J '
`
`09
`
`Vol. SJ, No.·8, August 1964
`
`definite bearing on the usefulness of any colWDn
`packing prepared. The performances of the seven
`supports mentioned previously were examined under
`the S9.I11e operating conditions. The supports that
`can be used for lightly loaded packings are: glass
`beads, Gas Cbrom-P, and Chromosorb W-HMDS.
`The other four supports cannot be used for lightly
`loaded column packing since their interaction with
`the antihistamines causes excessive peak tailing.
`The hydrogen fiame detector used in conjunction
`with the 0.010-in. stainless capillary column would
`not respond to compounds with boiling points above
`330°. This limitation prevented evaluation of this
`column for the analysis of these antilUstamines.
`The 100-ft. 0.065-in. copper open tubular column
`was coated with XF-1150 and evaluated using the
`above gtOUp Of antihistamines. The Sr9D ionization
`detector was used with a column flow of 36 ml./
`minute. The retention times obtained were com(cid:173)
`parable to the 6-ft.-XF-1150 packed column, but the
`peak base widths were considerably wider.
`:Because
`of this increase in base width, the 0.065-in. column
`was less efficient than the 6-ft. packed column.
`A 250-ft. 0.065-in. column wound on a l1/c-in.
`diameter mandrel has been reported to be more
`efficient than a packed column (15). There are two
`possible reasons why efficiency was less than pre(cid:173)
`viously reported: (a) the column was shorter (100
`ft.), and (b) the winding configuration was markedly
`different. The eolu.mn :was wound on a. 11/, X
`1/ rin. bar which resulted in a definite flattening of
`the tube around the edge of the bar.
`
`891
`
`CONCLUSIONS
`
`investigated, except for
`The antihistamines
`meclizine, can be separated, identified, and concen(cid:173)
`tration estimated using the Carbowiu 20M, PDEAS,
`and XF-1150 columns described. The PDEAS
`column is the most efficient of the three for the
`analysis of antihista.mlnes.
`The usefulness of the 0.010-in. capillary and the
`0.065-in. open tubular columns cannot be properly
`evaluated until the mentioned lim.ita.tions are re(cid:173)
`moved.
`
`REFERENCES
`
`{~ Ciepliz:sld, B. W., Anal. Chem., 35, 256(1068).
`
`(1) :Brochmann·Ranssen, B., a.nd Svendsen, A .B,. TJII5
`]o~=. 511 818(1962).
`3 Parker, K. D:l and Kirk, P. L., md., 33, 428(1963).
`Illid., 33, 1370(1961).
`:Bcochmann·Hanssen, B., and SveJJdsen, A. B., Tms
`(5
`]OUllNAL, 51, 938(1962).
`(6) Park~1_K. D., Fontan, C.R., B.lld Kirfc. P. L •• Anal.
`Chem., 34, 1Ml>(l062l.
`(7} Fale:i, H. M., IUld Pisallo, ]. ] ., Anczl. Biocllem., 3, 337
`(1962 •
`(8 P•rker, K. D., Fontan, C. R., B.lld Xirlr::, P. L., Anal.
`Chem., 34, 757(1962).
`(9) Anden, M. W., and :Manaering, G. ]., J. Cluomalog.,
`7, 258(1962).
`(10) Pa.rker, K. D., Fontan, C. R., a.ud Kirk, P. L., Anal.
`Chem., 35, 366(1963).
`(11) Fontan, C. R., Smith, W. C., a.ud Kirk, P. L., ibicl.,
`35, 691(1963).
`(1.2) Zubyk, W. J., and Comicr, A. Z., ibitl., 32, 912(1960).
`(13) Av~ W., "Progress iii Industrial Gas Chro.matog·
`raphy," edited by Szvniimsld. R. A., Plenum Press, New
`York, N . Y., 1961, J>- ~25.
`(14) MacDonald, A., Jr., and Pflawn, R. T., Tms Jotra(cid:173)
`NAL, 52, 816(1063),
`(15) Quiram, B. R., Anal. Chem., 35, 593(1963).
`
`Castor Oil as a Vehicle for Parenteral
`Administration of Steroid Hormones
`
`By C. RlFFK.IN, R. HUB~ and C. IL KBYSSER
`
`Steroid hormones may be administered parenterally in high concentrations as oil
`solutions. Ia this form they nh.ibit a prolonged action and .reduce the number of
`injections required. To accommodate the detnlllld for increasingly greater concen-
`. trations of hormones in solution, castor oil in combination with other suitable oil(cid:173)
`miscible solvents. has been found to fu1.fill a nee4. The development of several
`formulations together with the results of animal testing, as well as clinical trials in
`humans, attest to the acceptability of this oil fos: the purposes intended.
`
`FIXED OILS are included in the "United States
`
`Pharmacopeia XVI" as nonaqueous vehicles
`for injection and are characterized as being of
`vegetable origin, essentially odorless, and with(cid:173)
`out suggestion of rancidity. They must also
`comply with certain measurable physical limits
`specified for the saponification, acid, and iodine
`values.
`·
`p~ved September 91 1963, from the l'bannacology aud
`•--titu ccutical ResearcA & Development Sections, Squibb
`te for Medical Re.search, New Brunswick., N. J,
`.....,.
`Accepted for publication November 19, 1963.
`t The a.uthom ....e Indebted to the Chemical Control Labora(cid:173)
`~: ~r their assistance with the assays; to Dr. N . Coy and
`d elo erts of the Physical and AnalytiCnl Section, for their
`ev opmcnt of special a.ssays; to G. Lockwood of the
`P~togy Section for the animal muscle irritation tests•
`~ .. espf eaall;r to Dr. E. C. lleile.ast.eln, Jr., of the medl~
`....... , or the information co11~g the clinical trials.
`
`After subcutaneous injection, Deanesly and
`Parkes (1) observed the persistence of olive oil
`and castor oil in animal tissue. Comparing other
`ouS Brown, el al. (2), reported that sesame and
`com oils were superior to cottonseed and peanut
`oils because they were less irritating, less anti(cid:173)
`genic, more quickly released from tissue, and
`pQsses5ed superior physical properties.
`More recently the use of steroid hormone
`medication has expanded considerably. Due to
`llinited water solubility, hormones have been
`administered as aqueous suspensions or solu(cid:173)
`tions in oil. It has been claimed. that the latter
`provided the slow release preferred in cyclical
`
`MYLAN PHARMS. INC. EXHIBIT 1022 PAGE 1
`
`

`
`892
`
`Journal of Pharmace#tical Sciences
`
`T.ABLB L-J\NALYSIS OB CoMMBRCUL On.s A.ND CoHPARISON TO U.S.P. XVI 8PBcnnCA.TIONS
`
`ml. 0.02 N NaOH
`Bquiv. to Free
`Fatty Acid iD
`10..Cm. Sample
`35.Cl"
`14.0
`4.6
`7.9
`9.2
`s.o
`0.5
`1.4
`0.75
`0.45
`2.0
`
`Oil
`Castor Oil
`
`Sesame Oil
`
`Cotton.seed Oil
`
`Corn Oil
`
`Peanut Oil
`
`lodiDe Value
`83-88
`84.8
`87.0
`84.5
`84.2
`103-116
`106.9
`111.8
`104.7
`108.2
`109-116
`111.8
`113.1
`102-128
`119.1
`124.4
`120.0
`123.0
`84:-100
`94.4
`93.2
`87.8
`93.9
`
`Sapon. Value
`I.otNo.
`179-185
`U.S.P. specs.
`183.8
`23946
`179.8
`25589
`182.7
`23463
`180.4
`33742
`188-195
`U.S.P. specs.
`189.6
`23549A
`194.0
`26953
`189.6
`33646
`191.7
`29981
`190-198
`U.S.P. specs.
`195.9
`49684
`196.8
`44441
`187-193
`2.0
`U.S.P. specs.
`194.5
`1.0
`52148
`191.4
`1.2
`36716
`189.3
`1.2
`33436
`189.3
`1.0
`33715
`185-195
`2.0
`U.S".P. specs.
`192.0
`1.2
`22160
`191.7
`1.4
`20993
`193.1
`0.8
`33622
`190.4
`1.2
`26147
`"Tbc U.S.P. specilies that the titration of free (atty acids in ozsl grade castor oil shall not ucccd 7 ml. Of 0.1 N NaOH
`which is eqqa] to 35.0 ml .. of 0.02 N NaOH.
`T.ABLB ll.-SOLUBILITY OB Snmoms Dl U.S.P.
`OU.SAT 25°
`
`) ·
`
`Steroid
`17-llydroxyproge.sterone
`caproate
`Testosterone
`Estradiol valerate
`Progesterone
`
`Castor
`Oil
`
`55.6
`38.6
`60.6
`52.0
`
`mg./ml.
`Scsanic Peanut
`Oil
`Oil
`
`23.4
`5.4
`16.1
`22.9
`
`27.9
`8.1
`18.8
`23.5
`
`therapy (3). Using withdrawal bleeding in
`human females as the criterion, Master, ~ - oJ.
`( 4), compared the duration of act:ipn of an
`aqueous suspension of progesterone with an oil
`solution, and confirmed the superiority. of the
`latter. The prolongation of activity was gen(cid:173)
`erally related to storage in the fatty depots of
`the body (5).
`In 1952 Jnnkrnann (6) determined that a
`testosterone ester dissolved in sesame oil pro(cid:173)
`longed the androgenic · eifects in castrated rats.
`. Davis and Wied (7) demonstrated that pro(cid:173)
`longed activity was also obtained in humans
`when oil solutions of a progesterone derivative
`were injected. There was still a limiting factor,
`however, in that only a relatively small amount
`of hormone could be dissolved in the traditional
`oils. To increase the solvent power of the oil
`it was necessary to add compab."ble and non-·
`irritating cosolvents. Such additions consisted
`of benzyl benzoate, benzyl alcohol, ethyl lactate,
`ethyl oleate, etc. The U.S.P. recognized the
`·need for such "other vehicles," with the re(cid:173)
`strictions that they must be safe in the volume
`of injection administered, and that they should
`
`not interfere with the therapeutic efficacy of the
`preparation or its testing.
`Demand for increased hormone concentrations
`per dose. furthered the search for an acceptable
`oil with greater solubilizing power per se. Bos(cid:173)
`chann (8) in 1954, observed that 17-hydro:xypro(cid:173)
`gesterone caproate in a castor oil-ethyl lactate
`vehicle was well tolerated. In addition, private
`communications from clinicians in West Ger(cid:173)
`many1 reported good tolerance to Proluton-Depot
`containing a castor oil-benzyl benzoate vehicle.
`Since then other hormones have been used as
`so1utions in ricinoleic acid esters, ·as well as in
`castor oil (9-11). Accordingly, au investiga(cid:173)
`tion was undertaken into the suitability of cas(cid:173)
`tor oil as a vehicle for parenteral administration
`of steroid hormones.
`
`METHODS AND RESULTS
`Representative samples of -U.S.P. oils obtained
`from commercial sources were tested in accordance
`with the official method for free fatty acid content.
`saponification, and iodine values. The resul~ are
`listed in Ta.ble I along with the U.S.P. XVI specl1idl(cid:173)
`tions for these oils.
`Solubility of selected steroids in various oils w~
`determined in the following manner. AJJ. excess 0
`steroid was stirred for 4 hours at room temperatur~
`(25°) in the test oil, after which the undissolve
`solids were removed by filtration, and the clear solu(cid:173)
`tion assayed for steroid content. Table ll shows the
`results obtained.
`'ds
`An attempt was made to reduce the free fattY ae1
`in castor oil by treatment with alumina. and anhy·
`1 Dr. Napp Univcrsltats-R'.rnukenhaus, Ham!>urlfiJ?!:
`Pote, Rumbofdt-Unlvenitat-Charite Frau~J<lln1b~g· 11od
`Dr. Prill Univeraitats·FraueJJkllDik,
`':"' urz
`•
`Dr. RauSCher, Uoiveraltats-l'raucnkli.nill:, V1Clllla.
`
`MYLAN PHARMS. INC. EXHIBIT 1022 PAGE 2
`
`

`
`.•
`
`.I
`
`Vol. S3, No. 8, August 1964
`
`TABLB IIl.-.AllSOKPTION OF On. PROM ANnlAL
`MtrsCLB"
`
`Days
`after
`Illjectlou
`1-S
`1-S
`1-8
`
`Oil
`Castor oil
`(aged)
`Castor oil
`U.S.P.
`Sesame oil
`U.S.P.
`7~0 All oils
`
`ml. 0.02 N
`Na OH
`Bquiv.
`50
`
`13
`
`1.4
`
`Residual Oil iD
`Muscle (cstd.)
`
`I day -00,
`
`3 days-20
`1 day-30
`3days-10 0
`lday-30%
`a days-30 o
`DeclllUng 10
`to23
`
`" 1 ml. Injected into back muscle of rabbit.
`
`drous sodium sulfate. Three grams of dried, pow(cid:173)
`dered, amorphous aluminum oxide (Merck No. 1097)
`and 6 Gm. of anhydrous sodium sulfate, reagent
`grade, were suspended in 120 mt- of oil and heated
`at 80° under a blanket of nitrogen for 1.5 hours. ·
`After allowing the oil to cool to room temperature,
`the solids were filtered off and the acids titrated in
`the usual manner. A significant reduction in free
`fatty acid was not obtained.
`The absorption characteristics of oils with varying
`fatty aad content were e.xA!Jlined and compared on a
`biological basis. Aged castor oil with a high free
`fatty acid content was compared to fresh U.S.P.
`castor oil with a low acid content and U.S.P. sesame
`oil by injecting 1 ml of oil into the back muscles of
`rabbits, approximately 2 in. from the iliac crest. A
`rotational pattern of injection was used and the oil
`samples were stained to aid visibility in the tissues.
`The animals were sacrificed and the muscles ~ed
`and examined grossly. The results were averaged
`and appear in Table ill.
`The test disclosed that oil migrated or was carried
`to the fascia, and very smaU amounts remained for
`60 days. Localized degeneration produced by the
`high acid value castor oil was essentially healed in 7
`days, and the low acid value castor oil appeared to
`be no more irritating than sesame oil.1
`. In a specific test for irritation 0.25 ml of the above
`oil samples were also injected into the vcistu.s laleralis
`muscles of rabbits. After 2 days the animals were
`sacrificed and the injteted muscles examined grossly
`for evidence of irritation. It was found that the
`castor oil containing a high level of free fatty acid
`produced a lesion size measuring approximately 121
`mm. i. · The lesion itself was characterized mainly by
`degeneration of local tissue without necrosis. Cas·
`tor oil with low free fatty acid and sesame oil, on
`the other hand, produced no measurable lesion at the
`injection site.
`Combinations of benzyl alcohol and benzyl benzo(cid:173)
`ate with both castor oil and sesame oil were also
`injected: into the vaslus lateralis muscles of rabbits
`and Table IV lists the lesion sizes produced.
`Solutions which were formulated for clinical trials
`in humans were prepared by clissolving the steroid
`b.?rmones in appropriate vehicles at 60° under
`wtrogen. The solutions were then filtered through
`a coarse sintered-glass filter with the aid of nitrogen
`pressure., filled into vials, and sterilized by autoclav(cid:173)
`mg for 2 hours at 121° ( 15 lb. steam pressure). The
`Products were then submitted for assay, safety, and
`'Due to the apparent inc:rease In free tatty acids with
`aeing, tubscque:ut work utilized trcsh oils which requin:d for
`~euOtrallzaH)' tlou less than 3 ml. of 0.1 N NaOH (15 ml. of 0.2 N
`per 10 G111. of sample.
`a
`
`893
`
`animal muscle irritation testing prior to release for
`clinical investigation.
`DISCUSSION
`Throughout the investigation it was desirable to
`have a reference oil to serve as a basis for comparison.
`Since sesame oil is universally accepted as a paren(cid:173)
`.teral oil vehicle, it was chosen as the "standard''
`vegetable' oil to be compared to castor oil, with and
`without other cosolvents. The physical, chemical,
`and biological properties of sesame oil are well
`docnmented and require no comments here.
`Chemically. castor oil consists of the triglycerides
`of ricinoleic acid, together with small quantities of
`glycerides of other acids. The quantitative com(cid:173)
`position is given by Eckey (12) as follows: ricinoleic
`acid 87%, oleic acid 7.43, linoleic acid 3.13, dihy(cid:173)
`dro~cinoleic acid 0.63, and miscellaneous acids
`2.4o/o- Two grades are commonly recognized in this
`country--U.S. No. 1 which is cold pressed oil, and
`U.S. No. 3 which is oil extracted from the. pressed
`cake. Only the former is used for medkinal purposes.
`The high viscosity of castor oil compared to other
`vegetable oils is undoubtedly related to hydrogen
`bonding and it is probably the hydroxy groups
`which contribute to the greater polarity and superior
`solvent power of the oil As indicated in Table I,
`the saponification and iodine values of commercial
`castor oil appear to be slightly lower than the U.S.P.
`XVI limits for oils used for injection. On the other
`hand., the content of free fatty acids even in fresh oil,
`varies considerably and exceeds the traditional
`limits for injectable oils. The signific3.n.ce of this is
`somewhat obscure, although "Remington's Practice
`of Pb.arma.cy, 12th edition," page 387, states "a low
`free fatty acid content is essential since it indicates a
`fresh and pure product and not one that is likely to
`have become old and heavily contaminated with bac(cid:173)
`terial products."
`Despite better solubility of steroids in castor oil,
`other cosolve.nts were necessary to dissolve the
`
`IN
`lmuTA.TION PRODUCED
`IV.-LoCAL
`TABLB
`RAlrarr MllSCLB BY IN)'BCl'ION OF VARIOUS On.
`VEHICLES"
`
`Composi.tiou
`IdentiJicatioo.
`SHY-47-2 Sesame oil 983
`Benzyl alcohol 23
`·
`SBY-47-4 Castor oil 98 %
`Benzyl alcohol 2%
`SHY-47-8 Sesame oil 953
`Benzyl alcohol 53
`SHY-47-5 Castor oil 953
`Benzyl alcohol 53
`SHY-14-2 Sesame oil 653
`Benzyl benzoate 353
`SHY-14-5 Castor oil 653
`Benzyl benzoa.te 35%
`SHY-47·6 Sesame oil 683
`Benzyl benzoate 353
`Benzyl alcohol 23
`SHY-47-7 Castor oil 63 %
`Benzyl benzoate 35%
`Benzyl alcohol 23
`SHY-14-3 Sesame oil 503
`Denzyl benzoa.te 503
`SHY-14-6 Castor oil 503
`Benzyl benzoate 503
`
`Lesiou size,
`mm.I
`61
`
`ToosmaU to
`measure
`506
`
`106
`
`291
`
`184
`
`207
`
`262
`
`291
`
`158
`
`" A 0.25-ml. qWUltity of the oil vehicle was Injected. into the
`vaslu.t laleralll muscle of the rabbit. Two dar-i later the
`muscle wu ucl.sed aud th~ lesion size me11Bured m mm.•.
`
`MYLAN PHARMS. INC. EXHIBIT 1022 PAGE 3
`
`

`
`894
`increasingly higher concentrations required by
`therapeutic regimens. Often these materials con(cid:173)
`tn"buted additional advantages. For example, the
`addition of benzyl alcohol or benzyl benzoate to
`castor oil resulted in a lower and mare favorable
`viscosity, ma.king it easier to inject. Also, benzyl
`alcohol was an efiective preservative and local
`anesthetic.
`The nature of the irritative response depended on
`the particular hormone, its concentration in the
`formulations, and/or the composition of the vehicle.
`Although rabbit muscles are more sensitive than
`human muscles, they were selected primarily because
`·local changes in the muscle were observed easily.
`It was not always possi"ble, however, to coaelate
`muscle irritation in animals to that of humans.
`A numerical assignment to lesion size was used
`solely as a convenience for grading response. The
`numbers alone do not adequately describe the
`nature of the response, however. More completely
`it is characterized by the amount of hemorrhage and
`edema and the incidence. degree, and extent of local
`degeneration produced by the injection. A slight.
`reversible irritatjve respODSe may con:r a large area
`and a severe irreversible one may be comparatively
`small. A decrease in the size of the degenerated
`area indicates a reversible condition. The presence
`of necrosis, which is the most damaging sitnation,
`means that the cellular structure was destroyed and
`Tepair must ta.lee place.. The debris must be re(cid:173)
`moved and the original cellular mass in the area re(cid:173)
`placed with fibrous connective tissue. The extent
`of this fibrosis or formation of scar tissue gives an
`index of the amount of irreversible damage. For(cid:173)
`ttz?ately necrosis was not encountered, indicating the
`lack of permanent muscle damage. Since these
`changes take time, 1inal assessment of the effects of
`an injection in the muscle freq11ently required
`observation for 7 days or longer.
`It is unfortunate that pain cannot be measured by
`any known method of animal testing. The animal
`usually does not respond unless the painful stilllulus
`~ ~ked. Furthermor~ the pain caused by injec·
`. ~on mto hnman muscle is not usually proportionate
`to the irritation produced either in animal muscle
`or in human muscle. Realizing that these limita(cid:173)
`tions are inherent in animal test methods it re(cid:173)
`mained for final acceptability to be determined in
`man.
`When it was discovered that 17-bydroxyprogester(cid:173)
`one caproate possessed high progestational activity
`poten~es of the order of ~~ mg./ml. were used. B;
`mcreasmg the dose, additional prolongation of ac(cid:173)
`tion wa.s obtained, and eventwilly concentrations of
`the order of 250 mg./ml. were required. Such a
`solution in sesame oil produced acceptable animal
`muscle tolerance, but the pain and local reaction in
`humans was so great as to probJ"bit the adoption of
`the fotlllulation as a commercial product (see Table
`V, Lot Pr. 142-53/15-10).• Solutions were also
`prepared using castor oil as the vehicle, and Table V
`lists the formulations tested and the results ob(cid:173)
`tained.
`Information obtained from the clinical
`trials (14-21) attested to the acceptability and
`safety of the adopted formulatioDS.
`· Inherent in the development of an acceptable
`formulation of 17-hydroxyprogesterone caproate was
`
`Journal of Pharmauutical Scienca
`
`TABLB V.-Ev.AI.t7ATION OB 250 mg./ml 17-HY(cid:173)
`Dll.OX'iPllOGESTmlONB Cil>:ROATB SOLUTIONS JN
`v .AlUOUS OIL VEHICLES
`Animal
`Lot Number and
`Muscle
`Remarks on Clllliclll
`Lesion
`Testing
`Size, mm."'
`1049 Pr.142-53/15-7-238
`inje<:tions, 20.6%
`reactions, rejected
`Pr.142-53/15-8-270
`injections, 23.2%
`reactions, rejected
`
`691
`
`Vehicle
`Composition
`Sesame oil 503
`Benzyl benzoate
`50%
`Castor oil 58%
`Benzyl benzoate
`40%
`Benzyl alcohol 2%
`Sesame oil 60%
`J:lenzyl benzoate
`35%
`Benzyl alcohol 53
`Castor oil 543
`Benzyl benzoate
`46%
`
`Castor oil 52%
`Benzyl ben.zoate
`46%
`Benzyl alcohol 2%
`
`697
`
`258
`
`Pr.142-53/15-10-
`189 injections,
`10.7% reactions,
`rejected
`Pr.142-53/15-11-
`503 injections,
`4.2% reactions,
`accepted
`Pr.142-53/15-13-
`924 injections,
`· 1.3% reactions,
`accepted
`
`" Injection of 0.25 ml. hlto 11a.slus lokF11lis muscle of rabbits
`aud lesion siu determined 2 days after Uijcctio:a.
`TABLB V1-EvALt1ATION OF Es'rRADIOL V .ALRRATB
`IN V ARIO'US On, VBHICLBS
`
`Animal
`:Mu<cle
`Lesion
`Siz;e, =·"'
`197
`
`Composition
`20 mg.fml. in Cas-
`tor oil 78<7"o.
`Benzyl benzoa.te
`203, Benzyl al-
`cohol 23
`30 mg./ml. in Ses-
`ameoil60%.
`Benzyl benzoa.te
`40%
`30 mg./ml. in Cas-
`tor oil 80%,
`Benzyl benzoate
`203
`40 mg./ml. in Ses-
`a.me oil 65%,
`Benzyl benzoate
`30%, Benzyl al-
`=hol 53
`40 mg./ml. in Ses-
`ame oil58%,
`Benzyl benzoate
`40%, Benzyl al-
`. cohol 2%
`40 mg./ml in Cas-
`tor oil 58%,
`Benzyl benzoate
`40%, Benzyl al-
`cohol 2%
`
`306
`
`194
`
`803
`
`496
`
`250
`
`Lot NUD1ber and
`Remark.9
`Es.31-53/15-B(cid:173)
`C.Ommercially
`available
`
`DEK-98-2-Not
`tested clinically;
`. doSa.ge increased
`to 40 mg./ml.
`Es.31-53-V-Not
`tested clinically;
`dosage increased
`to 40 mg./rol
`SHX-94-4-Too
`irritating; not
`tested clinically
`
`Es.31-53-S-201 in·
`jections,
`23.2%
`reactions, rejected
`
`Es.31-53-A-826
`injections, .2.67%
`reactions (all
`mild), accepted
`
`" Injectiou of 0.25 ml into flllslu.r lakrlllis. mui;dc of
`iabbite and le.ion •ize deteruiincd 2 day:o after ii:IJ~D.
`
`the required development of a suitable assay xnethod.)
`Tb.is was accomplished by Roberts a.nd Florey (13
`using paper·strip chromatography.
`Since estrogens are more potent than progesto~
`and require less per dose, an ~ptable formulation
`of estradiol valuate was ea$ier to prepare. Be(cid:173)
`sides use in estrogen therapy, estradiol vale.rate baS
`found utility in the treatment of carcinoma, e.nd for
`that purpose high dosages were required. Concen·
`trations were increased f:rom 10 to 40 mg./rrll· and
`
`MYLAN PHARMS. INC. EXHIBIT 1022 PAGE 4
`
`

`
`Vol. 53, No. 8, August 1964
`
`again formulations containini; castor oil in the ve(cid:173)
`hicle proved to be less irritatiag than similar prepara(cid:173)
`tions containing sesame oil. Physically and chem(cid:173)
`ically both oil solutions were stable. Based on
`acceptable preliminary data, formulations such as
`those listed in Table VI were prepared and tested.
`.Acceptability in humans was confirmed by clinicians
`and described in the literature (22, 23) and iii case
`reports.'
`
`SUMMARY
`1. The development and testing of parenteral
`steroid hormone formulations has been described,
`using- castor oil as a vehicle.
`2. After ascertaining stability and animal muscle
`irritation, selected formulations were evaluated in
`humans. They exhibited a prolonged action, were
`eifective and well tolerated
`3. Examples of commercially available products
`are the estrogen, estradiol valerate' at 20 mg./mt
`and 40 mg./ml, and the progestogen, 17-hydroxy(cid:173)
`progesterone caproate' at 250 mg./ml
`'Case reports: estradiol valerate, 20 mg./ml. in castor oil
`783, bcneyl. b=att 203, beneyi alcohol 2'r.,-90 injec(cid:173)
`tions ill 46 patients. Two mild local reactions. Bstradiol
`valerate 40 JDg./ml. in castor oil 083, beaeyl bcnzoatc 403,
`bCDZYl alcohol 2o/'o-Sl patients. Numbcc of i.njectioltll not
`completely tabulated. One report is in press.
`I Marketed 119 Delest:rogcn by B. R. Squibb &: Sons, New
`York, N. Y.
`1 Marketed as Del.alutln by B. R. Squibb &: Sons, New
`York"N. Y.
`
`89S
`
`REFERENCES
`(1} Deancsly, R., and Parkes, A. S., J. Pht;riol., 78, loo
`(1933 •
`(2 Brown, W. E .. Wilder, V. M., Schwart:, P., J. Lob.
`Clin. Med.., 29, 259(1944).
`(3) ''Hormouc Therapy in Practice," 2ad ed., Schering,
`A. G.1 Erich Blaschkcr, Berlin, 1954, p. 109.
`(4J Master, W. EL, Grody, M. EL, and Magallon, D. T.,
`J. C/in. End«rinol., 121 1445(1952).
`(5) "Modem Treaaa in Endocrinology," R. Gardiacr(cid:173)
`Billf ed., P. B. Bocber, 1958, p. 233.
`6) Junlanmn, X:., Arch. Ezptl. Pathol. Pharmakol., 215,
`85( 952).
`(7) Davis, M. E., and Wied, G. L., J. Clin. Endacrinol.
`Mdllh., 15, 923(1955).
`(19
`•
`Richter, l1. S. pat. 2,822,816.
`
`~ Boschun, a W., Ard. Wochenschr., 9 (25) 591
`
`~l Brit. pat. 811,.241.
`
`1 Ercoll, A., U. S.
`t. 2,988,649.
`1 Bcltey, B. W. Fvcgctablc Fats and Oils," A.C.S.
`Monograph, Reinhold Publlsing Co., New York, N. Y.,
`Series No. 123, I'~· 587-591.
`(l~ lloberts, R.R., and Florey, X:., TmBJomm.u. 51, 794
`.(1962 • •
`(l Short, C. L., Am. Prad.ilioner Dig. Tre/Umenl, 11,
`149(1960).
`(15) GKcublatt, R. B., and Dutta. S. N., Aria. Med., 18
`(4), 107(1961).
`(16) Kelley R. M., and Baker, W. EL, Nt"fl! Engl. J. Med.,
`264, 216(1961,}.
`(17) KClllledy, B. J., J. Am. Med. A.s$0e., 184, 758(1963).
`(18) Danforth, D. N .. and Bw:kinghatll, J. C., Posluad.
`Med., 32, 345(0ct. 1962).
`(19! Kistner, R. W., Clin. Obstel. C,,ne~l .. 51 1166(1962).
`21 Pclle.rrbo. L., CurrenJ Therap. Ru .. 4, 301(1962).
`22 Mac:Oo:iald, I., and Yettra, M., Med. Clin. N. Am.,
`43, 971(1959).
`(23) Eichner, B.l. :Brown, M., and Sable, M., J. Inlern.
`Coll. Surgum.1, 32, 1194(1909).
`
`~20 Siegel, I., Obdel. ~., 21, 666(1963J.
`
`Isolation of Marmbiin, a Sterol, and a
`Sesquiterpene from Marrubium vulgare
`
`By HAROLD J. NICHOLAS*
`
`A simple column c;Jiro.aiatographic method for isolating the bicyclic diterpene
`marrubiin from acetone and ethanol extracts of M~rru6ium 1111/gare L. is described
`.An unsaturated sterol of the stigmastanol series, present in ester.Wed form, and a
`sesquiterpene (Ci~2102) have been isolated from the extracts.
`
`)
`
`JN PREPARATION for radioactive tracer work on
`the biosynthesis of marrubiln it was necessary ·
`to examine extracts of the plant for associated
`terpenoid substances. A convenient column
`chromatographic method was therefore devised
`for separating relatively pun~ marrubiln from
`crude acetone extracts. Two new terpenoid
`substances were detected in the extracts.
`
`EXPERIMENT AI.
`Materials and Methods.---Ground M. vulgare L.
`was obtained from the Wunderlich-Diez Corp.,
`ObR.~yed Ani:ust 15, 1963, from the Department of
`C~t~~~ <ffi1;.:ology, University of Kansas Medical
`~~d for pubUcation November 6, 1963.
`N ti mvertigatian was supported by a i;ro.nt from the
`B:th~~.1M:f~t~ of Bcaltli, U. S. Public licaltb Service,
`M!~e .author is Indebted to Fuad Jarjoura and Sharon
`• nar1ty for their tcchnical assistance.
`R Pnient address: Institute of Medical Education and
`StesearLo'*' St. Louis, Mo., and Department of :Biochemistry,
`· ws Unlvenity SchOol of Medicine, St. LouiB, Mo.
`
`Hasbrouck Heights, N. J. 1 This material was
`exhaustively extracted with hot acetone or: hot
`ethanol. Either solution on removal of solvent
`by distillation (the last stages in tiacuo) yielded
`black, viscous material which was used for further
`examination. Melting points were determined on a
`Fisher-Johns melting poip.t apparatus. Optical
`rotations (in CHC!a) and C-H analyses were
`determined by Drs. G. Weiler and F. B. Strauss,
`Microana.lytical Laboratory, Oxfor:d, England. An
`infrared spectrum of the unidentified diterpene was
`determined on a Perkin-Elmer spectrophotometer
`by the KBr disk method. s An infrared spectrum
`of the sterol was determined in chloroform solution
`in a 0.1-mm. sealed cell, compensated with CHCI.,,
`on a Beckman IR-4 recording infrared spectro(cid:173)
`photometer, 3 and by the KBr disk method. The
`
`t Tb.in lirm w given a""1llUDCC that the material i.nvcsli(cid:173)
`gattd was M. culgare or white horehound, not Ballola hirsula
`(blaclt horehound) •
`•We arc illdcbtcd to the Department of P1Lthology,
`Univuslcy of Kansas for this dctermication.
`• Determined by &dtler Research Laboratories, Phila(cid:173)
`delphia, Pa.
`
`MYLAN PHARMS. INC. EXHIBIT 1022 PAGE 5