MW, 1923
`
`AMERICAN PHARMACEUTICAL ASSOCIATION
`
`229
`
`THE BIOLOGIC STANDARDIZATION OF LOCAL ANAESTHETICS.
`WITH SPECIAL REFERENCE TO EFFECT on STERILIZATION ON SOLUTIONS OF COCAINE
`AND PROCAINE.
`
`(Continuation of a Previously Reported Paper.1)
`BY PAUL S. PITTENGER.
`
`In a paper1 read before this section last year, the author described a biologic
`method for the quantitative valuation of local anaesthetics.
`It was shown that
`the method described was sensitive to a variation of 1 mg. of cocaine and to 5 mg.
`of procaine.
`The sensitiveness of the method suggested its adaptability for checking the
`effect of aging and the various forms of sterilization upon solutions of cocaine and
`procaine. Accordingly, a series of experiments were started as follows:
`
`First.——Prepare a solution of "cocaine and one of procaine of such strength that 1 cc will
`contain the minimum amount of salt that will produce local anaesthesia in 5 minutes when tested
`by the foregoing method, reserving a sample of each of the salts used.
`Cocaine 0.6% solution
`
`} based on Experiments Nos. 1 and 2.1
`
`Procaine 7.0% solution
`Second.—Divide each solution into four portions, fill each portion into ampuls as follows:
`(/1) Fill into sterile ampuls without sterilizing or Berkfeldting.
`(B) Add 0.3% Three Cresols, Berkfeldt and fill into sterile ampuls.
`(C) Fill into ampuls and Arnold—sterilize on three consecutive days.
`(D) Fill into ampuls and autoclave for 15 min. at 115° C.
`TIzird.—-Test each lot to determine effect of sterilization.
`
`Fourth.——Test each lot every three months to determine rate of deterioration on standing.
`Fij'th.—At the end of six months make fresh solutions of the reserve salts and test to de-
`termine whether or not the salt deteriorates with age.
`
`The results of the 18 experiments reported last year showed that the amount
`of solution of cocaine or procaine required to produce complete anaesthesia was
`exactly the same before and after the different forms of sterilization, thus indicating
`that sterilization does not aflect the activity of solutions of cocaine or procaine.
`It was also shown that the unsterilized solutions, the solutions sterilized with
`heat, and the solutions sterilized without heat, all possessed exactly the same
`activity after 3 months as immediately after being prepared.
`Each lot of solution has again been tested 14 months after filling and sterilizing.
`results of this final test on each lot as compared with the two previous tests
`
`;Fl]l€
`
`0 lows:
`
`Experiments Nos. 3, 4 and 19.
`
`COCAINE SOLUTION 0.6%.
`Filled into sterile ampuls without sterilizing or Berkfeldting.
`
`Dose.
`0 .7
`0 -8
`
`1-6
`1
`1 -1
`~
`
`.
`
`Imme(i3i:i)£et:ly1\ai(f)te:1;;"fi1ling
`Sensitive
`Sensitive
`.
`Sensitive.
`*Not sensitive
`*Not sensitive
`Not sensitive
`Not sensitive
`
`.
`
`Results.
`3 !.'I1:CE):Sxpaii:t§IT'of:ll‘l1l.I]g'
`.
`Sensitive
`Sensitive
`Very slightly sensitive
`*Not sensitive
`*Not sensitive
`Not sensitive
`Not sensitive
`
`‘
`
`g_
`
`14 11ili(s»pt'aIf\i:'r 1%-llin
`,
`Sensitive
`Sensitive
`' Sensitive
`Sensitive
`Sensitive
`Sensitive
`*Not sensitive.
`
` O
`
`1 “The Biologic Standardization of Local Anaesthetics,” JOUR. A. PI-I. A., Vol. X, No, 10,
`ctober 1921.
`
`MYLAN ET AL. - EXHIBIT 1015
`
`MYLAN ET AL. - EXHIBIT 1015
`
`

`
`230
`
`JOURNAL on THE
`
`Vol. xn. No.3
`
`Minimum amount required to produce complete local anaesthesia is 1.0 cc
`for both Experiments Nos. 3 and 4, and 1-1 CC for EXPe1'iment N0‘ 19-
`
`Experiments Nos. 5, 6 and 20.
`
`T'/
`PROCAINE SOLUTIONS 7‘/O.
`_
`Filled into sterile ampuls without sterilizing or Berkfeldting.
`Results.
`
`Dose_
`0 8
`0.9
`0_g
`*1 _0
`as] 0
`1
`1
`
`.
`. N . 5.
`Expt. No. 6._
`Expt. No. 20.
`_
`1
`Immegigifily aiitcr filling.
`3 mos. after filling.
`14 mos. ‘after
`filling.
`Sensitive
`Sensitive
`Sensitive
`Slightly sensitive
`5'i‘T15ftfV‘3
`Sensftfve
`Slightly sensitive
`.
`Sensitive.
`.
`Sensitive.
`Not sensitive
`Not sensitive
`Not sensitive
`Not sensitive
`Not sensitive
`Not sensitive
`Not sensitive
`Not sensitive
`Not sensitive
`Minimum amount required to produce complete local anaesthesia is 1.0 cc
`for Experiments Nos. 5, 6 and 20.
`
`Experiments Nos. 7, 8 and 21.
`
`(
`COCAINE SOLUTION 0.6 (1,.
`
`0.3% Three Cresols, Berkfeldted and filled into sterile ampuls.
`Results.
`
`Dose,
`0 .8
`0.9
`0 ,9
`
`1 .0
`
`1 _0
`I .1
`
`Immegliftteléqgitzi filling.
`Sensitive
`Very slightly sensitive
`Sensitive
`
`*Not sensitive
`
`*Not sensitive
`Not sensitive
`
`3 mgifirgrosfiing.
`Sensitive
`Sensitive
`Slightly sensitive
`
`*Not sensitive
`
`*Not sensitive
`Not sensitive
`
`14 n§§.)t'a1ii:i ziilling.
`Sensitive
`Sensitive
`Sensitive
`
`Sensitive
`
`Slightly sensitive
`*Not sensitive
`
`Minimum amount required to produce complete local anaesthesia is 1.0 cc
`for both Experiments Nos. '7 and 8, and 1.1 cc for Experiment No. 21.
`
`Experiments Nos. 9, 10 and 22.
`
`PROCAINE SOLUTION 7%.
`
`0.3% Three Cresols, Berkfeldted and filled into sterile ampuls.
`
`Results.
`
`,
`
`Expt. No. 9.
`Immediately after filling.
`
`Expt. No. 10.
`3 mos. after filling.
`
`_
`Expt. No. 22.
`14 mos. after
`filling.
`
`Sensitive
`Sensitive
`Sensitive
`Not sensitive
`Not Sensitive
`N01 S€I1SitiV€
`
`Sensitive
`Sensitive
`Sensitive
`Not sensitive
`Not sensitive
`Not sensitive
`
`Sensitive
`Sensitive
`Sensitive
`Not sensitive
`Not sensitive
`Not sensitive
`
`Dosc.-
`
`0 .8
`0 .9
`0 . 9
`*1 .0
`*1 .0
`1 -1
`
`Minimum amount required to produce complete local anaesthesia is 1.0 CC
`for Experiments Nos. 9, 10 and 22.
`
`Experiments Nos. 11, 12 and 23.
`
`COCAINE SoLULioN i().6€’('_,
`Filled into ampuls and Arnold—sterilized on 3 consecutive days.
`Results.
`E t_ N 12
`3 mg: afte(i‘ifilli'ng.
`Sensitive
`Sensitive
`
`14
`
`t N _ 23,
`15:5‘? iaftgr
`filling.
`Sensitive
`’ Sensitive
`
`_
`l)ose.
`0 .8
`0 .9
`
`E_Xpt. NO. 11.
`Immediately after filling.
`Sensitive
`Sensitive
`
`

`
`March 1923
`
`AMERICAN PHARMACEUTICAL ASSOCIATION
`
`231
`
`Dose.
`09
`1' 0
`1.0
`1.0
`1.1
`
`Imme_g:€:jy1:?{e1r1fi11ing_
`Sensitive
`=o=N0t sensitive
`*NOt sensitive
`_
`,
`_
`,
`Not sensitive
`
`. 2.
`
`X
`
`.
`
`3 ‘rgosiatafiig fi1.lli.ng.
`Slightly sensitive
`Not sensitive
`Not sensitive
`.
`.
`.
`Not sensitive
`
`.
`
`.
`
`.
`
`14 ziiggtag.-ti gging.
`Sensitive
`Slightly sensitive
`‘_"N0t sensitive
`- Slightly sensitive
`*Not sensitive
`
`Minimum amount required to produce complete local anaesthesia is 1.0 cc
`for both Experiments Nos. 11 and 12, and between 1.0 and 1.1 cc for Experiment
`
`No. 23.
`
`Experiments Nos. 13, 14 and 24.
`
`PROCAINE SOLUTION 7%.
`
`Filled into ampuls and Arnold-sterilized on 3 consecutive days.
`
`Results.
`
`Expt. NO. 13.
`Immediately after sterilizing.
`
`Expt. No. 14.
`3 mos. after sterilizing.
`
`Expt. No. 24.
`14 mos. after sterilizing.
`
`Sensitive
`Sensitive
`
`Sensitive
`
`Not sensitive
`
`Not sensitive
`
`.
`
`.
`
`.
`
`.
`
`Not sensitive
`
`Sensitive
`Sensitive
`
`Sensitive
`
`Not sensitive
`
`Not sensitive
`
`.
`
`.
`
`.
`
`.
`
`Not sensitive
`
`Sensitive
`Sensitive
`
`Sensitive
`
`Not sensitive
`
`Slightly sensitive
`
`Not sensitive
`
`Not sensitive
`
`Dose.
`
`0 ,8
`0 _9
`
`0 .9
`
`*1 ,0
`
`*1.0
`
`1.0
`
`1.1
`
`Minimum amount required to produce complete local anaesthesia is 1.0 cc
`for Experiments Nos. 13, 14 and 24.
`
`Experiments Nos. 15, 16 and 25.
`
`COCAINE SOLUTION 0.6%.
`
`Filled into airipuls and autoclaved for 15 min. at 115° C.
`
`DOSE.
`0 -8
`0 -9
`:33
`*1'0
`1'1
`-
`
`Immediiiigiy lgrciérl iterilizing.
`Sensitive
`Slightly sensitive
`Not sensitive
`1IjI0t Sensitive
`0t Sensitive
`Not sensitive
`
`Results.
`
`3 I110§.xgitelfgteiiilizing.
`Sensitive
`Sensitive
`_
`Sensitive.
`Not sensitive
`Not sensitive
`Not sensitive
`
`14 mtgsxgitgizitgréilizing
`Sensitive
`Sensitive
`Sensitive‘ .
`Not sensitive
`Not sensitive
`Not sensitive
`
`f Minimum amount required to produce complete local anaesthesia is 1.0 cc
`OI Experiments Nos. 15, 16 and 25.
`Experiments Nos. 17, 18 and 26.
`
`PROCAINE SOLUTION 7%.
`Filled into ampuls, autoclaved for 15 min. at 115° C.
`EX!” N0- 17-
`R6521:Si No 18
`ImmSedi3~t_€1'Y after Stefiliziflg.
`3 mos. alfftier sterilizing.
`Sfinillgve
`.
`I
`Sensitive
`.
`'
`Sefsitiy Sensltive
`Slightly sensitive
`Not SeI\:€.t.
`SEIISIUVC.
`.
`Not Sen:t}V€
`Not sensitive
`Not SenSitfV9
`Not sensitive
`
`E t No 26
`14 mosfigfter sterilizing.
`Sensitive
`Sensitive
`.
`Sensitive.
`Not sensitive
`Not sensitive
`
`Not sensitive
`
`We
`
`Not sensitive
`
`Dos
`0.;
`0.9
`0.9
`:10
`#10
`1_1
`
`fOrEx1n1ff1um amount required to produce complete local anaesthesia is 1.0 cc
`Periinents Nos. 17, 18 and 26.
`
`

`
`232
`
`JOURNAL OF THE
`
`Vol. X11, u.,_3
`
`In order to determine whether or not the salts deteriorate with age, fresh solu-
`tions of the reserve salts were made after a period of 14 months and tested for com-
`parison with the tests of the original solutions.
`
`Experiment No. 27.
`
`COCAINE SOLUTION 0.6%.
`
`Results
`
`Dose
`0 ,7
`0 .8
`0 .9
`0,9
`*1_.0
`*1 .0
`1 .1
`
`Expt_ No 3
`Sensitive
`Sensitive
`Sensitive
`Not sensitive
`Not sensitive
`Not sensitive
`Not sensitive
`
`Expt NO. 27.
`Sensitive
`Sensitive
`Sensitive
`Sensitive
`Not sensitive
`Not sensitive
`Not sensitive
`
`Minimum amount required to produce complete local anaesthesia is 1.0 cc
`for both Experiments Nos. 3 and 27, showing that no deterioration takes place in
`cocaine during a period of‘ 14 months.
`
`Experiment No. 28.
`
`Made from reserve salt 14 months after solution tested under Experiment No. 5.
`
`PROCAINE SOLUTION 7%.
`
`Dose
`0 .8
`0.9
`0 .9
`*1 .0
`
`*1 .0
`
`1 . 1
`
`Results.
`
`Expt. No: 5.
`Sensitive
`Slightly sensitive
`Slightly sensitive
`Not sensitive
`
`Not sensitive
`
`Not sensitive
`
`Expt. No. 28.
`Sensitive
`Slightly sensitive
`Slightly sensitive
`Not sensitive
`
`Not sensitive
`
`Not sensitive
`
`Minimum amount required to produce complete local anaesthesia is 1.0 cc for
`both Experiments Nos. .3 and 28 showing that no deterioration takes place in pro-
`caine during a period of 14 months.
`
`EFFECTS OF REPEATED STERILIZATION.
`In the discussion of last year’s paper it was stated that in some operations
`surgeons use solutions of cocaine as high as 20% in strength and that owing to the
`general opinion that resterilization destroyed its activity,
`in most hospitals all
`unused portions of such solutions were discarded.
`Therefore, in order to determine the effects of resterilization upon the activiil)’
`of solutions of cocaine and procaine, I prepared a 20% solution of cocaine and a. 50%
`solution of procaine, and filled each into ampuls. One ampul of each lot was diluted
`to the proper strength and tested on dogs. The remaining ampuls were then au’£0‘
`claved at 115° C. for 15 minutes. Another ampul of each lot was then diluted to
`the proper strength and tested. The remaining ampuls were again autoclaved at
`115° C. for 15 minutes and another ampul tested. This procedure was I‘€Peated
`until each lot was sterilized and tested five times.
`
`The results of
`
`these experiments follow:
`
`

`
`M923
`
`Marc
`
`AMERICAN PHARMACEUTICAL ASSOCIATION
`
`.
`
`233
`
`_
`
`S°m.°i1° t- n
`Bef°‘° Sterfllii lotion
`After lst Sterllfaation
`After 2nd Sterliizzation
`After 3r:S:::1iZatiOn
`:31 Eterflization
`
`Solutiom
`Before Sterilization
`After lst sterilization
`After 2nd Sterilization
`After 3rd sterilization
`After 4th sterilization
`After 5th sterilization
`
`COCAINE 20% SOLUTION.
`
`Amount required to produce complete
`local anaesthesia.
`
`1.0 cc (0.6%)
`1 .0 cc (0 .6%)
`1 .0 cc (0 .6_%)
`1.0 cc (0 .6%)
`1 .0 cc (0.6%)
`1 .0 cc (0.6%)
`
`PROCAINE 50% SOLUTION;
`
`Amount required to produce complete
`local anaesthesia.
`1.0 cc (7.0%)
`1 -0 CC (7 -0%)
`1 .0 cc (7.0%)
`1 .0 CC (7 -0%)
`1 .0 cc (7 -0%)
`1 .0 CC (7 -0%)
`
`Physical appearance.
`
`Clear, colorless
`Clear, colorless
`Clear, colorless
`Clear, colorless
`A Clear, colorless
`Clear, colorless
`
`_
`Physical appearance.
`Clear, colorless
`C1931’: amber
`Clear, amber
`C1931‘: amber
`C1931‘, amber
`Clear» amber
`
`These results show that five successive sterilizations produced no apparent
`effect upon the activity of the above solutions of cocaine and procaine.
`It will also be noted that sterilization at 1 15 ° C. changes the procaine solution
`from colorless to an amber color, and that this change is not accompanied by any
`change in activity.
`'
`
`CONCLUSION.
`
`The activity of local anaesthetics can be quantitatively determined by the
`method proposed.
`The activity of solutions of cocaine and procaine is not affected by the addition
`of 0.3% Three Cresols or by sterilization by means of the Arnold sterilizer or auto-
`clave at 115° C. for 15 minutes.
`5,
`Unsterilized solutions of cocaine and procaine, solutions sterilized without heat
`and solutions sterilized with heat apparently lose no activity during a period of
`3months.
`'
`V
`
`~07
`9/0
`
`a
`
`Unsterilized solutions of cocaine and solutions of cocaine sterilized Without
`heat apparently lose about 10% of their activity during a period of 14 months.
`Solutions of cocaine which have been Arnold-sterilized apparently lose about
`0f their activity during a period of 14 months.
`Solutions of cocaine which have been autoclaved for 15 minutes at 115° C.
`pparenfly lose no activity during a period of 14 months.
`ti0nSUIt1Sterilized‘solutions of procaine, solutions sterilized without heat and solu-
`S erilized with heat, lose no activity during a period of 14 months.
`Etocffine fmd procaine in the dry form are apparently stable and show no signs
`emfat19n during 14 months.
`
`old
`
`times at 115° C. with
`out any apparent loss of activity.
`Finau
`Quicifor hy the author wishes to acknowledge his indebtedness to Mr. Arnold '
`1s able assistance in connection with the laboratory work.
`PHARMACODYNA
`MIC LABORATORY,
`H. K. MULFORD COMPANY,
`AUGUST 8, 1922.