clature we refer to these substances as plasma Ac-globulin and
`serum Ac-globulin. The properties of the two are so similar
`that no difference has been detected, ’even though both have
`been obtained in concentrated form.
`Thrombin is the substance which is responsible for the
`production of serum Ac-globulin, and calcium ion is not
`required fo~ that purpose. The most active thrombin prepara-
`
`B
`
`A
`
`With Serum
`AC- globulin
`
`With Plasmo
`Ac-qlobulin
`
`I I I ~ I I I I I I I I I
`8 12 16 20 24 28 52
`Time in minutes
`PROTHROMB|N + THROMBOPLASTIN ~ THROMBIN
`
`Fro. 1. Activation of purified prothrcmbin w|th serum Ac-globuiin
`(curve A) and with plasma Ac-globulin (curve B) in the pre~ence of excess
`thromboplastin and optimum calcium-ion concentratlo~.
`
`tions obtained to date have been added to large quantities of
`oxalated bovine plasma, and subsequently, concentrates of
`serum At-globulin have been obtained in quantity and quality
`equal to those obtained from bovine serum itself.
`The function of Ac-globulin in the clotting mechanism can
`then be outhned by use of the following equations:
`Ca++
`(1) Prothrombin + Thrcmboplastin ~ Thrombin
`(2) Plasma Ac-globulin Thrombln÷ Serum Ac-globufin
`Ca++
`Serum Ac-globulin"-->
`
`(3) Prothrombin + Thromboplastin
`Thrombin
`(4) Fibrinogen
`
`Thrombin
`.-~ Fibrin Clot
`
`The clotting reaction is initiated by thromboplastin which
`comes from platelets and tissue juices. Some of the newly
`formed thrombin alters plasma Ac-globulin so that it becomes
`serum Ac-globulin. The latter intensifies the interaction of
`prothrombin and thromboplastin. Thrombin thus accelerates
`its own formation through an intermediate. This may be
`regarded as co-autocatalysis. These conclusions differ dis-
`tinctly from these of Owren (3), but are in harmony with the
`old and well-known evidence presented in the literature to
`show that autocatalysis is involved in thrombin formation.
`This is, however, not autocatalysls but co-autocatalysis, be-
`cause an intermediate is involved.
`We have found that neither serum Ac-globulin nor plasma
`Ac-globulin can substitute for thromboplastin in the activation
`of prothrombin in the presence cf optimum amounts of cal-
`cium ion.
`The curves of Fig. 1 were obtained with the use of prothrom-
`bin prepared by (NI-I,)2SO, fractionafion as described pre-
`viously (4). This product possessed a maximum specific
`
`SCIENCE, December 19, 1947
`
`activity of 23,000 units/rag, of tyrosine. The activity was
`measured by the two-stage method (7). Plasma Ac-globulin
`was purified by the method briefly outlined (5). The same
`procedure was used for the preparation of serum Ac-globufin.
`
`References
`I. FANTL, P., and N.~Jqc~. M.. Natgr¢, Lond., 1946, 1~, 708.
`2.0~, P. A. La~¢~t, 1947, ~2, ~6.
`3. Ow~, P.A. The coag~laHon o] bl~d:
`]actor. Oslo: ]. Chr. Gunderson, Bok~kkeri, 1947.
`4. S~o~s, W. H., Looms, E. C., and V~g~¢,
`1945, 6, 85.
`5. W~, A. G., G~s~, M. M., and S~zozgs, W.H. ]. ~iol. C~m., 1947,
`169, 231.
`6. W~, A. G., G~s~, M. M., and Szzo~as, W.H. Science, 1947, 106, 41.
`
`1936, 114,
`
`The Action of Pteroylglutamic
`Conjugates on Man1
`
`SIDNEY FARBER~ ELLIOTT C, CUTLER~
`JA~Es W. HAWKINS, J, HARTWE~L HARRISON,
`E. CONVERSE PEIRCE, 2~O, and ~B~g~ G. LENZ
`
`The Childrenis Hospital, Pet~ Bent Brigham
`Hospital, N~ England Deaconess Hospital,
`BosUn, and Departments of P~hology and
`Surgery, Harvard M~ic~ StOol
`
`In i9~, ~uchtenberger, Lewisohn, Laszto, and Leuchten~
`berger (~ reported that a ".fore acid concentrate" and a
`fermentation L. easel fac~r inhibited the ~owth of sarcoma
`180 transplanted in female Rockland ~ce. Further studies by
`Lewisohn and his co-workers (5) in 1945 showed complete
`recession in about one-third of the single spontaneous breast
`cancers obser~ed in three different strains of ~ce treated with
`daily intra~’enous injections of 5 gg. of fe~entation L. casd
`factor. This substance was thought at that time to be fofic
`acid; it is now known that it was a conjugate of fohc acid,
`pteroyltriglutamic acid (3). Subsequent work showed that
`pteroylglutamic acid (fofic acid), when tested under similar
`conditions, was not effective in producing regression of these
`breast cance~ (~.
`In 19~, Hutchin~, a d. (3) reported the isolation of the
`fermentation L. casei factor. THs compound was shown to be
`~0 per cent as active when assayed with L. casd and 2-
`6 pe~ cent as active when assay~ with Str. faecalls ~ as was
`the previously isolated fiver L. cpsd factor, pteroylgluta~e
`acid (8).
`Degra~tive reactions have shown that the fermentation
`L. casei factor differs from pteroylglutamic acid in that the
`
`1We are grateful for the cooperation of Y. SubbaRow and his colleagues in
`the research division of the Lederle Laboratories Division, .American Cy-
`anamid Company, who are responsible for the chemical research which
`forms the foundation of these studies, and to Benjamin Carey, who made
`available these substances for experimental trial. The compounds were-
`furnished in the form of dry material, yellow-orange in color, in steril~
`vials, under the names teropterin and diopterin.
`Thanks are due to the staffs of The Children’s Hospital, the Peter Bent
`Brigham Hospital, and to Shields Warren a~d the staff of the New England
`Deaconess Hospital, Boston, for invaluable assistance and cooperation
`which will be acknowledged specifically in detailed reports to be published.
`The assistance of Elisabeth Blumenthal, R.N., is gratefully acknowledged.
`Supported in part by National Cancer Grant 250, U. S Public Health
`Service.
`
`619
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1107-0001
`
`

`
`former ~z0~r~pnund contains two additional moles of glutamic
`~cid (1), Consequently, pteroyl-3,-glutamyl--r-glutamylglu-
`~;mi, c acid (pteroyltriglutam~c acid or teropterln) was syn-
`thesized and found to have microbiological activities identical
`nvi~h ~hose of the naturally occurring fermentation L. easel
`factor (2). During the course of this synthetic work the com-
`pound pteroyl-a-glu~:amylglutamic acid (pteroyldiglutamic
`acid or di~pterin) was also prepared and found to be only
`slightly active when assayed with L. easel and Stx. faecalis
`R (7). This compound is not a naturally occurring substance.
`Its preparation indicated the possibility of further research
`with other niembers of the pteroylglutamic series.
`The synthesis of these two compounds by SubbaRow and
`his co-workers made possible experimental clinical studies
`with new substances of the glutamlc series of known chemical
`structure. Our decision to employ these compounds on patients
`with malignant disease was based on a critical analysis of the
`data in the cited reports of the animal experimental work by
`Lewisohn and his co-workers on the effect of the fermentation
`Lo ¢asei factor (now known to be pteroyltriglutamic acid),
`~I~ i~ the purpose of this note to report briefly some obser-
`vations made in conjunction with the administration of these
`~nd closely related substances to 90 patients with malignant
`<tisease. Only those patients for whom established therapeutic
`]procedures offered no hope of cure were selected for treatment
`with these compounds. This necessary restriction to patients
`with advanced neoplastic disease, most of them with metas-
`tases and many of them treated previously with X-radiation,
`makes difficult the interpretation of data and necessitates
`large numbers of observations. It is too soon to attempt any
`evaluation of the action of these substances on the Course of
`neoplastic disease in man. This note will therefore be limited
`chiefly to a consideration of toxicity, dosage, method of ad-
`ministration, and ~ertain general effects. Detailed clinical and
`pathological studies will be reported later.
`This series includes patients with acute leukemia; astrocy-
`toma; Ewing’s tumor; carcinoma of the rectum, colon,
`stomach, cervix, prostate, pancreas, esophagus, bladder,
`breast, gall bladder, kidney, and ovary; ttodgkin’s disease;
`lymphosarcoma; osteogenic sarcoma; ependymoma; spon-
`gioblastoma mulfiforme; seminoma; hypernephroma; leio-
`rayosarcoma of the stomach; chondro~arcoma; epidermoid
`carcinoma of the pharynx and of the tongue; and embryoma
`of the kidney.
`The patients varied conslderably in age, 8 being under 3
`years of age; 29 from 4 to 10; 4 from 11 to 20; 8 from 21 to
`~0; 10 from 31 to 50; 28 from 51 to 70; and 3 over 71.
`The duration of treatment varied from a few days to 5
`months; the average length of treatment was about 5 weeks.
`’ After cautious initial trials were made, pteroyltriglutamic
`add (t~ropter~n) was administered in daily doses varying from
`
`-10 t0’-lS0 rag. intramuscularly and in other patients from 20
`to S00 -rag. intravenously. Pteroyldiglutamic acid (dioptcr~n)
`.:Was given in amoUnts from 50 to 250 rag. intramuscularly and
`from 20 to 300 reg./day orally. One patient received 19,000
`rag. of pteroyltriglut~mlc acid over a period of 5 months, and
`12,740 rag. were given intravenously to another in the space
`of 6 weeks, in both instances without evidence of toxicity.
`On the basis of experience alone our present initial trcat-
`~nt calls for the a&ninistrafion of 20 rag. daily of either
`~l~tance intramuscularly for one week, after which the dose
`
`is raised to 50 mg.[day for two to three weeks longer. De-
`cision concerning further experimental study is then made
`after the status has been evaluated.
`Each substance was dissolved easily in from 1 to 8 cc. of
`saline for intramuscular or intravenous administration. When
`large amounts of the material were used intravenously, as
`large a volume as 20 cc. of normal saline was employed.
`There have been no reactions following ~ntra~enous ad-
`ministration of either substance. No important local reactions
`following intramuscular injection of pteroyltriglutaxaic acid
`have been observed. Intramuscular injection of the diglutamic
`compound has been followed in some, but not in all, patients
`by moderate local reactions described as local burning and
`slight aching~ lasting generally not more than several hours.
`This reaction was said to be usually no worse than that follow-
`ing intramuscular injection of penicillin. In no instance was
`there a local reaction which required special treatment.
`Systemic reactions have not been observed. No important
`changers in pulse, respiration, or temperature were noted,
`nor have there been any significant long-term variations in
`
`¯ blood pressure. There have been.no allergic reactions of either
`major or minor degree. Detailed clinical laboratory studies
`were made; analysis of these data revealed no evidence of a
`deleterious action of the substances administered.
`Twenty-seven of the patients with advanced neoplastic
`disease have died~ and, of these, 13 were examined post mot-
`tern. Exclusive of i1 patients with acute leukemia from whom
`biopsies of bone m~rrow were obtained, there were 11 patients
`from whom biopsies of the tumor were obtained both before
`treatment was instituted and after treatment had been carried
`out for a period of at least a few weeks. Study of the gross and
`histological material available from these patients revealed
`no change in organs and tissues which could be regarded as a
`deleterious effect of the substances employed. In no instance
`was there any evidence of pancytopenla, agranulocytosis,
`degeneration of the kidneys, liver, or myocardium~ or any
`suggdstion of a polyarterifis.
`The limited number and the short duration of these ob-
`servations, and the possible role of psychotherapy, necessitate
`postponement of any conclusion concerning general effects
`upon the patient. In general, adult patients experienced
`improvement in energy, appetite, sense of well-being, and
`appeared to demonstrate’ less irritability and apprehension.
`In many instances, but not in all, such improvement might
`be ascribed to improved morale resulting from frequent visits,
`more medical attention to details of their complaints, and a
`definite impression that something more than usual was being
`done for them. In a few instances there was a definite dimin-
`ution in pain which could be measured by a reduction in the
`amount of sedation or analgesia required.
`Analysis of the collected data oa the group as a whole
`showed that in a few instances conditions were such that a
`causal relationship was apparent between the administration
`of the glutamic compound employed and changes in the
`patient’s condition or in the histological appearance of the
`tumor obtained at biopsy or at autopsy. In a larger group of
`patients with a clinical picture complicated by the use of
`more than one therapeutic agent (such as radiation therapy)
`in addition to the glutamic compound employed, changes
`were observed under conditions which suggested that it was
`¯ the addition of the glutamic compound which played an
`
`December 19, 1947
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1107-0002
`
`

`
`important part in their appearance. Examples of these changes
`were: temporary (several weeks) decrease in the size of multiple
`subcutaneous nodules of an amelanotic carcinoma; temporary
`(several weeks) decrease in size of metastases to the lung from
`a carcinoma of the testis; degeneration and necrosis which on
`two occasions was massive, as seen on pathological exami-
`nation of tumors; reduction to normal on two occasions of
`several weeks each in the acid phosphatase level in the blood
`of a patient with multiple metastases to bone from a carcinoma
`of the prostate. Such changes have been by no means constant.
`They have occurred frequently enough, however, to warrant
`further experimental studies of the action of these and of
`related compounds on patients with cancer.
`This preliminary report of the action of pteroyltriglutarnic
`acid and pteroyldiglutamlc acid on man reveals that these
`substances, as employed in these studies, are nontoxic and
`may be’ gi~cen either intravenously or intramuscularly. The
`absence of evidence of toxicity, as shown by clinical, labo-
`
`ratory, and post-mortem studies, and the ease of administra-
`tion indicatethat these substances are suitable for clinical use.
`No evidence has been presented in this report to suggest that
`these substances should be employed in the routine therapy of
`patients with cancer. Enough has been learned from these
`studies, however, to indicate that further investigation of the
`action of these and related compounds would be of interest.
`
`:References
`1. AUOXER, R. B., et al. Science, 1946, 103, 667.
`2. BOOTnE, J. ~-[., et al. J. Amer. chem. Sot., in press.
`3. HUTCHINGS, B. L., STOKSTAD, E. L. R., ]~OHONOS, N., and
`N.H. Science, 1944, ~9, 371.
`4. LEUCHTENBERGER, C., LEWISOHN, R., LASZLO, 9., and LEUCHTENBER-
`GER, R. Proc. Soc. exp. Biol.Med.,1944,55,204.
`5. LEUCHTENBERGER, R., LEUCHTENBERGER, C., LASZLO, D., and LEWI-
`SOHN, R. Science, 1945,101, 46.
`6. LEWISOHN, R.,LEUCHTENBERGER, C., LE~CIITENBERGER, R., and
`ESZTESY, J. Sc4cnce, 1946, I~4, 436.
`7. MbWAT, J. H., et al. J. Amer. chem. Sot., in press.
`8. STO~CSrAD, E. L.R../. biol. Chem., 1943, 149, 573.
`
`IN THE LABO,RATO RY
`
`A Mincing Apparatus for the Preparation
`of Embryo Extract for Tissue Culture*
`
`around the cup and could be tightened by means of a screw
`in contact with a flattened area on the outside of the tubular
`container (A).
`
`ESTHER CARPENTER
`
`Smith College, Northampton, Massachusetts
`
`In order to obtain more uniformity in the preparation of
`embryo extract, a number of substitutes have been suggested
`for the tedious method of cutting with scissors until pieces of
`tissue are too small to be identified. One of the simplest of
`these is the method suggested by Earle (1) in which a piece of
`monel metal screen of known mesh size is inserted in the base
`of a syringe, enough pressure then being exerted on the
`plunger to force the embryonic material through the mesh.
`This works satisfactorily with young embryos. If, however, one
`is using chick embryos of I0. or more days of incubation, two
`problems arise: (i) it is very difficult to exert enough hand pres-
`sure on the plunger to force the material through the screen;
`and (2) the. increase in pressure is accompanied by danger of
`breakage of the syringe.
`To circumvent these difficulties the equipmen~ illustrated
`in Fig. 1 was devised and made of monel metal with the help
`of Russell Douglas, of the Physics Department. The tubular
`cup (A) is large enough to contain at least two 10-day chick
`embryos. At one end the cup is closed by a disc perforated by
`holes about 1 ram. in diameteL. At the other end the inside of.
`the cup is threaded to match the threads on the plunger (B).
`The latter is equipped with a horizontal handle by means of
`which the plunger can be screwed in far enough to force all
`the material in the tubular container through the holes in
`the base. For convenience, and also to avoid handling the
`
`equipment, when sterile, a holder (C) was made which fittDd
`
`This piece of equipment has proved very useful in our:
`laboratory during the last few years. There is no danger of-
`breakage, and the handle on the plunger and the screw arrange-
`ment make it possible to exert considerable pressure with a
`minimum of effort.
`
`Reference
`
`~ Contribution from the Department of Zoology, No. 216.
`
`I. EARLI~, W.R. Arch. ~xp. Zellforsdt~, 1958, f$, 510-511.
`
`SCIENCE, December 19, 1947
`
`621
`
`Sandoz Inc. IPR2016-00318
`Sandoz v. Eli Lilly, Exhibit 1107-0003