blisters/bullous disorders (friction, keratin abnormalities)
Diabetes
Barrier Abnormality Triggers Imrrmnologic Abnormality
Psoriasis (plaque type)
Irritant contact derrrcatitis (acute)
Occupational dermatitis (acute)
Diaper dermatitis
Allergic contact dermatitis
Barrier Abnormality Sustains Pathophysiology
Atopic: dermatitis
Irritant contact dermatitis (chronic)
Occupational dermatitis (chronic)
Psoriasis
Hypertrophic scars and keloids
Cheilitis
Lamellar ichthyoses; >=?pidermolytic hyperkeratosis; Harlequin ichthyosis
Immunologic Abnormality Triggers Barrier Abnormality
Phytotoxic reactions
Bullou.s allergic reactions
6
CA 02321406 2000-11-21
Erythrodermic, pust.ular, and guttate psoriasis
Optimal methods and frequency of administration will be readily apparent to
those skilled in the art or are capable of determination by routine
experimentation. Effective
results in most cases are achieved by topical application of a thin layer over
the affected area,
or the area where ones seeks to achieve the desired effect. Depending on the
condition being
addressed, its stage or degree, and whether application is done for
therapeutic or preventive
reasons, effective results are achieved with application rates ranging from
one application
every two or three days to four or more applications per day.
The invention is generally applicable to the treatment of the skin of
terrestrial
mammals, including for example humans, domestic pets, and livestock and other
farm
animals.
The following examples are offered for purposes of illustration, and are not
intended to limit or to define the invention. All literature citations in
these examples and
throughout this speci:frcation are incorporated herein by reference for all
legal purposes
capable of being served thereby.
EXAMPLES
Materials:
Castrated, sham-operated, and normal hairless male mice (Sk:hl) were
purchased from Charles River Laboratories (Philadelphia, PA). Adult mice, age
11-14
weeks, were used for all experiments. Serum testosterone levels were >1500
pg/mL in
control animals and less than 20 in castrated animals. Prepubertal male mice
were utilized
between four and five: weeks of age. Testosterone propionate and flutamide was
purchased
from Sigma Chemical Co. (St. Louis, MO). Subcutaneous testosterone and
flutamide were
solubilized in peanut oil. Topical testosterone and flutamide were solubilized
in propylene
glycol:ethanol (7:3 vols) at the final concentrations, volumes, areas and
routes of
administration described in the results and figure legends for each
experiment. Peanut oil and
propylene glycol:ethanol were used as vehicles, respectively.
A single 58-year old, hypogonadal male subject (post-transphenoidal
hypophysectomy in 1984) was studied at various points before and after
testosterone
replacement (testosterone cyprionate 200 mg IM at three-week intervals).
7
CA 02321406 2000-11-21
Measurements of Barrier Recovery and Stratum Corneum Integrity:
Trans-epidermal wader loss (TEWL) was measured with an electrolytic water
analyzer (Meeco, Warrington, PA). Barrier function was disrupted by sequential
cellophane
tape stripping until TEWL levels reached 3 to 8 mg/c:m2/h. TEWL was measured
immediately after barrier disruption a.nd at various time intervals after
acute disruption,
usually at one, three, six, and 12 hours after barrier disruption. Stratum
corneum integrity
was defined as the mean number o:F tape strippings required to abrogate the
barrier to >3
mg/cm2/h.
Lipid Synthesis Studies:
For the in vitro studies, animals were killed one hour after barrier
disruption
and skin samples of approximately 1 cm2 were incubated for two hours at
37°C in a 2-mL
solution of 10 mM ethylene diamine tetraacetic acid (EDTA) in Dulbecco's PBS,
calcium
and magnesium free, containing 40 pci [14C] acetate. After stopping the
reaction by
immersion in iced phosphate-buffered saline (PBS), the epidermis was separated
from the
dermis and the quantities of labeled fatty acids and cholesterol were
determined in the
epidermis after saponification, extraction, and thin layer chromatography
(Grubauer, G., et
al., J. Ljpid Res. 28:746-752 (1987); :Mao-Qiang, M., et al., J. Clin. Invest.
92:791-798
(1993)). Results were expressed as nanomoles incorporated per hour per gram of
epidermis.
Light Microscopy, Ultrastructural and Quantitative Morphometric Studies:
Skin biopsy samples vvere taken immediately before and three hours after tape
stripping of castrated mice, treated with either subcutaneous testosterone or
vehicle (n = three
from each group), and processed for Might and electron microscopy. Samples
were minced to
<0.5 mm3, fixed in modified Kaxnovsky's fixative overnight, and post-fixed in
either 0.5%
ruthenium tetroxide or 2% aqueous osmium tetroxide, both containing 1.5%
potassium
ferrocyanide, as described by Hou, S..Y.E., et al., J. Invest. Dermatol.
96:215-223 (1991).
After fixation, all samples were dehydrated in graded ethanol solutions, and
embedded in an
Epon-epoxy mixture. One-half micron sections, stained with toluidine blue,
were used for
light microscopic studies. Ultrathin ~cections were ea;amined, with or without
further
contrasting with lead citrate, in an electron microscope (Zeiss 10A, Carl
Zeiss, Thornwood,
NY) operated at 60 kV.
For quantitative studies, 10-15 pictures of the outer stratum granulosum (SG)
and the SG-SC interface at a constant original magnification of 1 S,OOOx were
selected at
8
CA 02321406 2000-11-21
random from each sample group. l.,a~mellar body (LB) density was calculated
using a point-
count intersect method (Elias, H., e;t al., Guide to Practical Stereology,
Basel:Karger (1983)).
A grid of test points was superimposed over non-overlapping regions of each
picture.
Intersect points over lamellar bodies vs. cytosol were totaled per cell and
for each
experimental group as a whole. Assignment of organelles as lamellar bodies
required the
presence of a trilaminar limiting merrcbrane; characteristic ellipsoidal
shape; and a 0.4-0.6-~m
long axis. The nucleus was excluded from calculations of cytosolic volume. The
volume
ratio of lamellar bodies in the outer SG layer comprises the number of LB
intersects/number
of cytosolic intersects x 100. The extent of lamellar body secretion was
quantified as the
cross-sectional area of the intercellular domain at the SG-SC junction/the
measured length in
p,m of the measured area. The cross-sectional area was determined by two
unrelated
methods: (a) by weighing paper overlays of the region (Rassner, U., et al.,
Tissue & Cell
31:489-498 ( 1999)); and (b) by integrating area measurement on the scanned
pictures with
NIH image software.
Statistical Analysis:
For statistical analyses, two-tailed Student's t Test and SPSS software (SPSS
Inc., Chicago, IL) were used. Data are expressed as mean ~ SEM with p<0.05
considered
significant.
RESULTS
To assess the impact of androgens on permeability barrier homeostasis, the
kinetics of barrier recovery in castrated vs. sham-operated hairless mice were
compared, with
barrier recovery studies performed eight weeks after castration or sham
operation. Barrier
recovery was assessed one, three, six, and 12 hours after acute disruption by
sequential tape
stripping. The results, expressed as mean ~ SEM, are shown in FIG. 1, which
indicates that
the kinetics of barrier recovery was accelerated significantly at all time
points in castrated
mice, with the greatest differences apparent at one and three bouts (FIG. 1; p
< 001 and <
0.05, respectively). Moreover, castrated animals displayed significantly
greater stratum
corneum integrity than testosterone-rc;plete, castrated and control animals.
The results are
shown in Table I.
9
CA 02321406 2000-11-21
TABLE I: STRATUM CORNEI1M INTEGRITY IN CASTRATED, TESTOSTERONE
REPLETE VS. CONTROL MICE
ANIMALS TAPE STRIPPINGS f SEMa
Non-Castrated 3.29 + 0.18b
(n = 7)
Castrated + Vehicle 4.27 + 0.14h
(n=11)
Castrated + Testosterone 2.27 ~ 0.14h
(n = 11;)
a Number of stripping,s required to abrogate barrier to TEWL >3 mg/cm2/h
b Differences between all groups are significant at p <0.001
These results show that surgically-induced hypogonadism leads to accelerated
barrier recovery and enhanced stratum corneum intei;rity in male mice.
To ascertain further wlhether accelerated barrier recovery after castration is
due
to androgen depletion, the effects of testosterone-repletion vs. vehicle-
repletion in castrated
mice were assessed by injecting castrated mice simultaneously with either
testosterone
propionate (5 mg/kg) diluted in peanut oil or peanut ;ail alone daily for
seven days, followed
by measurement of barrier recovery after tape stripping over a distant site on
the eighth day.
Whereas vehicle treatment did not influence barrier recovery rates,
testosterone
administration slowed barrier recovery at all time points (FIG. 2, showing
mean ~ SEM),
with the greatest difference at three hours (p< 0.001 ). These results show
that testosterone
reverses the acceleration in barrier recovery that occurs in castrated mice.
To determine whether the castration-induced acceleration of barrier recovery
is specific for surgical castration, or a more general attribute of
testosterone deficiency, the
effects of medical castration on barrier recovery in normal (non-castrated)
male mice were
assessed. For these studies, either the androgen-receptor antagonist,
flutamide (50 mg/kg), or
vehicle alone subcutaneously were administered once daily for seven days,
followed by
measurement of barrier recovery on the eighth day. ,4s seen in FIG. 3 (again
showing mean ~
SEM), barrier recovery accelerated at all time points in systemic flutamide-
treated animals.
In fact, the absolute recovery rates at each time point were virtually
identical for medical
castration to those in surgically-castraited animals. These results show that
testosterone
depletion has positive effects on perrrieability barrier homeostasis,
independent of the method
of production of hypogonadism.
CA 02321406 2000-11-21
To determine whether the negative effects of testosterone on the barrier are
due to systemic or cutaneous alterations produced by the hormone, a study was
performed to
determine whether the effects of testosterone are due to a direct effect of
the hormone on the
skin itself, measuring; barrier recovery after seven, once-daily applications
of flutamide (30
~,g/mL) to a 2.5 cm2 area on one flank of normal, non-castrated mice. The
contralateral flank
was treated daily with an equal volume of the propylene glycol:ethanol vehicle
alone.
Flutamide accelerated barrier recovery, while the vehicle applied to the
contralateral flank did
not alter barrier recovery rates relative to vehicle applied to non-flutamide-
treated mice (FIG.
4, showing mean ~ S EM)). Since flutamide-treatment only affected the treated
flank (the
contralateral flank did not change significantly), these results show that the
effects of
testosterone are due to local, rather than distant effects of the hormone.
A study was then performed to determine whether variations in endogenous
androgen levels are associated with pre- vs. post-pubertal differences in
barrier homeostasis.
Mice display low androgen levels while they are still pre-pubertal, i.e., less
than six weeks
old, with a post-pube~rtal increase in hormone levels ;zfter eight weeks of
age. As seen in FIG.
5, prepubertal mice (~E-5 weeks old) display more rapid barrier recovery rates
than do young
adult male mice (11-week-old) at botlh three and six hours after acute
disruption (p < 0.02).
Recovery rates in the eleven week-old mice again we're comparable to those in
the somewhat
older (12-14 week), non-castrated adults described above (see FIGS. 1-4).
These results
show that differences in male gonadal status are associated with post-natal,
developmental
changes in barrier function.
A study was then performed to ascertain whether the negative effects of
androgens are of potential relevance for humans. Barrier recovery kinetics
were assessed in
one hypogonadal subject receiving intermittent testosterone replacement.
Barrier recovery
measurements taken at times of the high testosterone period (two or three days
after muscular
injections of testosterone cyprionate, 200 mg) and low (immediately prior to
next injection).
Serum testosterone levels were taken at the same time. As seen in FIG. 6,
barrier recovery
rates were highest when serum testosl:erone levels approached their nadir,
while conversely,
the kinetics of recovery slowed in conjunction with high testosterone blood
levels. Although
all observations were from a single subject, they achieved statistical
significance (p = 0.011;
Student's t test applied to multiple me;asurements). These results indicate
that permeability
barrier homeostasis in humans also changes with alterations in serum
testosterone.
11
CA 02321406 2000-11-21
A series of studies was then conducted to identify potential mechanisms by
which androgens might compromise barrier homeostasis. These results are shown
in Table
II.
TABLE II: LIPID SYNTHESIS IN TESTOSTERONE- vs. VEHICLE-TREATED
('AST RATED l~rIICE
ANIMALS SYNTHESIS (nmoUg epidermal weight/h)
Castrated Chol BA TNS
+ Testosterone 3.40 i O.SSa 17.12 t 1.9b 4.56 ~ 0.82°
<0.01 <0.001 <0.02
+ Vehicle 1.37 ~ 0.23 6.72 t 0.60 1.59 t 0.29
Sham-Operated
+Testosterone 3.49 ~ 0.92a IS.66~4.336 4.31 ~ 1.260
<0.05 NS <0.05
+ Vehicle 1.21 t 0.25 6.95 ~ 0.85 1.40 ~ 0.30
a.b.c Differences are not significant; ZNS = total non-saponifiable lipids
The syntheses of both non-saponifiable epidermal lipids (primarily
cholesterol) and saponifiable epidermal lipids (fatty acids) were comparable
in castrated and
non-castrated animals. However, testosterone administration caused a
significant, but
equivalent increase in. epidermal lipid synthesis in both castrated and non-
castrated animals.
Since these results show that testosterone repletion stimulates epidermal
lipid synthesis,
modulations in lipid synthesis alone cannot explain the decline in barrier
homeostasis in
testosterone-replete animals.
Three ultrastructural rr~arkers of the lamellar body secretory system were
then
assessed as potential mechanisms that could explain the testosterone-induced
decline in
barrier recovery rates.. Both visual assessment in coded micrographs (observer-
blinded) and
quantitative stereological (morphornetric) measures were employed. Because the
morphology of castrated epidermis was comparable to that of untreated skin,
both
assessments were focused on baseline: morphology in testosterone-replete vs.
vehicle-treated
12
CA 02321406 2000-11-21
castrated mice, when TEWL rates were comparable, and on the morphology of
castrated,
testosterone-replete vs. vehicle-treated epidermis three hours after acute
barrier disruption
(when functional differences were maximal). Under basal conditions, a
reduction was
observed in the number of lamellae bodies (LB) in the cytosol of the cells in
SG layer in
testosterone-replete animals. These observations were supported by
quantitative
(stereological) measurements, which showed a significant reduction in the
volume fraction of
LB in the cytosol of outermost SG cells (Table II). Presumably as a result of
decreased LB
production, the amount of secreted contents at the stratum granulosum (SG)-SC
interface was
also reduced in testosterone-replete animals (Table I:f ). Further evidence of
reduced secretion
included the frequent presence of entombed LB within the cytosol of
testosterone-replete, but
not vehicle-treated.corneocytes. Furthermore, the processing of secreted LB
contents into
mature lamellar bilayers appeared to be delayed, as indicated by the
persistence of partially-
processed lamellar contents at the level of the SC 2-3 interface. Finally, as
a result of
decreased LB formation and secretion, the absolute quantities of extracellular
lamellae in the
SC interstices also appeared to be reduced, as evidence by decreased lamellae
between SC 1
and SC2.
The reductions in LB iEormation and secretion in testosterone-replete animals
were even more striking three hours after barrier disruption, observations
that were again
validated by quantitative studies (T'able II). Decreased LB secretion was
evidenced by a
diminution in the quantities of extracellular lamellae in testosterone-replete
animals, as well
as increased, intercellular lacunae in the SC interstice°s, which
displayed decreased lamellar
contents. Together, t:~hese results demonstrate a decrease in LB formation,
resulting in both
decreased secretion and a diminution in extracellular lamellar bilayers in
testosterone-replete
animals.
The foregoing is offered primarily for purposes of illustration. It will be
readily apparent to those skilled in the art that the concentrations,
operating conditions,
materials, procedural steps, and other parameters and protocols described
herein may be
further modified or substituted in various ways without departing from the
spirit and scope of
the invention.
13
