[CANCER RESEARCH 54. 1690-1694, April 1, 1994]
`
`A Phase I and Pharmacokinetic Study of Intravenous Phenylacetate
`in Patients with Cancer1
`
`Alain Thibault,2 Michael R. Cooper, William D. Figg, David J. Venzon, A. Oliver Sartor, Anne C. Tompkins,
`
`Maribeth S. Weinberger, Donna J. Headlee, Natalie A. McCall, Dvorit Sumid, and Charles E. Myers
`
`Clinical Pharmacology Branch [A. T., M. R. C., W. D. F., A. O. S., A. C. T., M. S. W., D. J. H., N. A. M., D. S., C. E. M/ and Bioslaiislics
`[D. J. V.l, National Cancer Institute, NIH, Belhesda. Maryland 20892
`
`and Dala Management
`
`Section
`
`ABSTRACT
`
`tumor growth and
`Phenylacetate has recently been shown to suppress
`promote differentiation
`in experimental models. A phase I trial of phe-
`ny(acetate was conducted in 17 patients with advanced solid tumors. Each
`patient received a single i.v. bolus dose followed by a 14-day continuous
`i.v. infusion of the drug. Twenty-one
`cycles of therapy were administered
`at four dose levels, achieved by increasing
`the rate of the continuous
`i.v.
`infusion. Phenylacelate
`displayed nonlinear pharmacokinetics
`|A,„=
`105.1 ±44.5 (SD) /Kg/ml, Vm.s = 24.1 ±5.2 mg/kg/h and Vd = 19.2 ±33 L].
`There was also evidence for induction of drug clearance. Ninety-nine % of
`phenylacetate
`elimination was accounted for by conversion to phenylac-
`etylglutamine, which was excreted in the urine. Continuous
`i.v.
`infusion
`rates resulting in serum phenylacetate
`concentrations
`exceeding A,,, often
`resulted in rapid drug accumulation
`and dose-limiting
`toxicity, which
`consisted of reversible
`central nervous
`system depression,
`preceded by
`emesis. Three of nine patients with metastatic, hormone-refractory
`pros
`tate cancer maintained stable prostatic
`specific antigen levels for more
`than 2 months; another had less bone pain. One of six patients with
`glioblastoma multiforme, whose steroid dosage has remained unchanged
`for the duration of therapy, has sustained functional
`improvement
`for
`more than 9 months. The use of adaptive
`control with feedback for the
`dosing of each patient enabled us to safely maintain stable phenylacetate
`concentrations
`up to the range of 200—300/ug/ml, which resulted in clinical
`improvement
`in some patients with advanced disease.
`
`INTRODUCTION
`
`is a small
`a product of phenylalanine metabolism,
`Phenylacetate,
`molecule (Mr 136) normally present
`in the mammalian circulation in
`low concentrations
`(1). It has been administered primarily to children
`with hyperammonemia
`due to inborn errors of urea synthesis
`(2, 3)
`and to patients with hyperammonemia
`resulting from the chemother
`apy of leukemias
`(4) or from portal systemic encephalopathy
`(5). In
`humans, phenylacetate
`is conjugated with glutamine by the hepatic
`enzyme phenylacetyl Coenzyme A: glutamine acyltransferase
`to yield
`phenylacetylglutamine
`(6, 7), which is then excreted in the urine. The
`mobilization of glutamine-associated
`nitrogen is believed to be the
`mechanism whereby hyperammonemia
`is improved. More recently,
`phenylacetate and related compounds have received attention for their
`ability to induce tumor cytostasis
`and differentiation
`in laboratory
`models (8-11)
`and fetal hemoglobin synthesis in patients (12). Inter
`est
`in phenylacetate
`as an anticancer
`agent was also generated by
`reports that antineoplaston AS2-1,
`a preparation which by weight
`is
`80% phenylacetate,
`displayed clinical antitumor activity (13).
`the
`Preclinical
`studies documented
`that phenylacetate modifies
`biology of various hematopoietic and solid tumors,
`including prostatic
`carcinoma and glioblastoma
`(10, 11). To achieve this effect requires
`that cells be exposed to phenylacetate
`concentrations
`in excess of 275
`
`Received 9/15/93; accepted 1/28/94.
`The cosi of publication of the article were defrayed in pan by the payment of page
`charges. This article must
`therefore be hereby marked advertisement
`in accordance with
`18 U.S.C. Section 1734 solely to indicate this fact.
`1This study was supported in part by a grant from Elan Pharmaceutical Research Co.
`2 To whom requests
`for
`reprints
`should be addressed,
`at Clinical Pharmacology
`Branch. National Cancer
`Institute, NIH. Building
`10. Room I2C103. Bethesda, MD
`20892.
`
`It appeared feasible to expose
`pig/ml for a minimum of two weeks.
`adults with solid tumors to similar concentrations
`of phenylacetate,
`which children with urea cycle disorders had tolerated (2, 3). Hence,
`a phase I trial was designed to deliver a CIVI3 of phenylacetate over
`a 2-week period. We herein report
`the clinical and pharmacokinetic
`results of
`this study and discuss
`an alternative
`schedule of drug
`administration
`for future trials.
`
`MATERIALS AND METHODS
`
`Patient Population. Patients were eligible for this study if they had ad
`vanced solid tumors
`for which conventional
`therapy had been ineffective,
`a
`Karnofsky performance
`status greater
`than 60%, normal hepatic transaminases
`and total bilirubin,
`a serum creatinine
`less than 1.5 mg/dl, and normal
`leuko
`cyte (>3,500/mm3)
`and platelet counts (>150,000/mm3). All patients signed an
`
`that had been approved by the National Cancer
`informed consent document
`Institute Clinical Research Subpanel.
`Seventeen
`patients,
`16 men and 1
`woman, with a median age of 57 (range: 36-75) were enrolled
`between
`January and June 1993. Disease distribution included progressive, metastatic,
`hormone-refractory
`prostate
`cancer
`(9 patients),
`anaplastic
`astrocytoma
`or
`glioblasloma multiforme (6 patients), ganglioglioma
`(1 patient), and malignant
`pleural mesothelioma
`(1 patient).
`Sodium phenylacetate for injec
`Drug Preparation and Administration.
`tion was prepared from bulk sodium phenylacetate
`powder supplied by Elan
`Pharmaceutical Research Co. (Gainesville, GA). The finished injectable stock
`solution was manufactured
`by the Pharmaceutical Development Service, Phar
`macy Department, Clinical Center, NIH,
`in vials containing
`sodium phenyl
`acetate at a concentration of SOUmg/ml
`in sterile water for injection, USP, with
`sodium hydroxide and/or hydrochloric
`acid added to adjust
`the pH to approx
`imately 8.5. Doses of sodium phenylacetate
`to be infused over 30 min to 2 h
`were prepared in 150 ml of sterile water
`for injection, USP. Doses of pheny
`lacetate to be given over 24 h were prepared similarly to yield a total volume
`of 1000 ml and were administered
`using an infusion pump.
`Clinical Protocol. The protocol as originally designed delivered an i.v.
`bolus dose of phenylacetate
`(150 mg/kg over 2 h) on the first day of therapy,
`to allow for the estimation of pharmacokinetic
`parameters. This was followed
`24 h later by a CIVI of the drug for the next 14 days. Cycles of 2-week drug
`infusions were repeated every 6 weeks. The rate of drug infusion was to be
`increased in sequential cohorts of at least three patients, and individual patients
`could escalate from one dose level to the next with sequential cycles of therapy
`provided they had experienced
`no drug-related
`toxicity and their disease was
`stable or improved.
`over the 6-month period, (a)
`several modifications
`The protocol underwent
`the size of the initial bolus dose was reduced from 150 to 60 mg/kg i.v. and the
`bolus infusion duration from 2 h to 30 min, after the first
`three patients were
`treated. This change resulted in drug concentrations
`optimal
`for estimating the
`pharmacokinetics
`of the drug (see below) within a 6-h time period, (b) after the
`nonlinear nature of the pharmacokinetics
`of phenylacetate was recognized (see
`below),
`the protocol was changed from a fixed dose escalation (dose levels 1
`and 2, 150 and 250 mg/kg/day,
`respectively)
`to a concentration-guided
`esca
`lation trial (dose levels 3 and 4, 200 and 400 fig/ml,
`respectively).
`In the latter
`format each patient was given an i.v. bolus dose of phenylacetate
`(60 mg/kg
`over 30 min)
`l week prior
`to beginning
`a 14-day CIVI of the drug. The
`patient-specific
`pharmacokinetic
`parameters
`estimated
`from the bolus dose
`were used to calculate
`an infusion rate that would maintain the serum phen-
`
`1 The abbreviations
`
`used are: CIVI, continuous
`
`i.v.
`
`infusion; CNS, central nervous
`
`system; CSF, cerebrospinal
`
`fluid; HPLC, high performance
`
`liquid chromatography.
`
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` on July 16, 2011cancerres.aacrjournals.org
`
`
` American Association for Cancer Research
` Copyright © 1994
`
`1690
`
`LUPIN EX. 1009
`
`

`

`PHARMACOKINETICS
`
`OF PHENYLACETATE
`
`at the targeted level during the 14-day infusion. Drug
`ylacetate concentration
`concentrations were monitored according to the sampling schedule described
`below. Serum samples were analyzed weekly, prompting weekly reestimation
`of individual pharmacokinetics
`and dosage adjustment
`(adaptive control with
`feedback).
`Sampling Schedule. With the initial 150-mg/kg i.v. bolus, blood samples
`were obtained through a central venous catheter at the following time points
`calculated from the beginning of the infusion: 0, 60, 115, 125, 135, 150, 165,
`180, 240, 360, 480, and 600 min. For the 60-mg/kg bolus given over 30 min,
`blood sampling was performed at 0, 30, 60, 75, 90, 105, 120, 150, 180, 270,
`and 390 min from the beginning of the infusion. During CIVI, blood samples
`were obtained by venipuncture. At dose levels 1 and 2, blood samples were
`obtained daily during the CIVI; while at dose levels 3 and 4, blood samples
`were obtained on days 1, 2, 3, 8, 9, and 10 of the infusion. Twenty-four-h
`urine
`collections
`for the determination
`of phenylacetate
`and phenylacetylglutamine
`excretion were obtained on days 1, 7, and 14 of therapy. Sampling of the CSF
`was performed only if clinically indicated.
`Analytical Method. Determination of sodium phenylacetate and phenyla
`cetylglutamine
`in serum and urine by HPLC. Blood was drawn into a Vacu-
`tainer
`tube free of anticoagulant
`and was then refrigerated.
`It was centrifuged
`at 1200 X g for 10 min in a Sorvall RT 6000D centrifuge
`(DuPont Co.,
`Wilmington, DE) at 4°C. Serum was
`then removed
`and stored in Nunc
`Cryotubes
`(Nunc Co., Denmark)
`at -70°C until
`the day of analysis.
`
`by adding known amounts of sodium
`curve was generated
`A standard
`(Elan Pharmaceutical Research Co.) and phenylacetylglutamine
`phenylacetate
`(a gift from Dr. S. W. Brusilow, Johns Hopkins University, Baltimore, MD) to
`a commercial
`preparation
`of pooled serum (Baxter Healthcare Corporation,
`Deerfield,
`IL). The standard
`values
`spanned
`the expected
`range of serum
`concentrations:
`0, 5, 10, 20, 50, 100, 250, 500, 750, and 1500 pig/ml.
`Two hundred fi\ of serum were pipeted into a 1.7-ml Eppendorf
`tube (PGC
`Scientifics, Gaithersburg, MD). Protein extraction was carried out by adding
`100 fj.\ of a 10% (v/v) solution of perchloric
`acid (Aldrich Chemical Co.,
`Milwaukee, WI). The tube was vortexed and then centrifuged at 4500 X g for
`10 min. Supernatant
`(150 /¿I)was transferred to a new 1.7-ml Eppendorf
`tube
`and 25 /xl of 20% KHCO3 (w/v) were added to neutralize
`the solution. This
`was centrifuged
`at 4500 x g for 10 min and 125 fil of supernatant were
`transferred to an autosampler
`vial and maintained at 10°Cuntil HPLC injec
`
`tion. Urine samples were processed in an identical manner after an initial 1:10
`dilution with water.
`The HPLC system (Gilson Medical Electronics, Middleton, WI) was com
`posed of two pumps
`(305 and 306), an 805 manometric module,
`an 811C
`dynamic mixer, a 117 variable wavelength UV detector, and a 231 autosampler
`fitted with a 20-/xl
`injection loop and cooled with a Grey Line model 1200
`cooling device. The column was a Waters
`(Millipore Corporation, Milford,
`MA) C]s Nova-Pak,
`3.9 x 300 mm, maintained
`at 60°C with a Waters
`temperature
`control module. The mobile phase solutions consisted of acetoni-
`trile (J. T. Baker Chemical Co.,
`Inc., Phillipsburg, NJ) and water, both
`acidified with phosphoric
`acid (0.005 M). An acetonitrile
`concentration
`gradi
`ent was used, which increased from 5% to 30% over 20 min.
`Twenty ju.1of the neutralized supernatant were injected onto the column and
`eluted at 1 ml/min. The progress of the elution was followed by monitoring the
`UV absorbance
`at 208 nm. Characteristic
`elution times for sodium phenylac
`etate and phenylacetylglutamine
`under these conditions were 17.1 and 9.8 min,
`respectively.
`by Ion-Exchange
`of Plasma Glutamine Concentrations
`Determination
`Glutamine concentration was measured in sodium hepa-
`Chromatography.
`rin-preserved plasma (-80°C storage)
`following a 1:2 dilution/deproteinization
`with 15% 5-sulfosalicylic
`acid/sarcosine
`hydrochloride
`(Sigma Chemical Co.,
`St. Louis, MO). A stock solution of L-glutamine
`(1000 /ig/ml)
`(Sigma) was
`diluted with Li-S buffer
`(Beckman
`Instruments,
`Inc., Palo Alto, CA)
`to
`generate a standard curve ranging from 0.78 to 100 fig/ml. A pooled plasma
`sample from 250 patients was used to make a quality control solution.
`Fifty-fil
`samples were
`autoinjected
`onto a 10-cm cation-ion
`exchange
`column integrated into a Beckman Model 6300 amino acid analyzer
`(Beck
`man). The solvent
`flow rate (2:1 water/ninhydrin) was maintained constant at
`0.5 ml/min. Column temperature was maintained at 33°Cuntil glutamine was
`eluted, at which time the column temperature was raised by 1.5°C/minto elute
`
`standard. The column was regenerated with lithium
`the internal
`sarcosine,
`hydroxide at 70°Cfollowing each injection. Absorbance was measured at 570
`
`color development with ninhydrin-RX
`postcolumn
`and 440 nm following
`(Beckman)
`at 131°C. Beckman System Gold software was used for data
`
`acquisition and data management.
`Pharmacokinetic Methods.
`Initial estimates of Vmaxand Km for phenyla
`cetate were obtained by generating Lineweaver-Burk
`plots from concentration
`versus time curves following i.v. bolus doses. These initial parameter estimates
`were refined by nonlinear
`least squares
`fitting to a single compartment,
`open
`nonlinear model, using the Nelder-Mead iterative algorithm, as implemented in
`the Abbottbase Pharmacokinetic
`Systems
`software package
`(Abbott Labora
`tories, Abbott Park,
`IL; version 1.0). Each data point was weighted equally.
`Statistical Methods. Student's t test was used to compare estimates of the
`pharmacokinetic
`parameters
`of phenylacetate
`derived from the Lineweaver-
`Burk plots with those obtained using nonlinear
`least squares regression. Serum
`phenylacetate
`concentrations
`observed on day 2 of the CIVI were compared
`with those observed on day 11, using the Wilcoxon signed rank test for paired
`data (14) to assess the significance of time-related changes
`in drug concentra
`tions. At dose levels 3 and 4, the CIVI rate varied with time and the presence
`of induction of clearance was assessed by comparing
`a single compartment,
`open nonlinear model with the same model modified
`to incorporate
`two
`additional
`pharmacokinetic
`parameters which
`allowed
`for
`time-dependent
`changes in the maximum velocity of drug elimination (Vm.,x). The performance
`of each model
`in describing a given set of dosing and concentration
`data was
`quantified
`by calculating
`the weighted sum of the errors
`squared following
`nonlinear
`least
`squares
`fitting. The
`standard
`deviation
`of
`the errors was
`modeled as a function of drug concentration multiplied by the coefficient of
`variation
`of
`the assay. The
`fitting procedure was used to maximize
`the
`likelihood of normally distributed variâtes,and the normality of the distribu
`tion of the standardized
`errors was confirmed by the method of Shapiro and
`Wilk (15). Confidence
`regions
`for
`the parameters were derived
`from the
`weighted sum of squares in the model
`incorporating
`the induction parameters,
`and approximate
`significance
`levels for testing between the two models were
`calculated using the F distribution (16); P < 0.05 was considered significant.
`The Spearman
`rank correlation method was used in an attempt
`to discern
`whether
`there was a relationship between the dose of phenylacetate
`adminis
`tered and the P value derived from the F distribution for each cycle of therapy.
`
`RESULTS
`
`Analytical Assay. The reverse phase HPLC assay allowed both
`serum phenylacetate
`and phenylacetylglutamine
`concentrations
`to be
`determined simultaneously
`from the same sample (see Fig. 1). The
`lower limit of detection for both compounds
`in serum and urine was
`5 ju.g/ml, based upon a signal:noise
`ratio of 5:1. The interassay
`coefficient of variation for serum concentrations was less than 6%
`within the range of 40-1000
`fig/ml
`(Table 1). The lower
`limit of
`detection for glutamine was 0.5 /xg/ml, with an interassay coefficient
`of variation that did not exceed 7%.
`
`0.4-
`
`eo
`
`0.2
`
`5.00
`
`15.00
`10.00
`Time (min)
`
`20.00
`
`25.00
`
`and phenylacetylglutamine.
`Fig. 1. Chromatogram of phenylacetate
`and 17.1 min represent phenylacetylglutamine
`and phenylacetate,
`concentration of 250 fig/ml
`in holh instances.
`
`The peaks at 9.X
`respectively.
`Serum
`
`1691
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`
`
` American Association for Cancer Research
` Copyright © 1994
`
`LUPIN EX. 1009
`
`

`

`PHARMACOHNETICS OF PHENYLACETATE
`
`Model Specification and Initial Parameter Estimation. Fig. 2
`shows
`representative
`concentration
`versus time curves
`for simulta
`neously measured serum levels of sodium phenylacetate
`and phenyl-
`acetylglutamine
`and plasma levels of glutamine following both 150-
`and 60-mg/kg bolus doses of sodium phenylacetate. The decline in
`serum phenylacetate
`concentration
`following the 150-mg/kg bolus is
`linear when plotted on a nonlogarithmic
`scale and consistent with
`saturable elimination kinetics. While useful for demonstrating a zero-
`order process,
`the magnitude of the bolus was inadequate for param
`eter estimation insofar as most of the phenylacetate
`concentrations
`obtained over the 6-h sampling period were above Km. In order
`to
`generate concentrations
`both above and below Km, the bolus was
`changed to 60 mg/kg i.v. over 30 min. Visual
`inspection of
`the
`concentration versus time curves following these boluses revealed no
`evidence of an initial distributive
`phase,
`suggesting
`that a single
`compartment,
`open nonlinear model would be adequate to describe
`the pharmacokinetics
`of the drug.
`Initial estimates of Km [90 ±30
`(SD) /j,g/ml], Vmax (26.0 ±10 mg/kg/h), and Vd (22.4 ±6.8 liters)
`were calculated in 13 patients using the Lineweavcr-Burk
`equation.
`
`Table 1 PA" standard
`
`cur\~e assay variability
`
`PA(Mg/ml)404001000CV(%)2.61.73.4PAG(fig/ml)404001000CV(%)4.64.33.1
`
`' PA. phenylacctate; PAG. phcnylacetylglutaminc:
`
`C'V. coefficient of variation.
`
`220 -
`
`180
`
`concentrations over time during CIVI (250 mg/kg/day)
`Fig. 3. Declining phenylacetate
`in one patient,
`suggestive
`of clearance
`induction. D. measured serum phenylacetate
`concentrations;
`bars,
`l)5% confidence
`limits of the model's
`til to the data.
`
`Days
`
`least
`estimates by nonlinear
`Refinement of these initial parameter
`squares fitting of the entire concentration versus time profile for each
`bolus dose yielded the following estimates: Km = 105.1 ±44.5
`fig/ml; Vnlax = 24.1 ±5.2 mg/kg/h; and Vd = 19.2 ±3.3 liters. The
`differences between the two methods of estimation were not statisti
`cally different, as measured by Student's
`/ test (P = 0.89).
`
`In some patients treated at
`Induction of Phenylacetate Clearance.
`dose levels 1 and 2, we observed a tendency for the serum phenyl
`acetate
`concentration
`to decrease with time. An example of
`this
`phenomenon is shown in Fig. 3. Considering the 12 cycles of therapy
`delivered at these levels, a comparison of the serum drug concentra
`tion measured on day 2 of CIVI to that observed on day 11 demon
`strated a 23% mean decline in concentration
`over
`this time period
`(Wilcoxon signed rank test, P = 0.016).
`targeted serum
`At dose levels 3 and 4, attempts
`at maintaining
`phenylacetate concentrations using adaptive control with feedback led
`to variable rates of drug infusion over time, which precluded a simple
`comparison of drug concentrations
`at the beginning and end of ther
`apy. We therefore analyzed all cycles of therapy at all four dose levels
`and compared the performance of the single compartment
`nonlinear
`model described above with the same model modified to allow Vmax
`to increase with time. The formula used to describe this increase was
`
`100
`
`200
`
`300
`
`400
`
`500
`
`600
`
`700
`
`Time (min)
`
`V„„uO = Vmax (/ = 0)X {1.0 + [(IF -
`
`1.0) X (1.0 - c'KX')]}
`
`01
`
`500
`
`400
`
`300
`
`200
`
`Co
`o
`
`100 , : I
`
`o*
`
`B
`
`200
`
`where I is the time elapsed (in h) since the initiation of therapy, IF is
`an induction factor representing the maximum-fold
`increase in Vmax
`at infinite time, and//? is a first order rate constant (h~')describing
`the
`
`rate at which Vnlax increases over time. Each cycle of therapy (n =
`21) was evaluated by comparing the difference in the weighted sum of
`errors squared generated by nonlinear
`least squares fitting with each
`model. The significance of the difference was evaluated using the F
`distribution.
`In 9 of the 21 cycles, allowing Vnlaxto increase with time
`yielded an improved fit (induction parameters,
`IF = 1.87 ±0.37,
`IR = 0.0028 ±0.003 h - 1). The Spearman rank correlation method
`did not demonstrate a correlation between rate of drug administration
`and the need to incorporate
`the two induction parameters
`into the
`model (rank correlation coefficient,
`-0.39; P = 0.084). The dose rates
`administered ranged from 450 to 1850 mg/h.
`revealed no
`Review of concomitantly
`administered medications
`of a time-
`association
`between specific drugs and the occurrence
`dependent
`increase in phenylacetate
`clearance.
`In the seven patients
`with primary CNS tumors,
`treatment with anticonvulsants
`always
`antedated the administration
`of phenylacetate by months to years.
`1692
`
`100
`
`300
`
`500
`
`Time (min)
`
`(•)and
`(•)and phenylacetylglulamine
`of phenylacetate
`Fig. 2. Scrum concentrations
`plasma concentrations of glutamine (A) following a 15(l-mg/kg i.v. bolus of phenylacetate
`over 2 h (A), and a 60-mg/kg i.v. bolus over 30 min (B).
`
`Downloaded from
` on July 16, 2011cancerres.aacrjournals.org
`
`
` American Association for Cancer Research
` Copyright © 1994
`
`LUPIN EX. 1009
`
`

`

`PHARMACOKINETICS
`
`OF PHENYLACETATE
`
`aphasia for more than 9 months. Although no change in the size of the
`tumor mass was noted,
`reduction in peritumoral
`edema was docu
`mented by magnetic
`resonance
`imaging (see Fig. 4). His steroid
`regimen had not been changed for 2 weeks prior to starting therapy
`with phenylacetate
`and has been kept unchanged since.
`
`DISCUSSION
`
`of phenylacetate
`the pharmacokinetics
`of
`Previous descriptions
`have been fragmentary. Simell et al. (3) reported the drug to have first
`order elimination kinetics with a half-life of 4.2 h following bolus
`dose administration (270 mg/kg) in children. The failure to recognize
`the nonlinear nature of phenylacetate
`pharmacokinetics
`probably re
`sulted from the smaller total doses given to these patients compared to
`those given in our study. The saturable pharmacokinetics
`of phenyl-
`
`Tahlc 2 PA" anil PA(ìconcentration.* per dote level during CIVl
`
`(fig/ml)90
`
`±34
`150 ±63
`188 ±55
`306 ±51
`
`Dose
`level1
`
`dose
`
`(mg/kg/day)150
`
`4PA"
`
`±19*
`
`104 ±40178
`250
`±85
`266 ±40
`397 ±244PAG
`374 ±95PA(/ig/ml)49
`" PA, phenylacetate; PA(i, phenylacetylglutamine.
`'' Mean ±SD.
`
`23
`
`of Phenylacetate Clearance. As shown in Fig. 2,
`Mechanisms
`underwent
`rapid conversion to phenylacetylglutamine.
`phenylacctate
`In the three patients who received 150 mg/kg of phenylacciaie over 2
`h, the peak serum concentration of phenylacetylglutamine was 224 ±
`81 /j-g/ml, 325 ±72 min postinfusion. After the 60-mg/kg boluses,
`the
`peak serum phenylacetylglutamine
`concentration was
`104 ± 33
`/xg/ml at 86 ±33 min.
`prior to bolus treatment with
`The plasma glutamine concentration
`(n = 16), similar
`to values
`phenylacetatc was 109 ± 29 fig/ml
`reported in the literature
`for normal volunteers
`(2, 3). The largest
`reduction in circulating plasma glutamine levels (46%) was observed
`¡na patient
`receiving a 150-mg/kg bolus. Since phenylacelate
`is
`conjugated with glutamine to yield phcnylacetate,
`the molar excretion
`of glutamine was found to increase in direct proportion to the dose of
`drug administered.
`was determined
`of phenylacetylglutamine
`The molar excretion
`from 24-h urine collections.
`It accounted for 99 ±23% (n = 18) of
`the dose of phenylacetate
`administered over the same period of time.
`The recovery of free, nonmetabolized
`drug was only 1.5 ±2.4% of
`the total administered dose. A strong phenylacetate odor was detect
`able on patients'
`clothes
`and on examiners'
`hands after physical
`
`examination. This suggests that phenylacetate may also be excreted to
`some extent
`transdermally.
`into
`and Phenylacetylglutamine
`Distribution of Phenylacetate
`the CSF. Clinical circumstances required evaluation of the cérébro-
`spinal
`fluid in two patients who had metastatic prostate cancer and
`were free of CNS métastases.The first had reached steady-state
`phenylacetate
`and phenylacetylglutamine
`concentrations
`of 141 and
`199 fxg/ml, respectively. The corresponding
`simultaneous CSF con
`centrations were 74 and 5 /xg/ml, respectively. At the time of simul
`taneous serum and CSF sampling,
`the second patient had not received
`further
`therapy for 6 h after having reached a scrum phenylacetate
`concentration of 1044 /j,g/ml. Measurements
`in serum and CSF were
`781 and 863 fig/ml
`for phenylacetate
`and 374 and 46 ^.g/ml
`for
`phenylacetylglutamine,
`respectively.
`Clinical Toxicities. No toxicity was associated with bolus admin
`istration of the drug. The highest peak serum concentrations were
`measured after the 150-mg/kg bolus over 2 h (533 ±94 fig/ml). Table
`2 lists the average serum phenylacctate
`concentrations per dose level.
`Although those achieved at dose levels 3 and 4 are close to their
`target,
`the large associated standard deviations
`reflect our inability to
`maintain serum phenylacetate concentrations within the desired range,
`even when using adaptive control with feedback.
`Drug-related toxicity was clearly related to the serum phenylacetate
`concentration. Three episodes of CNS toxicity,
`limited to confusion
`and lethargy and often preceded by emesis, occurred
`in patients
`treated at dose levels 3 and 4. They were associated with drug
`concentrations
`of 906, 1044, and 1285 jug/ml (1078 ±192 /Ag/ml),
`respectively. Symptoms
`resolved within 18 h of terminating the drug
`infusion in all instances.
`Antitumor Activity. Prostatic specific antigen (measured weekly)
`remained stable for more than 2 months in 3 of the 9 patients with
`prostate cancer
`treated at dose levels 2, 3, and 4. A fourth patient
`taking 180 mg of morphine daily experienced marked improvement
`in
`bone pain control and was able to substitute
`a nonstcroidal
`antiin-
`flammatory drug to his narcotic regimen. The mean phenylacetate
`concentration of the four responders was 244 ±33 /xg/ml (186, 197,
`269, and 325 j^g/ml). These values were not statistically
`different
`from those achieved in the other six patients with prostate cancer. One
`of six patients with glioblastoma multiforme
`that recurred after sur
`gery, standard radiation therapy and chemotherapy with bischloroeth-
`ylnitrosourea
`has maintained
`improvements
`in performance
`status
`(30% on Karnofsky's
`scale),
`intellectual
`function,
`and expressive
`
`brain magnetic resonance imaging in a
`Fig. 4. (A) Pretreatment gadolinium-enhanced
`patient with gliohlasloma multiforme,
`(fi) Posttreatment gadolinium-enhanced magnetic
`resonance imaging after 1 cycle of phenylacelate
`(15(1 mg/kg/day)
`illustrating resolution
`of peritumoral edema.
`
`Downloaded from
` on July 16, 2011cancerres.aacrjournals.org
`
`
` American Association for Cancer Research
` Copyright © 1994
`
`1693
`
`LUPIN EX. 1009
`
`

`

`PHARMACOKINLIÃ(cid:143)CS OF PHENYLACETATE
`
`in drug concen
`in the rate of drug infusion results in large changes
`tration. An alternative strategy is to deliver the drug by repeated short
`infusions. Our
`limited experience with the 150-mg/kg i.v. boluses
`suggests
`that
`serum phenylacetate
`concentrations
`occurring
`tran
`siently above 500 ng/ml are well
`tolerated.
`In addition,
`intermittent
`drug infusion should permit
`some drug washout
`to occur,
`thereby
`minimizing drug accumulation. A regimen of 200 mg/kg every 12 h
`(1-h infusion)
`is simulated in Fig. 5. It assumes that the pharmacoki-
`netic parameters determined from our 17 patients are representative of
`the cancer population at large and that Vmax does not change with
`time. It predicts that a wide range of peak drug concentrations will be
`observed. However,
`it
`is possible
`that
`these would be sufficiently
`transient
`so as not
`to produce CNS toxicity and the troughs not so
`prolonged as to abrogate the antitumor activity of the drug.
`Although dosing alternatives
`should be explored, our study indi
`cates that phenylacetate can be safely administered by CIVI and result
`in clinical
`improvement
`in some patients with hormone-refractory
`prostatic carcinoma and glioblastoma multiforme who failed conven
`tional
`therapies.
`
`ACKNOWLEDGMENTS
`
`radiological
`for his expert
`to Dr. Nicholas Patronas
`We are grateful
`tance during the conduct of this study and to Frank N. Konstantinides
`assaying glutamine
`levels in plasma.
`
`assis
`for
`
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`by ranking methods. Biometrics,
`
`1: 80-83,
`
`comparisons
`
`2
`
`1000
`
`900
`
`- 800
`
`3 700
`
`60°
`
`C I
`
`§ 500
`
`<S 400
`
`Å“ 300
`V)
`
`200
`
`100
`
`Q0
`
`4
`Days
`1-h
`regimen (200 mg/kg/dose,
`phenylacetate
`of an every-12-h
`Fig. 5. Simulation
`infusion)
`in a pharmacokinetically
`average patient. For simplicity,
`induction of clearance
`was not factored in. Burs. 95% confidence
`limits expressing
`the anticipated
`range of
`concentrations
`in a population of patients.
`
`6
`
`8
`
`acetate is consistent with an enzymatic process and our calculations
`from the 24-h urinary excretion of phenylacetylglutamine
`confirm that
`this is the major
`route of elimination. Evidence that drug clearance
`may increase with time was derived from the comparison of drug
`levels on days 2 and 11 of the CIVI, adding another
`layer of com
`plexity to the pharmacokinetics
`of phenylacetate. To explain this
`phenomenon, we first considered the potential
`role of concomitantly
`administered medications but failed to demonstrate
`any association.
`Our analysis of a possible relationship between an increase in drug
`clearance with time