Bull. Org. mond. Santé
`Bull. Wld Hlth Org.
`
`} 1970, 42, 445-449
`
`The Uptake of Tritiated Hycanthone by Male
`and Female Schistosoma mansoni Worms and
`
`Distribution of the Drug in Plasma and Whole Blood
`of Mice following a Single Intramuscular Injection *
`
`A. YARINSKY,‘ P. HERNANDEZ 2 & E. W. DENNIS 3
`
`Mice infected with Schistosoma mansoni were given an intramuscular injection of a
`single 80 mg/kg dose of randomly tritiated hycanthone. The uptake of the drug in male and
`female worms, as well as its concentration in the red blood cells and plasma of the mice,
`was followed for a period of 24 hours.
`Blood and plasma drug levels reached maximum values within 30 minutes. During the
`postmedication period, plasma concentrations were consistently higher than red blood cell
`concentrations. At 2-6 hours, when blood levels were declining, peak uptake was reached
`in the female schistosome warms; _the concentration in the female worm was 5 times
`higher than in the male worm. At 24 hours the schistosomes contained appreciably more
`drug than was present in the blood.
`
`Hycanthone (I), the hydroxymethyl analogue of
`lucanthone (II), has been reported to be schistosomi-
`cidal
`in the mouse, hamster and monkey (Rosi
`et al., 1965; Berberian et al., 1967; Pellegrino
`et al., 1967).
`
`0
`
`l|\THCH,CH,N(C,H,),
`
`Q S Q
`
`R
`
`(1)
`R=CH,OH
`
`(II)
`R=CH,
`
`investiga-
`A number of laboratory and clinical
`tions are currently under way to elucidate the action
`of the drug and to provide information concerning
`its characteristics.
`
`The experiment described herein was performed
`to determine the distribution of hycanthone between
`red blood cells and plasma in mice, and to determine
`the time relationship of drug levels in plasma and
`whole blood to drug levels in adult S. mansoni male
`and female worms during a 24-hour period after
`a single parenteral dose of drug.
`
`MATERIALS AND METHODS
`
`Assessment of the drug in man by administration
`of daily oral doses (2.5 mg/kg ;|; 0.5 mg/kg) for
`3-5 consecutive days or of a single intramuscular
`injection (3.0 mg/kg ;t 0.5 mg/kg) has demonstrated
`its eflectiveness against Schistosoma haematobium
`and S. mansoni (Katz et al., 1968; Maritz, 1968;
`Clarke et al., 1969).
`
`* From the Sterling-Winthrop Research Institute, Reus-
`selaer, N.Y., USA.
`1 Head, Parasitology Section.
`' Research Biologist.
`* Director, Biology Division.
`
`Female Swiss mice each weighing 32 g were
`infected with a Puerto Rican strain of S. mansoni
`
`according to the method of Berberian & Freele
`(1964); 48 days after infection the mice received an
`intramuscular
`injection of a single large dose
`(80 mg (base)/kg in 0.1 ml distilled water) of the
`methanesulfonate salt of randomly tritiated (173.1
`mCi/mmol) hycanthone. The selection of the dose
`was based on an earlier observation that it caused
`
`the worms in copula to be separated, and initiated
`a shift of worms from the mesenteric veins to the
`
`liver within 24 h (A. Yarinsky & B. A. Jackson-
`unpublished data, 1968). The ED5., of hycanthone
`
`2487
`
`_445_
`
`AstraZeneca Ex. 2131 p. l
`Mylan Pharms. Inc. V. AstraZeneca AB IPR20l6-01324
`
`

`
`446
`
`A. YARINSKY, P. HERNANDEZ & E. W. DENNIS
`
`base in the mouse is only 18.5 mg/kg ;l; 2.4 mg/kg
`(Berberian et al., 1967).
`At0.5h,1h,2h,4h,6h,8hand24hafter
`the medication, heparinized blood was obtained
`from 4 infected mice and pooled. A sample of
`0.1 ml of whole blood was pipetted on to a square
`of filter-paper and allowed to dry. The blood was
`then centrifuged and an aliquot of 0.1 ml of plasma
`was also placed on filter-paper. Following this,
`schistosome worms were removed from the mesen-
`
`teric veins (except at 24 h) and rinsed briefly in
`0.8% saline;
`the females were mechanically sepa-
`rated from the males and both sexes were given a
`second saline rinse. Eight hours after medication
`male and female worms were also collected from
`
`the hepatic sinuses because a major shift of worms
`to the liver had occurred. By 24 h, the worm shift
`to the liver was almost complete and all the males
`and females obtained were from the hepatic sinuses.
`The distribution of worms between the mesenteric
`veins and liver was recorded at 8 h and 24 h.
`
`At each time period, 6 males were blotted to
`remove excess moisture, weighedl as a group, and
`placed on 2 in X 2 in (5 cm X 5 cm) squares of
`filter-paper;
`6 or 7 females were placed directly
`into a weighing pan without blotting since the
`amount of residual moisture was small and evapo-
`rated prior to weighing. The females were weighed
`as a group and then placed on similar squares of
`filter-paper. The papers containing the samples of
`blood or worms were folded and made into pellets
`to ensure uniform and complete combustion in a
`Packard (Model 300) Tri-Carb Sample Oxidizer.
`After combustion the tritiated water formed and
`
`samples of counting solution were auto-
`10-ml
`matically dispensed into counting vials. The system
`was purged with nitrogen to eliminate oxygen
`quenching and the samples were counted in a Packard
`Tri-Carb Liquid Scintillation Spectrometer (Mod-
`el 874). The counting solution used contained 100 g
`naphthalene, 5 g 2,5-diphenyloxazole (PPO) and
`0.25 g 2,2-p-phenylenebis(5-phenyloxazole) (POPOP)
`dissolved in 1 litre of a mixture of 81% dioxane,
`4% absolute ethanol and 15 "/0 toluene.
`Under the conditions described above the range
`of counting efficiency was 30 %—36%; the recovery
`for tritiated hycanthone added to blood or placed
`directly on filter-paper and then burned was con-
`sistently greater than 97 %. Blanks were introduced
`
`1 h were
`‘The male and female worms collected at
`weighed in a Mettler balance. A Cahn electrobalance
`(Model M-12) was used for the other weighings.
`
`between samples to check the residual radioactivity
`in the system.
`It was found that after rinsing and
`purging only traces of radioactivity remained in the
`machine from the previous run.
`In one instance a
`blood sample that yielded more than 10'’ counts per
`minute was followed by a blank in which only
`100 counts per minute were recorded,
`indicating
`less than 0.01 ‘X, residual contamination of the system.
`For the determination of the nature of the radio-
`
`activity present in the worms, groups of 6 or 7 worms
`(randomly selected from among the worms taken
`from all mice in each batch of 4) were homogenized
`with a Potter-Elvelhjem homogenizer in 3 ml of
`0.2 In borate buffer, pH 9.5. The homogenates were
`extracted with 3 volumes of dichloromethane. After
`
`the organic phase
`separation by centrifugation,
`was concentrated at 50°C under nitrogen. The con-
`centrated extracts were spotted on 5 cm>< 20 cm
`precoated
`silica
`gel
`F-254
`thin-layer
`plates
`(E. Merck AG, Darmstadt, Federal Republic of
`Germany). The plates were developed in an ether-
`methanol—trimethylamine (8 : 1 : 1) system and the
`chromatographs were scanned in a Packard Radio-
`chromatogram Scanner (Model 7201). Non-radio-
`active hycanthone and available metabolites were
`placed on one side of each plate for comparison with
`radioactive rnaterial(s) extracted from the worms.
`
`RESULTS AND DISCUSSION
`
`The data in Table l confirm earlier work in
`
`which it was found that within 24 h after a single
`intramuscular injection of hycanthone methanesul—
`fonate at 80 mg/kg the worms normally found in
`copula become separated.
`In addition, there was a
`shift of worms from the mesenteric veins to the liver
`
`so that within 24 h 85% of the worms were in the
`hepatic sinuses. At
`the end of the experimental
`period all the worms found were still alive; however,
`they were sluggish and less resistant to mechanical
`separation than were the worms collected through
`the first 8 hours.
`
`Peak concentrations of hycanthone in blood and
`plasma were reached within 30 min, after which
`they declined to the end of the experimental period
`(Table 2, and figure). The ratios of hycanthone con-
`centrations of plasma : red blood cells were cal-
`culated (Table 2).
`It is clear that, although the
`ratios varied, plasma concentration levels were con-
`sistently higher at each point during the sampling
`period.
`The uptake of the tritium label by the male worms
`was rapid; within 30 min hycanthone was found
`
`Astrazeneca Ex. 2131 p. 2
`
`

`
`DISTRIBUTION OF I-IYCANTHONE IN MICE INFECTED WITH SCHISTOSOMA MANSON]
`
`447
`
`HEPATIC SHIFT OF SCHISTOSOMA MANSON! AFTER MEDICATION OF HOST MICE
`WITH A SINGLE INTRAMUSCULAR INJECTION OF 80 mo (base)lko OF RANDOMLY
`TRITIATED HYCANTHONE METHANESULFONATE“
`
`TABLE 1
`
`Average no. of worms per mouse 5
`Mesenteric veins
`Liver
`
`No.
`|
`%
`%
`|
`
`
`No.
`
`
`Average no. of
`unpaired worms
`No.
`[
`% _
`
`
`
`
`
`No. of worms
`.
`exar::.2fig per
`“‘"d "‘°°“’
`
`
`
`
`Time after
`InI<?l€_:';I0"
`‘
`
`8
`
`3-31 (15.0)
`8.4
`47.5
`10.8
`67.5
`
`
`12-23 (18.6)
`
`27.0 2.8 85.0 5.0
`
`
`
`
`
`
`
`“ In contrast to the shift of the worms to the liver shown in the table, In untreated infections
`e mouse.
`y
`igevvte: than 2 worms are found In the liver. or less than 10% of the total number of worms harboured
`I’ 5 mice were necropsled at each time period.
`
`to the extent of 60 pg/g wet worm tissue. No distinct
`drug peak was observed although between 30 min
`and 6 h the concentration of the drug increased
`slowly in the male. By 24 h, the males retained a
`high drug level (72 pg/g wet worm tissue) in contrast
`to amounts detected in the blood (1.1 pg/ml) and
`plasma (1.4 pg/ml).
`The female worms concentrated a larger amount
`of the drug than the males during the first 6 h after
`medication. A peak concentration of 392 pg/g
`was reached at 4 h which, on the basis of pg hycan-
`thone/g wet worm tissue, was more than 5 times
`that in the males. The enhanced uptake of a schisto-
`
`somicidal drug by female worms (compared with
`male worms) is in accord with the findings of Hess
`et al. (1966) for niridazole, of Khayyal (1964) and
`Molokhia & Smith (1968, 1969) for tartar emetic
`and of Browne & Schulert (1964) and Molokhia
`& Smith (1969) for sodium antimony dimercapto-
`succinate (TWSb/6).
`It is interesting to note that during the 30 min
`to 4 h postmedication period, during which time
`hycanthone levels in the blood and plasma were
`dropping, the female worms were accumulating the
`drug. By 6 h and continuing to the end of the
`experiment (24 h) the females showed a decrease
`
`TABLE 2
`CONCENTRATION OF HYCANTHONE“ IN MALE AND FEMALE SCHISTOSOMA MANSON! WORMS AND IN PLASMA AND
`WHOLE BLOOD OF THE MOUSE HOST
`
` .
`
`:a°ni§'ii:g'§§i's%3
`
`No. worms
`°°"°°ted I’
`
`I Female
`
`Male
`
`7
`
`6
`
`7
`
`6
`
`6
`6
`
`6
`
`6
`
`6
`
`6
`
`6
`6
`
`0.5
`
`1
`
`2
`
`4
`
`6
`8
`
`Source of worms
`
`Mesenteric veins
`
`,,
`
`,,
`
`,,
`
`,,
`
`.,
`
`,.
`
`..
`..
`Mesenteric veins
`and hepatic sinuses
`
`Hycanthone conc.
`(pg/g wet worm
`tissue)
`T Blood
`Female l Male
`|
`(uuIm|)
`
`Hycamhone
`
`Plasma
`luolml)
`
`Plasma : RBC
`ratio C
`
`201
`
`197
`
`318
`
`392
`156
`
`69
`
`60
`
`62
`
`73
`
`75
`81
`
`60
`
`30.0
`
`25.5
`
`15.5
`
`9.0
`2.8
`
`1.7
`
`39.8
`
`37.3
`
`22.9
`
`10.5
`3.8
`
`2.1
`
`2.47
`
`4.18
`
`4.52
`
`:53
`2.68
`
`1.81
`
`Hepatic sinuses
`6
`6
`24
`
`
`1.1
`
`1.4
`
`2.06
`
`-11
`
`72
`
`‘ Radioactivity expressed in terms of hycanthone base.
`I’ Worms weighed in groups of 6 or 7.
`5 Adjusted for average haematocrit value of 41.5%.
`
`Astrazeneca Ex. 2131 p. 3
`
`

`
`448
`
`A. YARINSKY, P. HERNANDEZ & E. W. DENNIS
`
`HYCANTHONE UPTAKE AND RETENTION BY 5. MANSON! RELATED TO CHANGING CONCENTRATION
`IN BLOOD OF HOST MICE
`
`,ugHyconthone/mlofBlood
`
`WetWormTissue
`
`2‘as
`
`cO4
`
`:O-
`
`c0u>
`
`.
`
`IU
`
`’
`3!
`
`K
`
`HOURS POST MEDICATION
`Hyconthone 80 mg/kg (IM)
`
`J
`
`in their concentration of hycanthone. Nevertheless,
`at the latter period the concentration of the drug
`in the female (41 pg/g wet worm tissue) far exceeded
`that in plasma and whole blood. Khayyal (1964)
`reported that S. mansoni worms from mice which
`had received 1“Sb-labelled tartar emetic showed
`
`higher drug levels than that found in the blood.
`Brown & Schulert (1964) reported similar findings
`in the case of S. mansoni-infected hamsters treated
`
`it is probable
`with 1"‘Sb-labelled TWSb/6. Thus,
`that the ability of the worms to concentrate schisto-
`somicidal agents is a contributing factor to the
`action of the drugs.
`
`When the worms were examined to determine the
`
`nature of the radioactive material, it was found that
`the males contained only unchanged drug. At the
`1-h postmedication period a sulfoxide derivative of
`hycanthone as well as unchanged hycanthone was
`found in the female worms. The sulfoxide derivative
`
`was present to the extent of approximately 20 %—3O %
`of the total radioactivity found in the worm. At
`subsequent sampling periods only unchanged hycan-
`thone was detected in the female. These observations
`
`imply that most, if not all, of the toxicity of hycan-
`thone for the adult schistosome may be attributable
`to the action of the unchanged drug.
`
`ACKNOWLEDGEMENTS
`
`The authors wish to thank Mr F. W. Gubitz for the preparation of the methanesulfonate salt of tritiated
`hycanthone and Mr E. F. Kurtik and Mr W. F. Banks for their competent technical assistance.
`
`RESUME
`
`ABSORPTION DE L’HYCANTHONE MARQUE AU TRITIUM PAR SCHISTOSOMA MANSON!
`MALE ET FEMELLE ET REPARTITION DU MEDICAMENT DANS LE PLASMA ET LE SANG COMPLET
`DE LA SOURIS APRES UNE INJECTION INTRAMUSCULAIRE UNIQUE
`
`On a administré par voie intramusculaire a des souris
`infectées par Schistosoma mansoni une dose unique de
`80 mg/kg d’hycanthone marqué au tritium, et suivi
`
`pendant 24 heures I’absorption du medicament par les
`parasites males et femelles ainsi que les variations de
`sa concentration dans le plasma et dans le sang complet.
`
`Astrazeneca Ex. 2131 p. 4
`
`

`
`DISTRIBUTION OF HYCANTHONE IN MICE INFECTED WITH SCHISTOSOMA MANSON!
`
`449
`
`La teneur du plasma et du sang complet en hycanthone
`est maximale aprés une demi-heure. Dans les schisto-
`somes,
`les concentrations les plus fortes sont observées
`aprés 2 a 6 heures; 2‘: cc moment, et a poids égal, les vers
`femelles renferment approximativement cinq fois plus de
`medicament que les vers males.
`
`Dans l’interval1e de 24 heures, plus de 95 % du produit
`marqué est éliminé du sang et du plasma. La quantité
`
`d’hycanthone présente dans les vers femelles diminue
`pour n’étre plus que du dixiéme environ de la valeur
`maximale enregistrée, mais elle reste trés supérieure aux
`concentrations résiduelles dans le plasma et dans le sang
`complet. Chez les vers males, en revanche,
`la teneur
`en hycanthone ne s’abaisse que trés faiblement durant
`la meme période. La concentration d’hycanthone est
`constamment plus élevée dans le plasma que dans les
`érythrocytcs.
`
`REFERENCES
`
`Berberian, D. A. & Freele, H. (1964) J. Parasit., 50,
`435-440
`
`Berberian, D. A., Freele, H., Rosi, D., Dennis, E. W. &
`Archer, S.
`(1967) Amer. J.
`trap. Med. Hyg., 16,
`487-491
`
`Browne. H. G. & Schulert, A. R. (1964) Amer. J. trap.
`Med. Hyg., 13, 558-571
`Clarke, V. dc V., Blair, D. M. & Weber, M. C. (1969)
`Cemr. Afr. J. Mea'., 15, 1-6
`Hess, R., Faigle, J. W. & Lambert, C. (1966) Nature
`(Lom1.), 210, 964-965
`Katz, N., Pellegrino, J., Ferreira, M. T., Oliveira, C. A.
`& Dias, C. B. (1968) Amer. J. trap. Med. Hyg., 17,
`743-746
`
`Khayyal, M. T. (1964) Brit. J. Pharmacol., 22, 342-348
`Maritz, J. C. (1968) In: Third South African Symposium
`on Infective Diseases, Department of Internal Medicine,
`University of Pretoria, 11 March 1968 (cited by:
`Schneider, J. (1969) Med. Proc., 14 June, p. 201)
`Molokhia, M. M. & Smith, H. (1968) Ann. trap. Med.
`Parasit., 62, 158-163
`Molokhia, M. M. & Smith, H. (1969) Bull. Wld Hlth
`0rg., 40, 123-128
`Pellegrino, J., Katz, N. & Scherrer, J. F. (1967) J. Para.s'1't.,
`53, 55-59
`Rosi, D., Peruzzotti, G., Dennis, E. W., Berberian, D. A.,
`Freele, H. & Archer, S. (1965) Nature (Lond.), 208,
`1005-1006
`
`Astrazeneca Ex. 2131 p. 5