THE VITAMIN BI=~ CONCENTRATION IN LIVER EXTRACTS
`AND A NOTE ON THE RELATIONSHIP BETWEEN CLINICAL
`RESPONSE AND BI~ DOSAGE
`By W. F. J. CUTHBERTSON AND JOAN F. LLOYD
`
`Research Division, Glaxo Laboratories, Ltd., Green[ord, Middlesex,
`and W. B. EMERY and K. A. LEES
`
`Fermentation Division, Glaxo Laboratories, Ltd., Barnard Castle, Co. Durham
`
`Received July 12, 1949
`VITAMIN Br_~ CONTENT OF LIVER EXTRACTS
`A NUMBER of different liver extracts have been assayed for vitamin
`activity by the cup-plate method of Cuthbertson1 employing Lactobacillus
`lactis ATCC 8,000. Some of these samples have also been assayed by
`the technique of Lees and Emery~ employing the tube assay with Lacto-
`bacillus leichmannii 313. The results obtained are summarised in
`Table I.
`
`TABLE I
`B~=, CONTENT OF LIVER EXTRACTS FOR PARENTERAL USE
`
`Distributor
`
`Manufacturer
`
`Extract
`
`/zg. Bt.o,iml.~-
`
`Potency
`
`British
`
`European
`
`American
`
`A
`
`B
`
`C
`
`D
`
`E
`
`G
`
`H
`
`I
`
`J
`
`K
`
`L
`
`M
`
`N
`
`0
`
`P
`
`i
`2
`
`3
`
`6
`7
`
`8
`9
`
`10
`
`! l
`
`12
`
`13
`14
`15
`16
`
`17
`
`18
`
`19
`
`20
`
`21
`
`22
`
`1 ’4
`5" 0
`
`6.0
`
`2"9*
`0" 8
`
`I ¯ 0*
`12*
`
`3*
`12*
`
`0.2
`
`6*
`3 "4
`0" 23
`
`2’ 5*
`
`0.5
`
`0- l
`O. 1
`
`(a)
`
`0.1
`p.g. Bm <0 "05~"
`
`2"7
`
`3" 5
`
`22
`
`l - 8
`
`11 - 8
`
`low
`high
`
`high
`
`high
`low
`
`medium
`high
`
`low
`high
`
`high
`
`low
`low
`low
`
`high
`
`high
`
`medium
`high
`
`medium
`high
`
`low
`
`medium
`
`high
`
`high
`
`high
`
`* Average of 2 to 8 different batches. ~" No B~.. detected even after chromatography.
`, lhe essay results given in TABLES I. III, IV and V represent the activity of one or more of the ~.itamin
`Bt2 gt’otlp of factors expressed as the concentration of standard vitamin Bta (~tg/ml) giving the same
`response aS the sample.
`
`Samples used for assay are all recent, having been obtained for the
`most part in June, 1949, except for samples from manufacturer G of
`
`705
`
`F
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`Sandoz Inc. IPR2016-00318
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`
`

`
`W. F. J. CUTHBERTSON et al.
`
`which (a) were obtained during 1936 to 1939 and (b) was captured during
`the war. Of the above samples N, O and P are known to have been
`manufactured in America. The other samples whose manufacturers are
`described as European or American were nevertheless presumably made
`in England, since we are informed that no liver extracts have been
`imported into this country for the last 6 years. The description used is
`thus the country of the manufacturers’ headquarters, but not necessarily
`of the extract’s origin.
`The extracts have been classified into groups of low, medium and high
`potency on the basis of information supplied with the extracts by their
`manufacturers. This classification, shown in Table II, is necessarily
`very rough in the absence of any standard method of describing potency,
`but in assigning the extracts to low, medium or high potency groups,
`the suggested dosing schedules, U.S.P. units and liver equivalents have
`been taken into consideration.
`
`TABLE II
`
`Potency
`
`Stated liver
`equivalent ml.
`
`Low .....................
`
`<10 g.
`
`Medium ..................
`
`I0 to 20 g.
`
`High .....................
`
`>20 g.
`
`;
`
`!
`
`U.S.P. units
`
`2
`
`10
`
`15
`
`No attempt has been made to distinguish between highly refined and
`highly concentrated extracts, although it is obvious that some manufac-
`turers have attempted to produce potent refined materials, while others
`appear merely to have concentrated their liver extracts without any great
`degree of purification.
`Table I demonstrates the wide differences between extracts from
`different manufacturers, the low potency extracts ranging from 0.23 to
`6 ~tg./ml., while medium and high potency extracts vary over the ranges
`of 0.1 to 3-6 ~g. and 0"1 to 22 ~tg. B1..,/ml. respectively, These variations
`between samples of different origins would hardly be expected if adequate
`clinical trials had been carried out on all batches. This variation may be
`
`TABLE IIi
`
`BATCH TO BATCH VARIATION OF SAME BRAND OF EXTRACTS FROM DIFFERENT
`MANUFACTURERS
`
`Manufacturer
`
`Extract Sample L p,g. Bll/ml.
`
`Manufacturer Extract Sample
`
`/~g. B~l/ml.
`
`X
`
`Y
`
`B
`
`C
`
`9
`10
`1I
`
`4.8
`19.8
`11"3
`
`2"7
`3"6
`2"1
`3-0
`3-3
`
`24.0
`6’0
`9’0
`
`706
`
`12
`13
`14
`
`15
`16
`17
`1S
`19
`
`20
`21
`22
`
`15 "0
`7"5
`I0 "0
`
`8 "0
`5 "0
`5 "0
`15 "0
`9.0
`
`17.5
`12.0
`12.0
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1109-0002
`
`

`
`VITAMIN Bt2 CONCENTRATION IN LIVER EXTRACTS
`
`ascribed to differences irt manufacturing technique, quality of raw
`materials and clinical control (if any) of the products. Different batches
`of the same extract made by the same manufacturer may show a wide
`range of B~_~ concentrations, but it cannot be said whether these may not
`be due to alterations in procedure and materials available for manufac-
`ture. Table III shows the degree of variation encountered.
`
`From the results it is clear that little value can be attached
`to estimates of potency unless better controI is used than apparently at
`present. A number of these samples have been assayed by both of the
`different techniques employed in these laboratories, with the results shown
`in Table IV.
`
`TABLE IV
`
`MICROBIOLOGICAL ASSAY OF PARENTERAL LIVER EXTRACTS BY CUP-PLATE
`
`(L. LACTIS) AND TUBE (L. LEICHMANNII~ METHODS
`
`Extract
`
`~ Tube assayp.g. Bl~/ml.
`
`Plate assay/~g. Bt~/ml,
`
`1 .....................
`2 .....................
`3 .....................
`4 .....................
`5 .....................
`6 .....................
`7 .....................
`8 .....................
`9 .....................
`10 .....................
`11 .....................
`12 .....................
`
`0’05*
`0’04*
`0’6*
`ca 0’1"
`0"14
`0’4
`0-7
`1"5
`2"8
`3 ’0
`3"3
`9"7
`
`<0- I*
`<0-05"
`<0" 1"
`<0" 2*
`0-22
`0"8
`1’0
`1.4
`2.7
`3"5
`3"3
`10"5
`
`High concentrations of desoxyribosides present in these samples.
`
`On the whole the agreement is reasonable for methods involving two
`different organisms and two different techniques (the cup-plate and tube
`method) having different sensitivities to interfering substances. The
`discrepancies encountered are being further investigated.
`
`RELATIONSHIP BETWEEN CLINICAL RESPONSE AND MICROBIOLOGICAL
`ACTIVrFY
`All liver extracts prepared in these laboratories are clinically tested
`before they are released for sale. For this purpose typical cases of
`Addisonian pernicious anaemia in severe relapse are used, preferably those
`showing a red cell count between 1 and 2 million/cmm. The patients
`receive, by intramuscular injection, a single test dose of the extract.
`During the following 14 days the blood picture is determined on alter-
`nate days, but daily observations are made at the time when the peak of
`the reticulocyte response is expected. In interpreting the results par-
`ticular attention is paid to the red cell response, which should increase
`at the rate expressed by the formula of I = 0’94 - 0"214 E0 (Della
`Vida and Dyke3), where I is the weekly increment in the red cells and Eo
`is the initial cell count. In assessing the response of an individual
`patient, consideration is given to other factors that may bear on the
`test, e.g., h~emoglobin levels, shape of the response curves, reticu-
`
`707
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`Sandoz Inc. IPR2016-00318
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`
`

`
`W. F. J. CUTHBER-FSON et al.
`
`locyte response, infection and possible iron deficiency or abnormal red
`cell destruction. Foods that might have an anti-amemic action are with-
`held before and during the period of test.
`
`The results on 14 different liver extracts with I8 patients in a number
`of different hospitals are summarised in Table V, which gives only the
`red cell response as a percentage of that to be expected from the Della
`Vida and Dyke formula and the Ba~. content of the different extracts, The
`other observations made on these patients have been omitted for clarity.
`If the responses of the individual patients are considered and if a
`response of 90 per cent. of that expected is taken as indicating activity of
`the liver extract, then it can be seen that samples containing less than
`
`RELATION BETWEEN CLINICAL RESPONSE AND BI2 ACTIVITY OF EXTRACT
`(1 ML. OF EXTRACT USED FOR THESE TESTS)
`
`TABLE V
`
`Sample
`
`Increase
`in
`red blood
`cells
`Ba.. activity expressed
`as
`/zg.i m[.
`percentage
`of
`expected
`response
`
`Sample
`
`B1.- activity
`/~g.iml.
`
`in
`red blood
`ceils
`expressed
`
`percentage
`of
`expected
`response
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`g
`
`2.0
`
`2-9
`
`3-0
`
`75
`
`9 .........
`
`nil)~
`114~ 57
`
`10 .........
`
`9.0
`
`9.0
`
`62
`
`58
`
`nil
`
`11 .........
`
`12.0
`
`144~) 91
`
`4 "0 76
`
`4" I
`
`4"5
`
`6 "0
`
`6-0
`
`nil
`
`54
`
`50
`
`109
`
`12’0
`
`13 .........
`
`15.0
`
`93
`
`17"5
`
`10 ~g./ml. are much less satisfactory than those containing more than
`10 ~g./ml. Of the 11 patients receiving less than 10 t~g. of BI: 9 gave
`unsatisfactory responses, while of the 7 patients who received more
`than 10 ~g. only 2 gave unsatisfactory responses. Several of these extracts
`were tested on more than one patient. The marked variation in response
`from patient to patient is very clearly seen in the results obtained with
`these. In particular the different patients receiving samples 2 and 11
`show very wide differences in response. If the results obtained with the
`two patients on each sample are averaged, then it can be seen that only
`one extract out of 10 containing less than 10 ,g./ml. would satisfy our
`criterion (and this result depends on the reaction of only one patient)
`while none of the four extracts containing more than 10 ~g. B,~,/ml.
`would have failed to do so.
`
`708
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1109-0004
`
`

`
`VITAMIN B~ CONCENTRATION IN LIVER EXrRACTS
`
`SUMMARY
`1. The vitamin BI~ contents of a number of liver extracts have been
`reported.
`2. Assay results using Lactobaccillus lactis and L. teichmannii haxe
`been compared.
`3. The clinical responses to a number of extracts have been compared
`with their Br_, contents, and the range of activity found by clinical test
`has been indicated.
`We wish to express our thanks to the pathologists without whose
`enthusiastic co-operation the standardisation of liver extracts would
`not have been possible.
`
`I. Cuthbertson, Biochem. J., 1949, 44, v.
`2. Lees and Emery, Biochem. J., 1949, 45, ii.
`3. Della Vida and Dyke, Lancet, 1942, 243, 275.
`
`REFERENCES
`
`DISCUSSION
`
`THE three papers dealing with the micro-biological assay of liver extracts
`by Mr. Shaw and by Dr. Cuthbertson, Miss Lloyd, Dr. Emery and Mr.
`Lees were discussed together; the last paper was read by Mr. Lees.
`MR. SHAW, in presenting his papers, stated that since submitting them
`he had found that on applying his assay to a commercial solution of
`crystaIline vitamin BI: prepared for injection, the indicated vitamin
`content appeared to be approximately three times as great as the labelled
`value, using as reference standard the solid liver preparation supplied by
`Dr. Rickes and standardised by him at 0,4r~g. per rag. This observation
`indicated that the results quoted in the paper represented not necessarily
`vitamin B~ as such but were a measure of the growth activity for Lacto-
`bacillus lactis Dorner. This discrepancy along with the extremely slow
`speed of migration of the main constituent on paper chromatography,
`and the American view that microbiological assay of liver extracts for
`vitamin B~,_, is not reliable unless the vitamin BI_~ content of the liver
`solids in the preparation under test is of the order of 50 per cent. suggests
`the possibility that the clinical action of liver extracts may be due to a
`complex or conjugate of vitamin Ba_~ more than to the presence of the
`free vitamin. It might well be that for the assay of liver extracts a
`standard liver preparation will be a more satisfactory reference standard
`than pure crystalline vitamin Bz2.
`The CHAIRMAN said that the three papers dealt with a subiect which
`had been developed very considerably in the last year or so. If the
`figures given for assays of commercial extracts really represented their
`content of vitamin BI~ then he thought that they gave a very disturbing
`picture of the state of affairs. The assay process required improvement
`before it was possible to place reliance on it, but the results given in the
`papers suggested that there might be some relationship between it and
`the clinical response. Mr. Shaw used a method of paper strip chromato-
`
`709
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1109-0005