[CANCER RESEARCH 36, 1453-1461 , April 1976)
`
`The Disposition and Pharmacokinetics in Humans of 5-Azacytidine
`Administered Intravenously as a Bolus or by Continuous Infusion1
`
`Zafar H. lsraili,2 William R. Vogler, Elizabeth S. Mingioli, James L. Pirkle, Robert W. Smithwick, and Jacob H.
`Goldstein
`
`Departments of Medicine (Clinical Pharmacology, Hematology, and Medical Oncology; Z. H. I., W. A. V., E. S. M.Jand Chemistry(J
`Emory University School of Medicine, Atlanta, Georgia 30322
`
`L P A. W. 5., J. H. GJ,
`
`SUMMARY
`
`The disposition of 5-[4-4C]azacytidine, administered i.v.
`as a bolus or continuous
`infusion, was studied in cancer
`patients. After bolus, plasma ‘4Clevels exhibited a multi
`phasic disappearance pattern; half-life (t1,2, /3 phase) =
`3.4 to 6.2 hr. Of ‘4Cin plasma, <2% was associated with 5-
`[4-'4C]azacytidine 30 mm after dose. The ratios of ‘4Clevels
`were:
`red cells/plasma,
`—0.8;leukocytes/plasma,
`1.1 to
`2.3; nucleic acids/leukocytes,
`0.2 to 0.43; sputum/plasma,
`0.05 to 0.17. Urinary excretion (3 days) accounted for 73
`to 98% of ‘@C,<1% in feces. The relative concentration of
`5-azacytidine in plasma with continuous
`infusion stayed
`higher than with bolus; urinary excretion was similar. Fewer
`side effects were observed with continuous
`infusion than
`with bolus. The stability of 5-azacytidine was determined
`in various media at several temperatures by thin layer
`chromatography
`and nuclear magnetic resonance. At 20°
`in Ringer's lactate (pH 6.2), the t1,2was 94 to 100 hr. Stability
`increased with lowering of temperature and pH. From our
`data we conclude that 5-azacytidine should be given by
`continuous infusion rather than as a bolus.
`
`INTRODUCTION
`
`5-AC,3 a pynimidine analog of cytidine (Chart 1) synthe
`sized4 in 1964 (25), was shown to possess marked antibacte
`nial (7, 24, 29) and cancerostatic properties (29). In mice, 5-
`AC decreased the number of circulating lymphocytes and
`mature bone marrow myeloid cells (30). The compound
`possesses remarkable inhibitory properties against rapidly
`proliferating
`tissues includ ing various experimental neo
`plastic growths (17, 33, 34). 5-AC inhibits the synthesis of
`DNA (17, 27) and RNA (5, 13). The mechanism of action
`probably
`involves phosphorylation,â€(cid:157)
`incorporation
`into
`
`in part by NIH Grants GM14270, GM 10848, and RR00039 and
`I Supported
`USPHS Research Grant CA-03227. Part of this work was presented
`at the Fall
`1974 Meeting of the American Society for Pharmacology
`and Experimental
`Therapeutics, Montreal, Canada (12).
`
`2 To
`whom
`reprint
`requests
`should
`be
`addressed,
`at
`The
`Department
`of
`Medicine, 152 Woodruff Memorial Building, Emory University, Atlanta, Ga.
`30322.
`3 The
`abbreviations
`used
`are:
`5-AC,
`5-azacytidine
`(4-amino-1-f3-D-ribofu
`ranosyl-s-triazin-2-(lH)-one; NSC- 102816); 5-[â€(cid:157)C)AC,5-[4-'4C)azacytidine;
`TLC, thin-layer chromatography;
`NMR, nuclear magnetic
`resonance.
`4 Cultures
`of
`Streptoverticillium
`ladakanus
`can
`produce
`5-AC
`(2,
`
`9).
`
`S The
`phosphorylation
`of
`5-AC
`is
`catalyzed
`is blocked by cytidine and uridine (16).
`Received August 26, 1975; accepted December 19, 1975.
`
`uridine-cytidine
`
`by
`
`kinase
`
`and
`
`newly synthesized nucleic acids followed by fission of the
`triazine ring (26). It was found that 5-['4C]AC was phospho
`rylated in all leukemic tissues studied (17). Further, the drug
`has been shown to exhibit profound
`antitumor
`activity
`against munine L-1210 leukemiain vitro (17) and in vivo (30),
`against Walker 256 carcinoma (3), and against acute lym
`phoblastic leukemia in AK mice (30).
`it
`Studies of the phase specificity of 5-AC revealed that
`acted predominantly in the S phase of the mitotic cycle (17)
`and was lethal to L-1210 cells grown in tissue culture (17).
`Lloyd et a!. (18) noted that continuous exposure to low
`doses of 5-AC was more effective than was short exposure
`to larger doses and that the drug was relatively inactive in L
`1210 cells maintained in nonproliferating
`state. However,
`there are some suggestions that the drug may not be purely
`cell cycle-phase specific and may impair cell structures
`even in the resting state (23).
`5-AC has been found to be promising in the treatment of
`acute leukemia (1, 11, 14, 15, 35). The apparent
`instability of
`5-AC (26) in solution has necessitated administration
`by
`rapid iv.
`injection.
`In clinical
`trials (6, 15, 20, 21, 31, 32, 35,
`37), this has resulted in severe nausea and vomiting. Kanon
`et a!. (15) found that,
`in children,
`if the drug was infused
`over a period of 10 to 15 mm or given in divided doses,
`gastrointestinal
`toxicity was reduced. Moertel et a!. (21)
`found that, by dividing the dose,
`less nausea and vomiting
`occurred.
`Thus, the in vitro data suggesting that continuous expo
`sure was more effective and the clinical data indicating that
`intolerable gastrointestinal
`toxicity was a frequent side ef
`fect with bolus doses prompted us to neexplore the stability
`of 5-AC in solution. Furthermore, we compared the phanma
`cokinetic data obtained with continuous infusion and single
`injections using 5-['4C]AC in patients with metastatic cancer
`and leukemia.
`
`MATERIALS AND METHODS
`
`Patients, whose prior written informed consent was ob
`tamed, were selected for study on the basis of metastatic
`cancer or
`leukemia,
`life expectancy of at
`least 6 weeks,
`normal bone marrow and blood counts (except for leukemic
`patients), normal
`renal
`function, stable hepatic function,
`and no other existing disorder. Liven function tests included
`serum glutamate-oxalacetate transaminase, alkaline phos
`phatase, pnothrombin time, and serum protein. Renal func
`
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`z.H.Israilieta!.
`
`@
`
`NH2
`1*
`N
`
`N
`
`0
`
`N
`
`HOC@@@
`
`HO OH
`
`Chart 1. 5-AC (NSC-102816),*, position of “C.
`
`tion tests included blood urea nitrogen and cneatinine clear
`ance. The details of the patients and the doses administered
`(mg/sq m and pCi) are given in Table 1. After admission,
`each patient was observed during a control period of 1 to 6
`days. This was followed either by a treatment period of 1 day
`(for single bolus injection studies) or 6 days (for continuous
`infusion studies) and a follow-up period of 1 to 3 days. The
`diet was normal except
`that
`the patients were fasted from
`midnight until
`the morning of the treatment period.
`The single i.v. dose of 5-[14C]ACwas given at 8:00 am. For
`continuous
`infusion,
`the dose of 5-['4C]AC was started at
`8:00 a.m. and continued over a 12-hr period. This treatment
`was then followed with 5-AC every 12 hr for 9 doses; other
`medications were allowed after the 1st 12-hr period. After a
`single i.v. dose, 10-mI samples of hepaninized blood (with
`1000 units of bovine lung hepanin; Upjohn Co., Kalamazoo,
`Mich.) were obtained at predetermined intervals and imme
`diately chilled in ice. Plasma was separated by centnifuga
`tion (500 x g, 30 mm, 0-4°).Urine collections were made
`every 4 hr on the 1st day and every 24 hr on the 2nd and 3rd
`day. Daily collections of feces were made for several days.
`Vomitus,
`if any, was collected; specimens of sputum were
`obtained at specified times.
`In 1 case, leukocytes were also
`isolated (see below). With the continuous infusion protocol,
`20-mI blood samples (hepaninized) were collected at various
`time intervals after the beginning of
`infusion of 5-[14C]AC.
`Urine collections were made every 2 hr for the 1st 12 hr and
`then every 3 hr for the next 12 hn; thereafter, 24-hr collec
`tions were obtained for the next 3 days. Feces collections
`were made for 0 to 24 and 24 to 48 hr. In 1 case, a sample of
`cerebrospinal
`fluid was also obtained. All biological sam
`pIes were stored below 0°and analyzed as soon as possible.
`
`Dose Formulation
`
`5-AC was obtained in a dosage form consisting either of
`(a) 50 mg of the drug and 100 mg of polyvinylpyrrolidone
`(plasdone-C-1 5), or (b) a 1:1 mixture of the drug with manni
`tol for iv. use (Division of Cancer Treatment, National Can
`cer Institute, NIH, Bethesda, Md.). The labeled compound
`(5-['4C]AC)6 was dissolved in distilled water, and the solu
`tion was sterilized by passing through a disposable sterile
`filter
`(Swinnex 0.22 @m;Millipore Corp., Bedford, Mass.)
`
`I 5-(â€(cid:157)CJAC
`(Lot
`No.
`219-3C,
`49.1
`mCi/mmole)
`was
`synthesized
`by
`Mon
`santo Research Corp., Dayton, Ohio, and was supplied by the Drug Research
`and Development Chemotherapy
`Branch, National Cancer
`Institute, NIH,
`Bethesda, Md. The radiochemical purity (>95%) was shown by the manufac
`turer by descending
`paper chromatography
`in 1-butanol
`saturated with water
`(RF0.22). The labeled material as well as carrier were stored at —20°.
`
`and added to carrier 5-AC dissolved in sterile distilled water.
`This final solution was either injected immediately (in 8 to 10
`mm) as a bolus or added to 500 to 1000 ml of Ringer's
`lactate (Hartmann ‘sMcGaw Laboratories, Glendale, Calif.)
`for continuous
`infusion. The 1st 100 ml of solution were
`injected over a 20-mm period and the remainder
`in the next
`11 hrand40min.
`An aliquot of the dose solution was tested in rats for the
`absence of pyrogens (19). Male Wistar rats (197 to 217 g, 4 in
`each group) were given s.c. injections (20 mI/kg) of either:
`Group A, sterile distilled water; Group B, a 20% suspension
`of brewers' yeast in sterile water; or Group C, 1.4 x 10@M 5-
`[14C]AC (0.3 pCi/animal)
`in sterile water passed through a
`sterile Swinnex filter. Rectal temperatures were measured
`by a thermocouple at 0, 4, and 24 hr after injections. The
`average temperatures for Groups A, B, and C for 0, 4, and 24
`hr were as follows: Group A, 37.1°,37.5°,36.9°;Group B,
`36.6°,38.1°,37.6°;and Group C, 37.1°,37.8°,369°. The
`stability of
`the solution of 5-AC in Ringer's lactate was
`determined prior to infusion (see below).
`
`TLC
`
`TLC was carried out on: (a) Silica Gel G-coated glass
`plates without
`fluorescent
`indicator
`(Analtech Uniplate,
`Newark, Del.), and (b) Silca Gel G-coated plastic sheets with
`fluorescent
`indicator
`(Eastman Chromagram 6060). The
`plates were
`developed
`in
`System 1
`(1-butanol:-
`ethanol:waten, 49:11:19, v/v/v) at room temperature. Radi
`ochromatography was carried out on glass plates and 1-cm
`segments of silica gel were scraped and transferred into
`counting vials; 0.5 ml of methanol was added prior to count
`ing fluid. 5-AC and related compounds
`(1 p1 of 3.5 mM
`aqueous solutions) were spotted on fluorescent
`plates
`(6060) and, after development, the spots were visualized by
`quenching of fluorescence under UV light (254 nm), The RF
`values for 5-AC, 5-azacytosine, and 5-azauracil were 0.35,
`0.24, and 0.22, respectively.
`
`Measurement of Radioactivity in Biological Materials and
`Extracts
`
`Aliquots (up to 2 ml) of plasma or urine were mixed with
`18 ml of counting fluid (prepared by mixing 7 g of PPO, 0.36
`g of POPOP,
`200 ml of Beckman
`Biosolv BBS-3,
`and 1 liter
`of toluene). They were counted alone or in the presence of
`known amounts of 5-['4C]AC in a Beckman LS-255 liquid
`scintillation spectrometer
`(Beckman Instruments,
`Inc. , Ful
`lerton, Calif.) (counting efficiency, 90%). Similarly, ‘4Cwas
`measured in vomitus, sputum, WBC, and spinal
`fluid;
`for
`ABC, 0.3-mI aliquots were oxidized in a Beckman-Harvey
`combustion instrument, and the resulting ‘4C02was trapped
`in “Harveycanbon-14 cocktailâ€(cid:157)(A. J. Harvey Instrument
`Corp., Hillsdale, N.J.) (15 ml; counting efficiency, 70 to 75%).
`Recoveries (85 to 90%) of ‘4Cafter combustion were deter
`mined by oxidizing known amounts of 5-['4C]AC added to
`sucrose.Feces were homogenized with 100 to 500 ml of
`95% ethanol. Ten-mi aliquots were centrifuged and 1 to 2 ml
`of the clear supernatant were counted. This procedure was
`found to be satisfactory since practically no radioactivity
`was detected in the residue upon oxidation.
`
`1454
`
`CANCERRESEARCHVOL. 36
`
`
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`Disposition of 5-AC in Humans
`
`5-('@CJACPa
`
`Table 1
`Description of patients anddoses of
`
`tient°Age(yr)Wt
`m)Diagnosis1
`
`area(sq
`
`(kg)Surfacem)Dose
`
`(mgi
`sq
`
`Adenocarcinomaof lung
`200
`Adenocarcinomaof lung
`200
`Acute myeloblastic leukemia
`250
`150Mesothelioma
`
`103
`66
`56
`67186
`
`2.27
`1.70
`1.67
`190150
`
`23 45
`
`49
`44
`27
`Melanoma6
`2173
`44
`
`cell carcinoma of lung
`Acute myeloblastic leukemia
`50
`Adenocarcinomaof colon
`75Squamous
`
`64
`2459
`
`46
`521.80
`
`1.50
`1.5025
`
`78
`
`50
`
`a Patients
`
`1 to
`
`5 received
`
`5-['4C]AC
`
`as
`
`a single
`
`iv.
`
`bolus;
`
`the
`
`total
`
`dose
`
`of
`
`5-AC
`
`per
`
`week
`
`was twice the indicated value. For Patients6 to 8, who received5-['4C]ACby continuous
`infusion overa 12-hrperiod,the 24-hrdoseof 5-ACwastwice the indicated value;the total
`dose of 5-AC administered in 1 week was 10 times the 12-hr dose. Patients 1 and 2
`received25 j.@Ci;all others were given 50 @Ciof the labeleddrug. Patients3, 4, and 8 were
`females.The creatinine clearancesin patients 1 to 5 and 7 were 80, 124,119,90, 73, and
`101 mlimin,
`respectively.
`
`Measurement of 5-[4C]AC in Plasma
`
`Fresh plasma (3 ml) obtained from patients receiving 5-
`[‘4C]ACwas diluted with 5 ml of 10@ M Tnis buffer, pH 6.5.
`Then, 3 drops of 10% w/v oxalic acid were added (final pH, 6
`to 6.5). After shaking,
`the mixtures were immediately shell
`frozen and lyophilized (VirTis Co., Gandiner, N.Y.). The resi
`due was tnitunated with 10 ml of methanol, sonically ex
`tracted for 5 mm, and centrifuged for 10 mm at room tem
`perature. The precipitate was reextracted with 10 ml of
`methanol. The final residue was discarded, since essentially
`no ‘4Cwas found in it. The methanol extracts were pooled
`and evaporated to dryness in a vacuum at room tempera
`tune, and the resulting residue was redissolved in 1 ml of
`methanol. Aliquots of this solution were chromatographed
`(Silica Gel G-coated plates, without
`fluorescent
`indicator;
`AnaltechUniplate)usingSystem 1.
`
`Isolation of Leukocytes and Nucleic Acids
`
`Leukocytes were isolated by the method of Hirsch (10),
`Aliquots ofblood samples (10 mI)from Patient 4were mixed
`with 10 ml of
`ice-cold 3% w/v dextran (Phanmacia Fine
`Chemicals,
`Inc., Piscataway, N.J.; MW. 400,000) in 0.85%
`NaCI containing 100 units of hepanin per ml, The mixture
`was kept at 0°,and the ABC were allowed to sediment
`for 20
`to 30 mm. The leukocyte-nich suspension was aspirated and
`centrifuged (10 mm, 2000 x g, 4°),The resulting pellet was
`washed with 10 ml of 0.85% NaCI as above, Contaminating
`ABC were lysed for 10 mm at 0°(in 5-mI
`lyse solution
`consisting of 1 g sodium citrate dihydrate and 2.5 g NaCI in
`1 liter of distilled water; final pH adjusted to 5 with HCI). An
`equal volume of neutralizing solution (7 g of sodium citrate
`dihydrate and 15 g of NaCI in 1 liter of distilled water) was
`added, and the WBC were centrifuged as above and washed
`twice with 2 ml of 0.85% NaCI solution. The pellet was then
`suspended in 1 ml of 0.85% NaCI solution and weighed. An
`aliquot of this suspension was counted and was found to
`have 98% leukemic blast cells, and another aliquot
`(0.3 ml)
`
`was mixed with 0.3 ml of 0.8 M perchlonic acid at 0°.After
`standing for 10 mm the mixture was centrifuged (27,000 x g
`for 10 mm at 0°).The supennatant was decanted and ali
`quots were counted. The precipitate was washed twice with
`0.2 M perchlonic acid (5 ml) at 0°and resuspended in 1 ml of
`0.8 M penchloric acid. After heating (100°for 20 mm) and
`centnifugation, aliquots of the resulting supernatants were
`counted.
`
`Isolation and Measurement of [‘4C]Ureafrom the Urine of
`a Patient Receiving 5-['4C]AC
`
`To 2 ml of urine (Patient 1) were added 2 ml of an aqueous
`solution of urea (100 mg/mI)
`followed by 1.5 ml of 95%
`ethanol. The mixture was heated (100°for 5 mm) and then
`quickly filtered while hot. The filtrate was cooled to room
`temperature and again filtered. The remaining precipitate
`was washed 4 times with 1 ml of hot ethanol. The combined
`filtrate and washings were evaporated to dryness in a vac
`uum at 60°.The residue was tniturated with hot ethanol and
`filtered. The filtrate was cooled to —20°,and the resulting
`crystals of urea were collected by filtration. After 2 recnystal
`lizations from ethanol, urea was dried in a vacuum at 80°.An
`aliquot of the product
`from each crystallization was sub
`jected to TLC on Silica Gel G glass plates and developed in
`System 1. Segments of silica gel (4 mm) were scraped and
`counted. For visualization,
`the plates were dried overnight,
`sprayed with a solution of 0.05% bromcnesol green (in 95%
`ethanol, adjusted to pH 6.5 with dilute NaOH), followed by
`exposure of the plates to HCI vapors. A blue spot (RF0.46)
`corresponding to urea could be briefly seen against a yellow
`background.
`The presence of ‘4Cassociated with urea was specifically
`determined by treating urea samples isolated from urine
`with unease and measuring the evolved ‘4C02(8). A twin
`armed Warbung flask was charged as follows: body, 3 ml of
`urea solution (1.9 mg/mI)
`in 5 mM phosphate, pH 6.5, con
`taming 0.5 mM disodium EDTA; 1st side arm, unease (pne
`pared from Jack Bean meal) on water for control; 2nd side
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`arm, 0.5 ml 1 N HCI; center well, paper wick plus 0.5 ml 1 N
`NaOH. The flask was tightly stoppered and the contents of
`the 1st side arm were tipped and mixed with the urea solu
`tion. After a 2-hr incubation at 37°,HCI was added to the
`reaction mixture to release ‘@CO2.After 24 hn, the contents
`of the central well were transferred into a scintillation vial.
`The well was washed with several small aliquots of water
`and the washings were also transferred into the scintillation
`vial and mixed with counting fluid, and ‘4Cwas measured.
`
`Measurement of Partition Coefficient and Binding of 5-
`[‘@C]AC
`
`The partition coefficient of 5-['4C]AC was measured at
`room temperature between M/15 SOrensen buffer, pH 7.4
`(28), and various organic solvents (chloroform, n-heptane,
`and peanut oil). An aqueous solution of 5-[14C]AC(10 ml, 10
`i.&g, 0.01 MCi) was shaken with buffer-saturated
`organic
`solvents (20 ml) for 40 mm. After the phases were separated
`by centnifugation, ‘4Cwas measured in 2 ml of the buffer
`phase, 1 ml of peanut oil, and 2 ml of chloroform on n
`heptane (after evaporation).
`The binding of 5-[14C]AC to human albumin was meas
`ured at 37°and pH 7.4 by 2 methods: (a) equilibrium
`dialysis, and (b) molecular sieve (22).
`Equilibrium Dialysis. Visking dialysis tubing (VWR Scien
`tific, Atlanta, Ga.; 5/8 inch, size 20) was washed twice and
`then kept in distilled water for at least 24 hr and blotted dry.
`In 1 set of experiments, 2 ml of 5% w/v human serum albu
`mm (crystallized , Pentex; Miles Laboratories, Kankakee,
`Ill.) were placed inside the dialysis bags. These were then
`placed in test
`tubes containing 10 ml of Sorensen buffer
`containing 100 @g,0.01 @tCiof 5-['4C]AC (freshly prepared).
`In another set of experiments, 200 @g(0.02 pCi) of 5-['4C]AC
`were added to 2 ml of albumin solution, which was then
`placed in the dialysis bag and dialyzed in 10 ml of Sorensen
`buffer.
`In these experiments, equilibration was achieved by
`shaking in air (100 cycles/mm; Metabolyte Bath, New Bruns
`wick, N. J.) at 37°for 4 hr. Leakage of protein through
`the dialysis tubing was tested with 40% w/v aqueous tn
`chloroacetic acid. Radioactivity in the inside and outside
`phases was determined and the binding was calculated.
`Molecular Sieve Method. Since 5-AC is not very stable in
`solutions at 37°,the binding of the drug to human serum
`albumin (5% w/v in Sorensen buffer, pH7.4) at 37°was also
`measured by the molecular sieve method (which consumes
`much less time). The procedure used was essentially that
`described by Mu et a!. (22).
`
`Stability Studies
`
`The stability of 5-AC in various media at several tempera
`tunes was studied by 2 methods:
`(a) TLC using 5-['4C]AC,
`and (b) NMR using 5-AC. For Method a, the compound was
`dissolved in water on buffer at various pH's and stoned at
`appropriate temperatures (0-37°)for specific times. Aliquots
`were analyzed by quantitative TLC (silica gel plates, System
`1) as described above. Radioactivity
`in the zone come
`sponding to 5-['4C]AC was determined. These values (as
`percentages) were plotted versus time and half-lives (t112's)
`were calculated.
`
`For Method b, 5-AC was dissolved in the appropriate
`medium and NMR studies were carried out at 9-37°on a
`Bruker Scientific HFX-90 spectrometer
`(Bruker Scientific,
`Inc., Elmsfond, N. Y.). The spectra were taken at a width of
`120 Hz and were time averaged over multiple scans (Nicolet
`1074 computer). Tetramethylsilane and benzaldehyde in a 5-
`mm coaxial capillary served as external references. The rate
`of decrease (relative to the reference peak) in the height of
`the C-6 ring proton resonance of 5-AC was taken to be the
`rate of decomposition. The relative height of
`resonance
`peak for C-6 proton was calculated (sample peak height/
`reference peak height). Multiple regression analysis of the
`data was performed using 1st-order
`rate kinetics, and t112's
`at a 95% confidence level were determined.
`NMR spectra at 40°were taken on a Vanian A-60A spec
`trometer
`(Vanian Associates, Palo Alto, Calif.). The rate of
`decrease of the area under the C-6 ring proton resonance of
`5-AC (relative to the reference resonance) was assumed to
`be the rate of decomposition of 5-AC. The t112'swere deter
`mined as described above.
`
`RESULTS
`
`Studies In Patients
`
`Single i.v. Dose. Five patients received- 5-['4C]AC as a
`single bolus dose; plasma levels of ‘4C(expressed as @g
`equivalents of 5-[14C]AC pen ml of plasma) are shown in
`Table 2. The data were analyzed by computer, using a
`program developed by Dr. William Olson (Department of
`Medicine, Emory University). The plasma ‘4Clevels (Cu, 0 to
`12 hn) fit the expression:
`cp= Ae@°'+ Be_k@@
`
`where A and B are the intercepts and k0 and k8 are the
`slopes. The calculated values forA and B, the t12 of a and f3
`phases, and the apparent volumes of distribution (V,@)are
`given in Table 2. The t112of distribution
`phase (a) ranged
`from 16 to 33 mm, the t112of fJ phase ranged from 3.4 to 6.2
`hm,and Vd ranged from 0.58 to 1.15 liters/kg.
`Preliminary studies indicate that
`the concentration of 5-
`[‘4C]ACin plasma declined much fasten than total ‘4C;thus
`in 1 patient (Patient 1), at 5, 10, 15, and 20 mm after dose,
`the levels of 5-['4C]AC were 40, 37, 27, and 7%, respectively,
`of
`the total ‘4Cin plasma. After 30 mm the values for 5-
`[‘4C]ACwere <2% of ‘4Cin plasma. At least 2 metabolites
`and/or decomposition
`products of 5-AC were found in
`plasma by TLC.
`The ratio of ‘@Clevels in ABC to plasma was about 0.8
`(ABC/plasma ‘@Cconcentration ratios for Patient 3 at 5, 15,
`and 30 mm and 1 hr were 0.81, 0.82, 0.85, and 0.80, nespec
`tively). The ratio of concentration
`of ‘4Cin leukocytes to
`plasma ranged from 1.1 to 2.3, while ‘4Cassociated with
`nucleic acids of WBC was 20 to 43% of ‘4Cin leukocyte
`(Table 3).
`The level of 14Csecreted into the sputum was from 5 to
`17% of plasma concentrations;
`the vomitus
`contained
`<0.1% of the dose (Table 2). The patients vomited about 6
`times (range, 2 to 11) starting 1 hr after the dose (until 14 hr
`in some cases). The vomitus varied in colon from light yellow
`
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`Table2
`
`Plasma levels of 4Cin patients after administration of5-('@CJACConcentration
`
`pg/mI).
`
`of ‘4C(5-AC equivalent,
`
`fusionTimeâ€(cid:157)Patient
`
`After single iv. doseb
`
`.
`
`During
`
`and
`
`after
`
`continuous
`
`iv.
`
`in
`
`85
`
`1Patient
`
`2
`
`Patient 3
`
`Patient 4
`
`Patient 5
`
`Patient 6
`
`Patient 7
`
`Patient
`
`Disposition of 5-AC in Humans
`
`mm11.910.6
`13.910mm9.39.715
`
`mm8.99.3
`0.3230
`0.3845
`mm8.08.6
`mm7.07.8
`8.01
`hr6.57.2
`0.442
`hr4.66.1
`0.793
`hr3.75.1
`4.74
`hr2.84.3
`0.785
`hr2.53.86
`hr2.23.2
`0.768
`hr1.5
`0.6212
`hr1.01.7
`1.33@l6hr0.4
`1.220
`hr0.224
`0.1232hr0.1148
`hr0.440.44
`
`hr0.46
`
`15.7
`
`12.1
`9.8
`
`7.1
`5.0
`4.2
`3.8
`
`2.3
`
`0.7
`
`10.2
`
`7.5
`
`6.3
`5.2
`
`3.9
`
`2.8
`
`1.7
`
`0.11
`
`0.2
`
`0.89
`
`0.61
`
`8.5
`
`6.1
`4.4
`
`3.0
`
`2.0
`1.2
`0.61
`
`0.07
`
`0.23
`0.22
`
`0.22
`0.28
`
`039
`
`0.38
`0.44
`0,61e
`
`0.13
`
`0.45
`0.39
`
`0.39
`
`0.50
`0.44
`0.93'
`
`0.14
`
`continuous
`
`infusion,
`
`it
`
`is
`
`from
`
`start
`
`of
`
`‘5For
`infusion.
`
`time
`the
`dose,
`iv.
`single
`For doses, see Table 1.
`
`indicated
`
`is after
`
`drug
`
`administration;
`
`for
`
`6 Computer
`
`analysis
`
`of
`
`the
`
`0-
`
`to
`
`12-hr
`
`data
`
`of
`
`Patients
`
`distributionphase(a)forthese patientswere:Patient1,33 mm;Patient2,28 mm;Patient3, 16mm;Patient4, 22 mm;
`and Patient5, 17mm.Thet2's for /3phasewere: 4.7,5.5,3.4,6.2,and 3.5 hr, respectively.Thecorrespondingapparent
`volumesof distribution were 0.62,0.58,0.60, 1.15,and 0.58 liters/kg, respectively.The vomitus contained 0.01,0.09,
`0.03, and 0.02% of dose of ‘4Cin Patients 1, 2, 3, and 4, respectively; ‘@C(5-AC equivalent) in the sputum obtained at the
`indicatedtimewas as follows:Patient1, 1.5 hr, 0.27 jig/mI; Patient2, 0.5 hr, 0.8 @g/ml,and 1 hr, 0.55 @tg/mI;and
`Patient 3 1 hr, 1.22 @,tgiml.
`C Infusion
`stopped.
`
`1
`
`to
`
`5
`
`fits
`
`a
`
`2-compartment
`
`model
`
`(r
`
`>
`
`0.95).
`
`The
`
`t112's
`
`of
`
`Table 3
`Distribution of “Cin leukocytes°and nucleic acids isolatedfromthe
`
`
`blood of a leukemicpatient (Patient4)after administrationof5-(‘@CjAC‘4C
`
`(5-AC equivalent)
`
`levels
`
`Time after
`
`dose (hr)cytes1
`
`Plasma (@.Lg/
`ml)
`
`Leukocytes
`(/Lg/g)
`
`‘4Cin nucleic
`acids as % of
`‘4Cin leuko
`
`272
`314
`206
`3024
`
`43a
`
`6.3
`5.2
`3.9
`2.8
`0.9
`
`98%
`
`leukemic
`
`blast
`
`cells.
`
`7.0
`6.6
`4.3
`4.0
`2.1
`
`to green; pH ranged from 7.5 to 7.9. Nausea was pro
`nounced and was partially controlled by chlorpnomazine on
`prochlonpenazine.
`Most ofthe administered ‘4Cappeared in urine: 69 to 91%
`and 73 to 98% in 1 and 3 days, respectively, while less than
`1% of
`the dose was present
`in the feces (Table 4). The
`calculated amount of ‘4Cremaining in the body showed
`considerable individual variations in these patients;
`in 1
`case about 27% of the dose was apparently present even
`after3 days.
`for the 1st
`The renal clearance of ‘4C(drug + metabolite)
`12 hr after bolus dose varied from 74 to 210 mI/mm (Table
`4).
`
`Table 4
`5-Time
`Cumulatiye urinary excretion of ‘4Cin patients adminl
`(‘@C)ACas single iv. doseâ€(cid:157)‘stered
`inPatient
`of dose excreted
`
`50-4
`(hr)%
`
`1
`
`Patient
`2
`
`Patient
`3
`
`Patient
`4Patient
`
`22
`44
`38
`60
`50
`73
`58
`67
`0-8
`66
`58
`82
`71
`78
`0-12
`69
`0-24
`69
`91
`85
`90
`71
`77
`94
`93
`96
`0-48
`73
`8047
`95
`95
`98
`0-7244
`aPercentageof doseof ‘@Cexcretedin feceswasasfollows:
`Patient1, 0.01(0 to 2 days);Patient2, 0.36 (0 to 1 day);Patient3,
`0.98 (0 to 2 days); and Patient 4, <0.01 (0 to 2 days). The renal
`clearance(mlimin) of ‘4C(5-['4CJACand metabolites)for 0- to 4-, 4-
`to 8-, and8- to 12-hrintervalswereas follows:PatientI , 107,128,
`and 210; Patient2, 115,118,and 160; Patient 3, 125,178,and 127;
`Patient4, 74, 175,and 93; Patient 5, 127,77, and 79.
`
`Urea was isolated from the urine of Patient 1 and was
`crystallized twice from 95% ethanol. By TLC, this fraction
`was shown to be contaminated with materials cocrystallized
`with urea.
`[‘4C]Uneawas quantitatively measured by con
`verting it to ‘4C02by unease.Of the excreted ‘4Cin 0- to 4-, 4-
`to 8- to 12-, and 12- to 24-hr unines, 0.8, 1.6, 2.9, and 5.7%,
`respectively, was associated with urea.
`Continuous Infusion. Three patients received 5-['4CJAC
`
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`z. H.!srai!ieta!.
`by continuous infusion. Plasma levels and urinary excretion
`of ‘4Care presented in Tables 2 and 5. In 1 case (Patient 7, at
`6 hr after beginning of infusion)
`the spinal
`fluid to plasma
`concentration ratio of ‘4Cwas 0.08. At 15 and 30 mm and 1,
`2, 4, and 6 hr after beginning of infusion, plasma 5-['4C]AC
`levels were 70, 46, 42, 34, 15, and 13%, respectively, of the
`total ‘@Cconcentration in plasma. Urinary excretion of ‘4C(0
`to 24 hn) amounted to 85, 83, and 94% of the dose (Table 5)
`in the 3 patients, respectively.
`
`Partition Coefficient and Binding of 5-['4C]AC
`
`The partition coefficient of 5-['4C]AC (concentration in the
`organic phase/concentration
`in the aqueous phase) was
`found to be <0.005 for all 3 systems studied.
`The binding of 5-['4C]AC to 5% w/v human albumin in
`Sorensen buffer at pH 7.4, 37°,was found to be <1% both by
`equilibrium dialysis and molecular sieve procedures.
`
`StabIlIty of 5-AC in Solutions
`
`Freshly prepared solutions of 5-['4C}AC were incubated in
`various media at several temperatures and analyzed by TLC.
`For NMR the solutions of 5-AC were allowed to remain in the
`probe for the duration of the experiment.
`TLC Method. Almost all of the spotted radioactivity at time
`0 was found to be in the zone corresponding to 5-AC. This
`
`value was assumed to be 100%. The radioactivity (as per
`centage of control) associated with zones corresponding to
`5-[14C]ACwas measured at 1/2,1, 2, 4, 6, 24, and 48 hr after
`incubations at 0°,25°,and 37°in various media. The decom
`position of 5-['4C]AC followed 1st-order kinetics;
`the t12's of
`decay are given in Table 6. 5-AC was more stable in Tnis
`
`Table 5
`Cumulative urinary excretion of @Cin patients during and after
`5-('4CJACTimeâ€(cid:157)
`continuous
`infusion
`of
`of dose excreted inâ€(cid:157)
`
`(hr)%
`80-2
`
`% of dose
`infused
`
`0-4
`0-6
`0-8
`
`0-122351.60-24
`
`39
`54
`69
`100
`
`Patient6
`3.1
`10.9
`20.8
`30.2
`50.7
`
`Patient 7
`5.0
`11.0
`13.0
`27.6
`46.4
`
`0-48
`0-72
`0-96
`0-12085.4
`
`82.7
`91.7
`94.7
`96.2
`96.9
`
`Patient
`21.8
`36.6
`42.4
`42.7
`
`93.5
`97.2
`98.4
`99.0
`99.5
`
`a Time
`
`from
`
`beginning
`
`of
`
`infusion
`
`(lasting
`
`12
`
`hr).
`
`1@The
`
`renal
`
`clearances
`
`(mlimin)
`
`of
`
`‘4C (5-['4C]AC
`
`plus
`
`metabo
`
`lites)for 0-to 4-and 4-to 8-hr intervalswereasfollows: Patient6, 73
`and 96; Patient7, 88 and 103;Patient8, 84 and 92, respectively.
`
`Table6
`Stability studies of 5-AC
`Tern
`
`Mediumâ€(cid:157)Methodpera
`pointsDistilled
`waterNMR40°6.54.4
`0.6'@15EDTA
`(10â€(cid:157)M)/N2â€(cid:157)NMR406.38.5
`2.0'19Tris
`buffer(102 M)-EDTA(10@NMR406.36.3
`1.211M)Ringer's
`
`lactateNMR406.25.6
`1.3@13Ringer's
`lactate-EDTA(10â€(cid:157)M)iNMR406.25.8
`1.09N2Human
`
`turepHt@,2
`
`plasma(fresh)'NMR377.4°5.2
`0.814Human
`plasma(fresh)'TLC377,4°1
`.45Human
`plasma(stored)'NMR377,405,4
`0.812Human
`
`plasma(stored@TLC377.4â€(cid:157)2.05SOrensen
`buffer(Mi15)TLC377.45.05Sorensen
`
`
`buffer(M/15)TLC257.4175Tnis
`
`buffer (102M)TLC256.3686Ringer's
`lactateTLC256.2657Human
`plasma(fresh)'TLC257.4°7.05Human
`
`urineTLC256.5°707Ringer's
`lactateNMR206.294
`532Ringer's
`lactateNMR206.2100
`774Tris
`lactateNMR96.2170
`
`buffer(102M)TLC06.35287Human
`plasma(fresh)'TLC07.4â€(cid:157)537Human
`
`urineTLC06.5°2887
`
`625Ringer's
`
`of
`
`of decomposi
`tion of 5-AC(hr)â€(cid:157)No.
`±
`±
`±
`
`±
`±
`
`±
`
`±
`
`±
`
`±
`
`±
`
`a Concentration
`
`of
`
`5-AC
`
`=
`
`30
`
`mM
`
`(NMR)
`
`and
`
`5-['4C]AC
`
`=
`
`3.5
`
`m@i
`
`(TLC).
`
`S Each
`
`t112 determination
`
`carried out for 170to 190mm (0.5to 2.0 half-lives).Forthe TLC, incubationswere carried out up to
`48 hr.
`
`represents
`
`a single
`
`experiment.
`
`In
`
`the
`
`NMR
`
`studies,
`
`experiments
`
`were
`
`C p112
`
`±
`
`95%
`
`confidence
`
`level.
`
`EDTA.
`disodium
`aqueous
`d EDTA,
`e The R2 values for these 2 determinations
`I Fresh
`plasma
`for
`each
`experiment
`was
`stored
`at 0°for 1 to 10 weeks.
`U pH
`was
`not
`controlled.
`
`were between
`obtained
`from
`
`(NMR).
`>0.96
`for others,
`0.90 and 0.93;
`different
`individuals.
`Stored
`plasma
`was
`
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`

`buffer (pH 6.3, 102 M) or Ringer's lactate at pH 6.2 and least
`stable in fresh hepaninized plasma.
`NMR Method. The resonance due to the proton at C-6 of
`5-AC occurred at 8.7 ppm (singlet) in D2Osolution at 20°;the
`aldehyde proton of benzaldehyde resonated at 9.8 ppm
`relative to the reference,
`tetramethylsilane in the capillary.
`In Ringer's lactate at 20°,the resonances due to C-6 and
`benzaldehyde protons occurred at 9.0 and 9.9 ppm, respec
`tively. These chemical shifts varied slightly with temperature
`and the composition of the medium. The decomposition of
`5-AC (in D2O)is accompanied by the progressive disappear
`ance of the resonance at 8.7 ppm and the appearance of a
`new peak of 8.5 ppm (Chart 2). The addition of formic acid
`to the partially decomposed solution of 5-AC increased the
`intens