Note: Descriptions are shown in the official language in which they were submitted.
This invention xelate~s to the area of compositions
containing synthetic conjugated estroyens and to the fields of
compositions containing synthetic conjugated estrogens to be
employed for replacement therapy of estrogen deficiency
associated with menopausal syndrome, female hypogonadism
(hypogenitalism), amenorrhea, female castration, or primary
ovarian failure.
In U.S.. Patent 2,834,712, a process is described
for preparing a mixture of conjugated estrogens from the urine
of pregnant mares by absorption a.nd extraction. The mixture
consists primarily of water-soluble sulfates of a number of
conjugated estrogens including estrone, 17~-estradiol, equilin,
and equilenin. The mixture in the same proportion can also be
duplicated essentially with synthetic estrogens. Whether
synthetic or achieved from natural sources, mixtures of con-
jugated estrogens are applied by those skilled in the art
therapeutically to relieve mental and bodily complaints occurr-
ing in the menopausal syndrome.
In relatively crude extracts containing conjugated
estrogens from natural sources, natural but unknown stability
constituents are apparently present, since it has been found
that these products have a relatively high order of stability
as compared to more highly purified estrogenic compositions, or
those prepared from the synthetic conjugated estrogens. These
:~ natural conjugated estrogens are primarily derived from the
urine of pregnant mares. Unfortunately, this extraction pro-
: cess makes the natural con]ugated estrogens relatively expens-
ive and, accordingly, they are available in limited quantities.
The pure synthetic estrogenic conjugates, while ~
relatively inexpensive, have been found to be highly unstable, ~:
particularly in the presence of moisture. When moisture is
present, even to a slight extent, as is the case with most ~
tablets, an acid environment forms which quickly destroys the .: :
'
product. Obviously, the purified estrogenic conjugates are
unstable when carried in aqueous media such as might be em-
ployed in injectable preparations. ~le instability of the
estrogens is thought by those in the art to be due to hydroly-
sis which liberates hydrosulfuric acid and free estrogens which
are for the most part inactive in the intended applications.
To combat the hydrolysis problem, it was proposed
in U.S~ Patent 2,834,712 to add buffering agents which were
capable of maintaining the pH of aqueous estrogen solutions
at 6.5 to 7.5, with a preferance for a slightly alkaline pH.
While this approach might be satisfactory with conjugated
estrogens derived from natural sources, it has been found
that with synthetic estrogens, the presence of a buffer is
only sufficient to provide stability for approximately six
months when the estrogens are in dry tablet form. Unfortun- -
ately, it has been found that where the synthetic conjugates
are present in an aqueous carrier buffered between pH 6.5
and 7.5, substantial hydrolysis has been found to occur al-
most im~ediately. A substantial need arose - the need for a
synthetic conjugate which would not only be inexpensive but
would retard hydrolysis and be stable for periods of time for
beyond six months in order to give the corresponding pharma-
ceutical preparations acceptable shelf life.
The relevant prior art in the field of synthetic
conjugated estrogens for use in treating menopausal syndrome,
etcO, was unfortunately thin. It is to be emphasized that
several fields of art could be confused with the present fields
at issueD In steroid chemîstry, one field of art deals with
the conjugated estrogens which are water-soluble, and another
field deals in the chemistry of oil-soluble steroidsa One
field of art is concerned with conjugated estrogens used in
curing vitamin deficiencies for patients with general inade- -
-- 2 --
quate or broadly restricted diets (for example, see MODERN DRUGENCYCLOPEDIA AND THERAPEUTIC I~DEX, (9th Edition, 1963) at
280-281; 517-521, 553; 1112-1114, 1376, 1389-1390; 1441), while
another distant field of art is directed toward the use of con~
jugated estrogens for ~he replacement therapy (see Beall, U.S.
Patent 2,834,712~ of estrogen deficiency associated with meno-
pausal syndrome, female hypogonadism (hypogenitalism), amenorr-
rhea, female castration, or primary ovarian fallure. One art
might employ salts as preservatives in non-conjugated estrogens
10 (See MODERN DRUG, supra, at p~ 1441, TRI-GENIK , whereas another
art might employ the same as buffers for pH control. Chemists
within a particular field of art (in this instance the field
of synthetic and water-soluble conjugated estrogens for use
in treating menopausal syndrome, etc.~ do not pay the same
attention to art outside their own field for good reason -
generally the chemistry is so entirely different from field
of art to field of art as to render the other teachings in-
effectual.
While the following art will be discussed in some
detail, most of it falls outside the purview of the present
invention and is deemed by Applicant to be marginally, if at
all, relevant. MODERN DRUG ENCYCLOPEDIA A~ IERAPEUTIC INDEX
(9th Edition, 1963) at 280 - 281 contains a reference to
CL W ISOL GERIATRIC , which is a thexapeutic combination of
potent nutritional elements with steroids to meet the chall-
enge of waning metabolic e~ficiency in old patients, with
- vi-tamin deficiencies, having a conjugated equine estrogens
('PREMARIN '' by Ayerst Company) and a small frac-tion of Vit~nin
E in an amount faIling outside ~he ranges prescribecl by the
present invention. The vitamin E is present for purely thera-
peutic purposes, not as an antioxidant, as part of a "rnulti-
vitamin".
* - Trademark
_ 3 _
, ~
- -
See also PHYSICIANtS DESK REFERENCE at 584 ~1977, by Medical
Economics Co., C.E. Baker, Jr., Puba ) ~
Also, in MODERN DRUG at 517 is listed the prescrip-
tion of ESTOPHEROL TABLETS tPitman-Moore Company~ having
ethinyl estradiol and Vitamin Eo However, ESTOP~ROL TABLETS
do not contain a conjugated estrogen, and in fact ethinyl
estradiol is stable without Vitamin E in numerous oral contra-
ceptives. In the same art as ESTOPHEROL is ESTRADURIN
(Ayerst Company), employing a non-conjugated estrogen (poly-
estradiol phosphate) and a small amount OL nicotinamide as astabilizing element (MODERN DRUG at 518). A number of com-
panies market various ESTROGENIC SUBSTANCES (MODERN DRUG at
518-519), none of which mention the use of stabilizers.
MODERN DRUG at 553 lists FORMATRIX (Ayerst Company)
.. . . . ~
containing in each tablet 1.25 mg of conjugated estrogens
("Premarin ") and a very large amount of Vitamin C (not
intended as an antioxidant) for the treatment of protein
depletion and ascorbic acid deficiency (see PHYSICIAN'S DESK
REFERENCE, supra, at 587).
~ _
"Premarin " of Ayerst Company is a preparation of
orally-active, water-soluble and natural, conjugated estrogens
derived rom pregnant mares1 urine, with no mention of any
stabilizers being employed. MODERN DRUG, supra, at 1112-1114.
TESTROGYN , another preparation of Ascher Company,
contains estradiol and testosterone(not conjugated estrogens),
with sodium bisulfite as a preservative. MODER~ DRUG, supra,
1376.
TMEELIN (Parke-Davis Company) is a suspension of
estrogenic steroids employing suitable preservatives. MODERN
DRUG, supra, at 1389-13g0. TRX~GENIK ~Savage Company~ con-
tains the non-conjugated estrogens estradiol, progesterone,
and testosterone proprionate. A small amount of sodium meta-
* - Trademar~
- 4 -
bisulfite is added as a preservative.
U.S. Patent 2,324,348 (Anderson) teaches the use of
ascorbic acid as an antioxidant in the unrelated arts of soaps
and perfumes.~ No mention is made of employing Vitamin C in
conjugated estrogens.
Louis C. Schroeter, "Sulfurous Acid Salts as Pharma-
ceutical Antioxidants" ~50 J~ PHARMACEUTICAL SCIE~CÆS (No. 11)
pp. 891 et seq. (1961) is a general article on the chemistry
of the sulfurous acid salts as antioxidants. No mention i5
made of applying these salts to synthetic conjugated estrogens,
and Schroeter never teaches any particularities of employing
the salts, finding proper concentrations for applications con-
trolling pH, etc. (See also Lachman, "antioxidants and Chelat-
ing Agents as Stabilizers in Li~uid Dosage Forms", D&Cl at pp.
36-46 (196~).
Perhaps the most pertinent reference is British
Patent 806,779 to Scheringl which discloses a concentrated
aqueous solution of estrogens and certain acids (sodi~un estrone
sulphate, sulphurous acid and glacial acetic acid or lactic
acid) adjusted to a pH of 8.0 to 8.5, with buffers present to
prevent discoloration of the estrone solution for use in cos-
metic preparations.
Ludwig in U.S. Patent 3,666,865 ~1972) seeks to
stabilize trans-diethylstilbestrol ttrans - "DES"), a non-
conjugated estrogen, with phenolic antioxidants such as 2,4,5-
trihydroxybutyrophenone, urea, and sodium carbonate D ~0 men-
tion is made of an alkaline antioxidant formulation with the
DES. U.S~ Pat. 3,674,869 (1972) further claims to stabilize
trans-DES with sulfur-containing compounds, e.g., thiophenol
or ammonium sulfide~
Beall in U.S. Patent 2,884,712 seeks to control pH
between a range of 6.5 to 7.5 with certain buEferlng mixtures
such as sodium or potassium dihydrogen phosphate and sodium or
- 5
potassium hydroxide, etc. No mention is made of the use of any
materials as antioxidants.
A. E. Smith in 41 C.A~ 7484e (1974) and in "The
Instability of Oestrogens ~n Solution", 5 J. ENDOCRINOLOGY pp.
152 et seq (1947) describes attempts to stabilize the non-
conjugated estrogens dienestrol and stilbestrol with hydro-
quinone. Further listed are the estrone estradiol and hexestrol
in sesame oil. See MERC~ I~DEX (9th Edition) at 5~7 (1968)o
D. J. Nazir et al, 56 C~A. 2743i (1962) describes the
use of ~-tocopherol in vegetable oils as an antioxidant to
decrease peroxide values. No mention is made of employing ~-
tocopherol in water-soluble estrogens. H. P. Kaufmann et al,
in 58 C.A. 12785h-12786a (1963) describes the efficiency of sex
hormones in retarding the oxidation of buffered K linoleate.
The efficiency of the hormones was admitted to be less than ~-
tocopherol.
~.A. Zakhorova et al, in 65 C.A. 8981h-8982a (1966)
investigated the antioxidant properties of certain naturally-
occurring hormones. A. G. Stren-Kovskaya in 73 C.A. 1844t-
1844a (1970) estimated the amounts of selected estrogens in
animal fats and oils.
In U.S. Patent 3,696,195 (1972) to Crivellaio et al,
there are described p~armaceutical compositions which are
stabilized with thioglycerol and thioglycolic acid.
Surprisingly then, it was found in the instant case
that when the synthetic conjugated estrogens were mixed in
proper proportions with selected antioxidants to form a novPl
composition with the pH maintained at a level of not less than
about 7.0 J that the composition obtained was stable and pre-
~ented oxidation and hydrolysis for extremely long periods
(at ieast two yearsj.
For the first time, it is now possible to initiate
~ .
and maintain therapy for several female problems and diseases
with synthetic conjugated eskrogens, USP that have originated
in the laboratory and not from equine wastes~
.. ~he present invention is a stabilized composition
comprising an alkali metal sulfate salt of a steroid selected
from the conjugated estrogens, i.e., the group consisting of
estrone, equilin, 17~-dihydroequilin, equilenin, 17~estra-
: diol, 17~-dihydroequilin, 17~-dihydroequilenin, 17~-estradiol
and mixtures thereof, and an antioxidant in a neutral or
alkaline environment.
m e present invention relates to a stabilized thera-
peutic preparation exhibiting no oxidation and less thana~out 5%
hydrolysis up to at least 24 months, said preparation adapted
for treatment of menopausal syndrome, female hypogonadism,
amenorrhea, female castrati.on, primary ovarian failure, ab- .
normal uterine bleeding due to hormonal imbalance, and senile
vaginitis, consisting essentially of: at least one alkali metal
sulfate salt of a synthetic conjugated estrogen selected from
the group consisting of estrone, equilin, 17~-dihydroequilin,
; 20 17~-hydroequilin, 17~-estradiol, 17~-estradiol, equilenin,
and 17~-dihydroequilenin in estrogenically effective propor-
tions, and an antioxidant effective amount of at least one
suitable antioxidant for said alkali metal sulfate salt of
said synthetic conjugated estrogen selected from the group
consistins of sodium sulfite, potassium.sulfite, sodium meta-
bisulfite, potassium metabisulfite, sodium bisulfite, potassium
bisulfite, sodium thiosulfate, potassium thiosul~ate, thio-
glycerol, thiosorbitol, cysteine hydrochloride, and ~-toco-
pherol, wherein the preparation is maintained at an alkalinity
corresponding to a pH of not less than about 7Ø
Preferable alkali metals are sodi~n and potassium.
Suitable antioxidants which can be employed include
,l ~
.= ~ .
, : .
sodium and potassium sulfite, metabisulfite, bisulfites, thio
sulfates, thioglycerol, thiosorbitol, cysteine hydrochloride
and ~-tocopherol (Vitamin E). Of course, as those skilled in
the art will appreciate other antioxidants having different
cations in complexes (ammonium, for example) instead of those
in Groups IA and IIA of the Periodic Table may be used.
Preferably antioxidants are sodium and potassium sulfite, meta
bisulfite and ~-tocopherol. While the pH of the composition pH
should be at least about 7.0, it has been found that the likeli-
hood of hydrolysis is further decreased by use of even morebasic conditions. Therefore, it is preferred that the com-
positions exhibit a pH of at least above about 7.5. By the
phrase "at least about 7.0" we mean all pH levels from slightly
below 7.0 up to 14Ø
Effective amounts of alkaline antioxidants vary with
the typa of administration. To provide stable estrogen com-
positions (i.e., tablets, parenterals, atc.), generally from
0.25 to 6 moles of antioxidant are present for each mola of
alkali metal synthetic estrogen sulfate, although smaller
amounts may be effective for specific combinations. For paren-
teral preparations containing from 5 to 25 mg of alkali metal
synthetic estrogen sulfate (i.e., estrogen conjugates) from 1
to 6 moles of antioxidant are employed per mola of the synthetic
estrogen conjugates. For oral tablets, from about 1 to 5 moles
of antioxidant per mole of estrogen conjugates are employed.
The synthetic conjugated estrogens can also be employed in
the form of gelatin capsules wherein from 0.25 to ~ moles of
antioxidant are present for each mole of synthetic estrogen
conjugates~ Generally, oral dosage forms such as tablets or
gelatin capsules contain from 0.2 to about 20 mg of estrogen
conjugates in each dose ~e.g., tablet or capsule). Most pref-
erably, a combination of sodium estrone sulfate, sodiwm equilin -
(;I 7
sulfate, and 17~-dihydroequilin sodium sulfate is employed in A
weight ratio of about 6:3:1, wherein the total conjugated
estrogen content per dose is administered in a tablet or gela-
tin form of a multiple of about 0.625 mg. These weight tablets
are marketed under the name "GENESIS " by Organon Inc., of
West Orange, New JerseyO
In addition to the conjugated estroyens and anti-
oxidants, the compositions of the invention also include other
amounts of components commonly employed in pharmaceutical com-
positions and known to those skilled in the art, such as: inert
fillers (e.g., lactose, calcium phosphate, microcrystalline
cellulose~, dyes and colorants, disintegrating agents (hydrated
silica), binding agent (e.g., methyl cellulose, amylopectin),
anticaking agents (e.g., silicon dioxide), lubricants (e.g.,
stearic acid, magnesium stearates), and suspending agents
(vegetable oil).
The stabilized estrogenic compositions of the inven-
tion can be prepared either in solution or dry form. Solu-
tions of conjugated estrogens are useful in injectable composi-
tions in the treatment of abnormal functional uterine bleeding.
m e dose required to produce hemos~asis is relatively large and
may range ~rom 2.5 mg to 12.5 mg of conjugated estrogen in the
novel composition or more daily given in divided dosesn Bleed-
ing will usually stop in 2 to 5 days assuming adequate dosage.
The effective dose should be continued for the next twenty (20)
,
days with concomitant progestin therapy during the last five (5)
days in order to recycle the patient~ If subsequent treatment
cycles are required, one may treat the patient as for amenorrhea
below.
3V For ~emale hypogonadism (hypogenitalism)/ the dosage
requirements and duration of therapy required to prime a poten-
tially responsive endometrium will vary depending on the degree
* - Trademark
- 9
of estrogen deficiency. Cyclic therapy is recommended using
doses of 2.5 to 7~5 mg in divided daily doses for twenty (20)
days of a thirty-day cycle. If bleeding does not occur by
the end of the ten-day rest period, then cyclic and sequential
therapy with the novel compositions of my invention and a suit-
able progestin, given on the ~ifth day after bleeding has
started, should ~e instituted and continued.
For amenorrhea, do~age recommendations are the same
as above except that the progestin is given concomitantly from
the sixteenth to the twentieth day of each cycle to mimic the
natural pattern. In the event that breakthrough bleeding
occurs, therapy should be discontinued at the point and resumed
again on the fifth day of bleeding.
It is to be noted that for parenteral preparations,
an especially desirable range of antioxidant is between 20
and 40% by weight of the total con}ugated estrogens. In pre-
paring the injectable product, the conjugated estrogens are
combined with the antioxidant and other commonly employed mat-
erials, all dissolved in an aqueous vehicle. The pH of the
solution is carefully adjusted to from about 7.0 to about 8.0,
taking care that the pH does not fall below about 6.9. The
solution is filtered through a bacteriological membrane filter
and filled in vials or ampoules and freeze-dried. Such solu-
tions can have any pH above the range of 7O0 or 8 0 set forth
above, as only at the lower end of the p~I range, e.g., pH 7.0,
has been found to be critical for stability. The freeze-dried
preparations have been found to be stable for periods as long
as twenty-four ~24) months and even longer. They are recon-
stituted prior to use with an isotonic saline solution.
In its most important application and in adclition
to the utilities noted above, the stabilized synthetic con
jugates may be employed orally in the form of tablets for the
10 -
~J
treatment of menopausal syndrome. Generally, the tablets will
contain from about 0.3 to about 2.5 mg. of total conjugatecl
estrogens, althou~h much higher amounts can be employed. (The
dose required to bring symptoms under control is relatively
large and may even range from 1.25 mg. to 3 75 mg or even more
daily in divided doses). When this has been accomplished, dos-
age should then be established~ This will usually be 0.625 mg
or 1.25 mg or even less~
*
GENESIS may also be indicated for female castration,
primary ovarian failure, senile vaginitis and kraurosis vulvae
(0.3 to 1.25 mg daily adequate in most patients), for pallia-
tion of inoperable breast cancer in women with progressing or
roentgen resistant disease who are more than five (5) years
post-menopausal (10 mg thrice daily is operable), and palliation
of prostatic cancer (1.25 to 2.5 mg thrice daily) when castra-
tion is not feasible or when castration failures or delayed
escope following a response to castration have not occurred.
A very convenient method of preparing the tablets is
to prepare aqueous solutions from the conjugated estrogens
and the antioxidant. The resulting solutions are used to granu-
late the variou~ powdered components of the formulation. The
granules are dried, lubricated and compacted into tablet form
which may receive any of the customary coatings such as sugar
or film coating. Coatings are generally applied to protect the
active ingredients. However, and unexpectedly, it has been
found that evPn in the absence of a coating, the tablet form
is stable for extended periods (e.g., 24 months or longer~
and, therefore, a coating need not be employed~
For the preparation of a soft gelatin capsule, the
water-soluble conjugated estrogen-containing compositions are
dispersed in a vegetable oil, e~g., for example, soybean oil.
A liquid and suitable organic antioxidant, preferably dl-
~* - Trademark
- 11 -
~3
tocopherol is used as an antioxidant and microfine precipitat-
ed silica is added as dispersing agent. The oil suspension is
sealed in a soft gelatin capsule of the desired size and
shape.
The mechanism by which the antioxidants function to
stabilize the conjugated estrogens is not completely under-
stood. However, it is well-known that acid conditions tend
to cause hydroly~is of the conjugates. In this regard, U.S.
Patent 2,834,712 teaches that buf*ering agents should be em-
ployed to maintain pH levels between 6.5 and 7.5. However, itmust be stated even in the presence of appropriate buffers, if
the suitable antioxidants are not present J the estrogen con-
jugates are stable only for periods up to approximately siX
months. Use of antioxidants in addition at a pH of not less
than 7 provides stabilization both to solutions and "dry"
preparations for up to at least about 24 monkhs~ which is the
limit of testing so far conducted. It is hypothesized here
that the primary cause of instability is not acid hydrolysis
but rather oxidation of components in the estrogen preparation~
Oxidation may create acidic conditions whlch cause hydrolysis.
Hydrolysis is, therefore, minimized by minimizing oxidation.
The above-described invention is more particularly
set forth in the following Examples which are to be construed
for purposes of illustxation only and not for limitation of my
invention. Obvious modifications from the following Examples
can be made to accommodate various synthetic conjugated estro-
gens and antioxidants in various administrations.
EXAMPLE I
Preparation of a batch of 1,000,000 tablets of 2.5 ~g of -
synthetic conjugated estrogens per tablet.
- 12 -
The following conventional ingredients are admixed
and blended, screened, dried, and reduced to a fine micro-
granulation~
Lactose (anhydrous) 97.117 kg.
Microcrystalline cellulose (Avicel by the
FMC Corp. of Marcushook, Pa., 19061) 62.00 kg.
Corn Starch 24.80 kg.
Methyl cellulose (Methocel 60 HG, 50 cps) 3.75 kg.
Amylopectin 3.75 kg.
A blend containing 2.750 kg. of selected synthetic
conjugated estrogens~ 833 kg. of "Tris"~, and 1.50 kg of
anhydrous sodium sulfite is added to a microgranulation of the
- above ingredients. Magnesium stearate (1~0 kg.) and 1.5 kg.
of Silicon Dioxide (Syloid 244 ) are added as lubricants. The
resulting blend is compressed into tablets of 200 mg.
1 _ The synthetic conjugated estrogens here are a
mixture con~isting of (on a weight basis) 10% 17~-dihydro-
equilin sodium sulfate, 30% equilin sodium sulfate and 60%
estrone sodium sulfate, the ~ame as in `'GENESIS '`.
2 _ "Tris'` is 2-amino-2 hydroxymethyl-1.3-propane-
diol. ~he estrogen conjugates are prepared synthetically and
the "Tris'` functions as an initial stabilizer temporarily until
the sodium sulfite of the invention is added.
; Percentaqes of Constituents in 2.50 mq of Active
Synthetic Coniu~ated Estroqens
+ 10% of ~ 30% of + 60% of gO - 110% of
Sodium 17~- Sodium Sodium Total Conju-
Time Dihydroequilin Equilin Estrone gated Estrogen
Period Sulfate Sulfate Sulfate ~
(Excess of con- -
jugatecl estrogen
above 2.5
mg per tablet)
Initial 8.9% 29.3% 61.8% 109%
6 weeks- 9.8% 29.9% 60.3% 107%
45C
* - Trademark
' ~' - 13 -
D7
Percenta!~es of Constltuents in 2.50 1~_ f Active
Synthetic Conluqated Estroqen~ - Cont1d.
+ ~ ~
_ 10% o~ _ 30% of _ 60% of 90 110% of
Sodium 17~- Sodlum Sodium Total Conju-
Time Dihydroequilin Equilin Estrone gated Estrogen
Period Sulfate _ _ Sulfate Sulfate 2.50 mq~ablet
(Excess of con-
jugated estrogen
- above 2.5
mg per tablet)
13 weeks- 7.7% 30.5% 61.8% 99/O
37C
6 months- 9.8C/~ 29.1% 61.1% 105%
Rm. Temp.
(22C)
12 months- ~.9% 29.7% 60.4% 108%
Rm. Temp.
(22C)
24 months~ 8.6% 28.7% 62.7% 104%
Rm. Temp.
(22C)
The percentages represent the percentum weight of
the active (or estrogen) ingredients in the approximated 2.5 mg.
of the entire cornposition.
EXAMPLE III
Conju~ated Estroqens + Non-antioxidant Buffer
Examples III t~rough V were conducted ~y using
aqueolls solutions of conjugated estrogens of the following com-
position:
Sodium 17O~-dihydroequilin sulfate 18 mg.
Sodium equilin sulfate 54 mg.
Sodium estrone sulfate 108 mg.
Tris 120 mg
.
Water 30 ~1.
The aqueous conjugated solution was buffered to p~
7~0 using a phosphate buffer of pH 7Ø To a seconcl aqueous
solution, 300 mg. of sodium sulfite was added and the solution
maintained at pH of 7O0. After storage for two weeks at 37C,
the huffered solution exhibited a red color indicating the - -
14 -
presence of oxidized equilin and 17~-dihydroequilin. The
sodium sulfite solution remained colorless (no oxidation)
and clear (no hydrolysis). Analysis by gas chromato~raphy
indicated that no free steroids were present. By contrast,
analysis of the first buffered solution without antioxidants
showed that a precipitate of free steroids was present indi-
cating that in the two-week period approximately 50% of the
estrogen conjugates had decomposed. This Example indicates
that for a given short period of time (less than the full two-
year period mentioned above), the solution with the suitableantioxidants was significantly more stable than prior art
compositions.
EXAMPLE IV
Stability at pH 7.0-7.5
An aqueous solution (30 mlO) of conjugated estro- -
gens as in Example III was neutralized to a pH of about 7~0
to about 7.5 with one normal sulfuric acid. 300 mg~ of
sodium sulfite was added which brought the pH to about 9.7.
Using one normal H2SO4, the p~ was then adjusted to 7~0.
Similar solutions were prepared at pH increments of 0.2, i.e.,
7.2, 7.4, and 7.6. After storage for five days at 60C, all
solutions remained clear and only a trace of free steroids
could be detected using thin layer chromatography to analyze a
chloroform extract of the aqueous solution.
; Repeating the above procedures, conjugate solutions
were found to be stable at pH 6.5-7~0. Only at pH of less
than about 4.5 did substantial hydrolysis occur. For example,
at pH 3.16, about 20% of the conjugated estrogens had been
hydrolyzed after the five-day storage period. Thi9 Example
30 indicates that even at abusive low pH ranges not within the
scope of our invention, the novel composition of our invention
have surprisingly good stability, although for practical - -
c~ - 15 -
applications the pH should be maintained at a pM of more than
about 7~0.
EXAMPLE V
To determine the e~fect of "Tr:is" alone, solutions
were prepared as in Example IV at a pH of about 7~0 with the
difference that one solution contained sodium sulfite whereas
in the second solution only Tris was used as the stabilizer.
Both solutions were stored for two weeks at 37~C. The second
solution (without sodium sulfite) had a slight precipitate
and a chloroform extract indicated the presence of free
steroids when ~nalyzed by TLC. The first solution ~with
sodium sulfite) was clear and chloroform extraction was nega-
tive as to free steroids. This Example indicates that ordinary
constituents such as "Tris" added which control pH only within
the ranges of this invention will not operate to extend shelf
life in the absence of an effective amount of a suitable anti-
oxidant.
The above Examples were illustrative only, were not
intended to limit the scope of the inventionl but to give some
guidance to those skilled in the art in the preparatlon of the
desired novel compositions containing synthesized conjugated
estrogen sulfate salts and effective amounts of one or more
suitable antioxidants.
While the novel compositions represent a benefit for
those women afflicted with certain conditions, use of the novel
compositions of my invention, like all estrogen-containing
pharmaceuticals, is not without some unknown risks as will be
appreciated by those skilled in the art~
An increased risk of thromboembolic disease assoc-
33 iated with the use of oral contraceptives containing estrcgensand progestins has now been conclusively establishecl. Retro-
spective studies have shown a statistically significant - -
- 16 -
association between thrombophlebitis, pulmonary embolism, and
cerebral thrombosis and embolism and the use of these drugs.
There have been three principal studies in &reat Britain and
one in the United States leading to this conclusion:
1. Royal College of General Practitioners: Oral Contraception
and Thrombo-Embolic Disease. J. Coll. Gen. Pract. 13:267-
279, 1967.
2. Inman, W. H. W. and Vessey, M.P., Investigation of Deaths
from Pulmonary, Coronary and Cerebral Thrombosis and
Embolism in Women in Child-Bearlng Age, Brit. Med. J~ 2:
193-199, 1968.
3. Vessey, M. P. and Doll, R., Investigation of Relation
Between Use of Oral Contraceptives and Thromboembolic
Disease. A further Report. Brit. Med. J., 2:651-657, 1969,
and
4. Sartwell, P. E., Masi, A. T., Arthes, F. G., Green, G. R.,
and Smith, H. E., Thromboembolism and Oral Contraceptives:
An Epidemiological Case-Control Study. AmO J. Epidem.
90:365-380, (~ovember) 1969.
As a result of these studies, it has been estimated that users
of oral contraceptives containing estrogens are 4 to 7 times
more likely than non-users to develop thromboembolic disease
without evident cause. m e American study also indicated that
the increased risk did not persist after discontinuation, nor
was it enhanced by long continued administration. Although
the American study was not designed to evaluate a difference
between products, it did suggest that there might be an in-
creased risk of thromboembolic disease in users of sequential
products. Confirmation of -this finding requires further study.
In a more recent analysis of data derived from several
national adverse reaction reporting systems, British investi-
gators concluded that the risk of thromboembolism, including
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:~o~
coronary thrombosis, i5 directly related to the dose of
estrogen used in oral contraceptive products. (See Inman, W.
H. W., Vessey, M.P., Westerholm, B., Engelund, A., Thrombo-
embolic Disease and the Steroidal Content; of Oral Contra-
ceptives, Brit. MedO J., 25th April, 1970.)
Their analysis did suggest, however, that the quan-
tity of estrogen may not be the sole factor involved. Never-
theless, in view of this study, as well as others that have
demonstrated a positive relationship between estrogens and
thromboembolism, it would ~eem prudent and in keeping with
basic therapeutic principles, to utilize, whenever feasible,
the smallest effective dose of estrogen in treating patients.
Risks associated with certain other known adverse
reactions, such as elevated blood pressure, liver dysfunction,
and reduced tolerance to carbohydrate, have not as yet been
quantitated.
Long term administration of both natural and synthet-
ic estrogens in subprimate animal species in multiples of the
human dose increases the frequency for some animal carcinomas.
~hese data cannot be transposed directly to man. The possible
carcinogenicity due to the estrogens can neither be confirmed
nor refuted at this time. Close clini~al surveillance of all
women taking estrogens must be continued.
It has been reported in a recent study done in the
United States -that the maternal ingestion of diethylstilbestrol
during pregnancy appears to increase the risk of vaginal
adenocarcinoma developing years later in the offspring exposed:
Herbst et al--Adenocarcinoma of the Vagina--New England Journal
~ of M dicine, Vol~ 284, Number 16 (April 22, 1971).
Of course, it must be emphasiæed that no t:oxic side
effects have so far-been found with the novel compositions of
; matter of this inventionO Of course, in the administration of
:
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the novel compositions of our invention should certain mani-
festations occur of the following type, medication should be
withdrawn.
Estrogens may be excreted in the mother's milk and
an estrogenic effect upon the infant has been described. The
long range effect on the nursing infant cannot be determined
at this time.
Hypercalcemia may occur in as many as 15% of breast
cancer patients with metastases and this usually indicates
progression of bone metastases. This occurrence depends
neither on dose nor on immobilization~ In the presence of un-
toward effects such as progression of the cancer or hypercal-
cemia, the effect of estrogen medication should be stopped.
Enteric coating retards absorption from the gastro-
intestinal tract and this form of therapy should not be used
when rapid action is desired.
A statistically significant association has been
reported between maternal ingestion during pregnancy of
diethylstelbestrol and the occurrence of vaginal carcinoma
developing years later in the offspring. Whether such an
association is applicable to all estrogens is not known at
this time. In any event, estrogens are not indicated for use
during pregnancy.
It must be noted that a statistically significant
association has been demonstra-ted b~tween use of estrogen-
progestin oral contraceptives and the following serious re-
actions: thrombophlebitis, cerebral thrombosis, pulmonary
embolism.
Although available evidence is suggestive of an
association, such a relationship has been neither confirmed
nor refuted for the following serious reactions: coronary
thrombosis, neuro-ocular lesions ~e.g. re-tinal thrombosis - -
and optic neuritis).
19-
The following adverse reactions are known to occur
in patients receiving estrogens: nausea, vomiting, anorexia,
gastrointestinal symptons (such ac abdomi;nal cramps or bloating),
edema, breakthrough bleeding, spot-tiny or withdrawal bleeding,
breast tenderness and enlargement, change in body weight
(increase or decrease), headache, increa,sed cervical mucus,
allergic rash, loss of libido and gynecomastia in the male,
reactivation of endometriosis, aggravation of migraine head-
aches, hepatic cutaneous porphyria becoming manifest, chole-
static jaundice, rise in blood pressure in susceptible indivi-
duals, mental depression, cystitis-like syndrome, loss of
scalp hair, erythema nodosum, hemorrhagic eruption, pre-
menstrual-like syndrome, changes in libido, changes in appe-tite,
nervousness, dizziness, fati~ue, backache, erythema multiforme,
itching, possible diminution in lactation when given immedi.ately
post-partum, irritability, malaise.
Therefore, although no relationship has been shown
to exist between the compositions of my invention and the above
adverse reactions, the following precautions should be observ- :
ed by those administering the compositions of my invention:
1. Because normal endogenous hormone production varies
individually,certain patients may be unusually res-
ponsive to estrogenic therapy and may respond with un-
desirable manifestation of excessive estrogenic stimula-
tion, such as abnormal or.excessive uterine bleedin~, -
masto~dynia, edema, etcO
2. Because of estrogen induced salt and water retention,
these drugs should be used with caution in patients with
epilepsy, migraine, asthma, cardiac or renal disease.
3. Patients with a history of psychic depression should
be carefully observed and the drug discontinuecl if the
depression recurs to a serious degree. - - ~
20 - :
4. In the event that any unexplained or excessive
vaginal bleeding would occur while on estrogen therapy,
nonfunctional causes should be borne in mind. The drug
should be discontinued and a thorough investigation made
as to the cause, being certain to rule out the possibil-
ity of malignancy.
5. Pre-existing uterine fibromyomata may increase in
size while using this product, therefore, patients
should be examined at regular intervals while receiving
estrogenic therapyO
6. Women with a strong family history of cancer, recur-
rent chronic cystic mastitis, or abnormal mammograms
should be administered estrogens with caution.
7~ ~ecause of a possible decrease in glucose tolerance,
diabetic patients should be followed closely~
8. Because estrogens influence the metabolism of cal- `~
cium and phosphorus, they should be used with caution
in patients with certain metabolic bone diseases that
are associated with hypercalcemia or in patients with `
renal insufficiency.
9. The pathologist should be advised of estrogen therapy
when relevant specimens are submitted.
10. Because of the effects of estrogens on epiphyseal
closure, they should be used judiciously in young
patients in whom bone growth is not complete.
11. A pre-treatment physical examination should include
special reference to breast and pelvic organs as well as
a Papanicolaou smear since estrogens have been known to
produce tumors, some of them malignant, in five species
of animals.
12. When large doses o~ estrogens are used, urinary
stress incontinence may occur in nonpregnant females.
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13. Prolonged high doses of estrogens will inhibit
anterior pituitary functions. This should be borne in
mind when treating patients in whom fertility is desired.
140 Continuous use of estrogens will result in prolonged
stimulation of the endometrium and breastO In order to
avoid this, oral estrogens should be administered cyclic-
ally in the menopausal or hypogonadal patient.
150 The age of the patient constitutes no absolute
limiting factor, although treatment with estrogens
~ay mask the onset of the climactericu
16. Certain endocrine and liver function tests may
be affected by treatment with estrogens. If such tests
are abnormal in a patient taking these drugs it is
reco~mended that they be repreated after the drug has
been withdrawn for two months.
17~ Any possible influence of prolonged estrogen
therapy on pituitary, ovarian, adrenal, hepatic, or
uterine function awaits further study.
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