-
molecules that could regulate tubulinassembly and function in
cells. It isimportant to elucidate the conformationand structure of
these binding sites tocomprehend the cellular control mecha-nisms
for microtubules. In addition, suchinformation may prove essential
forunderstanding the chemotherapeuticproperties of taxol.
References
(/) SCHIFF PB, HORWITZ SB: Taxol stabilizesmicrotubules in mouse
fibroblast cells. ProcNatl Acad Sci USA 77:1561-1565, 1980
(2) MCGUIRE WP, ROWINSKY EK, ROSENSHEINNB, ET AL: Taxol: A
unique antineoplasticagent with significant activity in
advancedovarian epithelial neoplasms. Ann Intern Med111:273-279,
1989
(3) HOLMES FA, WALTERS RS, THERIAULT RL, ETAL: Phase II study of
taxol, an active drug inthe treatment of metastatic breast cancer.
JNatl Cancer Inst 83:1797-1805, 1991
(4) EINZIG Al, HOCHSTER H, WERNIK PH, ET AL:A phase II study of
taxol in patients withmalignant melanoma. Invest New Drugs9:59-64,
1991
(5) ROWINSKY EK, BURKE PG, KARP JE, ET AL:Phase I and
pharmacodynamic study of taxolin refractory acute leukemias. Cancer
Res49:4640-4647, 1989
(6) SCHIFF PB, HORWITZ SB: Taxol assemblestubulin in the absence
of exogenousguanosine 5'-triphosphate or microtuble-associated
proteins. Biochemistry 20:3247-3252, 1981
(7) KUMAR N: Taxol-induced polymerization ofpurified tubulin.
Mechanism of action. J BiolChem 256:10435-10441, 1981
(8) SCHIFF PB, FANT J, HORWITZ SB: Promotionof microtubule
assembly in vitro by taxol.Nature 277:665-667, 1979
(9) PARNESS J, HORWITZ SB: Taxol binds to poly-merized tubulin
in vitro. J Cell Biol 91:479-487, 1981
(10) MERRILL BM, WILLIAMS KR, CHASE JW, ETAL: Photochemical
cross-linking of theEscherichia coli single-stranded
DNA-bindingprotein to oligodeoxynucleotides. Identifica-tion of
phenylalanine 60 as the site of cross-linking. J Biol Chem
259:10850-10856, 1984
(11) NATH JP, EAGLE GR, HIMES RH: Direct pho-toaffinity labeling
of tubulin with guanosine5'-triphosphate. Biochemistry
24:1555-1560,1985
(12) WOLFF J, KNIPLING L, CAHNMANN HJ, ET AL:Direct
photoaffinity labeling of tubulin withcolchicine. Proc Natl Acad
Sci USA 88:2820-2824, 1991
(13) SHELANSKI ML, GASKIN F, CANTOR CR:Microtubule assembly in
the absence ofadded nucleotides. Proc Natl Acad Sci USA70:765-768,
1973
(14) RINGEL I, HORWITZ SB: Taxol is converted to7-epitaxol, a
biologically active isomer, incell culture medium. J Pharmacol Exp
Ther242:692-698, 1987
(75) GASKIN F, CANTOR CR, SHELANSKI ML: Tur-bidimetric studies
of the in vitro assembly anddisassembly of porcine neurotubules. J
MolBiol 89:737-755, 1974
(16) LAEMMLI UK: Cleavage of structural proteinsduring the
assembly of the head of bac-teriophage T4. Nature 227:680-685,
1970
(17) WANI MC, TAYLOR HL, WALL ME, ET AL:Plant anlitumor agents.
VI. The isolation andstructure of taxol, a novel antileukemic
andantitumor agent from Taxus brevifolia. J AmChem Soc
93:2325-2327, 1971
(18) ZAREMBA TG, LEBON TR, MILLAR DB, ET AL:Effects of
ultraviolet light on the in vitroassembly of microtubules.
Biochemistry23:1073-1080, 1984
(19) MANDELKOW EM, HERRMANN M, RUHL U:Tubulin domains probed by
limited pro-teolysis and subunit-specific antibodies. JMol Biol
185:311-327, 1985
Pharmacokinetics ofPamidronate in Patients WithBone
Metastases
S. Leyvraz,* U. Hess, G. Flesch,J. Bauer, S. Hauffe, J. M.
Ford,P. Burckhardt
Background: Pamidronate is a second-generation bisphosphonate
used in thetreatment of tumor-induced hyper-calcemia and in the
management ofbone metastases from breast cancer,myeloma, or
prostate cancer. Thepharmacokinetics of pamidronate isunknown in
cancer patients. Purpose:To determine the influence of the rateof
administration and of bone metabo-lism, we studied the
pharmacokineticsof pamidronate at three different infu-sion rates
in 37 patients with bonemetastases. Methods: Three groups of11-14
patients were given 60 mg pam-idronate as an intravenous
infusionover a period of 1, 4, or 24 hours.Urine samples were
collected in thethree groups of patients. Plasma sam-ples were
obtained only in the 1-hourinfusion group. The assay of
pamidro-nate in plasma and urine was per-formed by high-performance
liquidchromatography with fluorescencedetection after the
derivatization ofpamidronate with fluorescamine.Results: The body
retention (BR) at0-24 hours of pamidronate represented60%-70% of
the administered doseand was not significantly modified bythe
infusion rate. In particular, theBR at 0-24 hours was not reduced
atthe fastest infusion rate. Amongpatients, a threefold variability
in BRat 0-24 hours occurred, which wasrelated directly to the
number of bone
metastases and, to some extent, tocreatinine clearance. At 60
mg/hour,the plasma kinetics followed a multiex-ponential course
characterized by ashort distribution phase. The mean (±SD)
half-life of the distribution phasewas 0.8 hour (±0.3), the mean
(±SD)of the area under the curve for drugconcentration in plasma x
time at0-24 hours was 22.0 ± 8.8 (xmol/L xhours, and the mean (±SD)
of themaximum plasma concentration was9.7 (xmol/L (±3.2) .
Pharmacokinetievariables remained unchanged afterrepeated infusions
applied to fourpatients. Clinically, the three infusionrates were
equally well tolerated with-out significant toxicity.
Conclusions:The 1-hour infusion rate could be pro-posed as
kinetically appropriate forthe administration of pamidronate
topatients with metastatic bone diseases.[J Natl Cancer Inst
84:788-792, 1992]
Bisphosphonates are structural ana-logues of pyrophosphate, a
natural reg-ulator of bone mineral precipitation anddissolution.
Pamidronate is a second-generation bisphosphonate that
stronglyinhibits bone resorption without interfer-ing with bone
mineralization (7,2). Itsactivity, measured by inhibition of
boneresorption, is more potent when com-pared with the activity of
etidronate andclodronate, probably because of a directaction on
osteoclast precursors (1-4).
Pamidronate has been used widely inbenign clinical conditions
such asPaget's disease (5) and osteoporosis (6).In malignancy, it
has been administeredto treat tumor-induced hypercalcemia(7-9) and
to reduce morbidity caused bybone metastases (10,11). The
intra-venous route of administration is gener-ally preferred for
the treatment ofmalignant hypercalcemia or bone meta-
Received September 11, 1991; revised January21, 1992; accepted
January 30, 1992.
S. Leyvraz, J. Bauer (Centre Pluridisciplinaired'Oncologie), P.
Burckhardt (Department of Inter-nal Medicine), University Hospital
Lausanne,Switzerland.
U. Hess, Kantonsspital, St. Gallen, Switzerland.G. Flesch, S.
Hauffe, J. M. Ford, Ciba Geigy
Limited, Basel, Switzerland.*Correspondence to: Serge Leyvraz,
M.D.,
Centre Pluridisciplinaire d'Oncologie, UniversityHospital—Niveau
10, 1011 Lausanne, Switzerland.
788 Journal of the National Cancer Institute
-
stases, because of poor absorption by thegut (12) and gastric
irritation caused byoral administration. The optimal sched-ule and
infusion rate have not yet beendefined, however, and might be
clarifiedby pharmacokinetic information.
The pharmacokinetic properties ofbisphosphonates have been
determined inlaboratory animals {13,14) in which halfof the dose
was accumulated in the skel-eton and half excreted unchanged in
theurine. Accumulation of bisphosphonatesin the reticuloendothelial
system of ani-mals varies between species (15) andbetween
individual bisphosphonates(16), suggesting different
distributionkinetics—factors that mandate cautiousinterpretation
when extrapolating datafrom animals to humans.
Pamidronate pharmacokinetics werestudied in rats following oral
and intra-venous administration of l4C-labeledcompound (17). Bone
accumulation wasconstant at 25%-35% of dose. The up-take of labeled
pamidronate by thereticuloendothelial system was, however,dose
dependent, with less than 3% in theliver at 0.01 mg/kg and 30% in
the liverat 10 mg/kg. Because the liver is animportant storage
compartment with ashort half-life (ti/2) of retention,
furtherincrease of bone accumulation occurredwith time. Release of
pamidronate fromthe skeleton was extremely slow with anestimated tm
of at least 300 days.
In humans, pharmacokinetic data onbisphosphonates are limited.
In healthysubjects, means of 73% and 81% of theadministered dose of
clodronate wererecovered unchanged in the urine within24-48 hours,
with no difference betweenthe three doses tested (18,19). In
sixpatients with metastatic breast cancer,total urinary excretion
was similar at75% (20). However, in patients withPaget's disease,
mean urine excretionwas lower at 58%. Because clodronatewas
administered as a slow infusion overa 5-day period, it was
concluded thatslow infusion increases accumulation inthe body and
decreases urine excretion(21). It is not known by how much
thepharmacokinetics of bisphosphonatescould be affected by the rate
of admin-istration or by bone metabolism.
In initial clinical studies with pami-dronate, multidose
regimens have beenused for a period of 6-9 days, but,recently,
single-day infusions have beenshown to be equally effective and
arecurrently recommended (7). We report
the pharmacokinetic study of pami-dronate given to cancer
patients withbone metastases. Pamidronate wasadministered as a
single infusion, at con-stant dose, but at three different
infusionrates, to test the influence of infusionrate on
pharmacokinetic variables.
Subjects and Methods
Study Subjects
The study was conducted at the CentrePluridisciplinaire
d'Oncologie, Univer-sity Hospital, Lausanne, and at
theKantonsspital, St. Gallen, Switzerland.It was approved by the
ethical committeeof both institutions. Informed consentwas obtained
from every patient. Entrycriteria included an age limit greater
thanor equal to 18 years, a histological diag-nosis of cancer with
radiological evi-dence of bone metastases, serum cre-atinine level
less than 150 (xmol/L orbilirubin level less than 25 (Jimol/L,
andnormal levels of liver enzymes. Exceptfor one patient with a
serum calciumlevel at 2.91 mmol/L, the serum calciumlevel was not
above 2.75 mmol/L. Thepatients were not treated previously
withbisphosphonates. No new medicationsand no chemotherapy were
started for atleast 72 hours prior to entry or at anytime during
the study. Patients on hor-mone therapy were eligible, providedthey
did not have a change in treatmentschedule for at least 2 weeks. A
total of37 patients were enrolled in the study;their
characteristics are summarized inTable 1.
Treatments
The study was performed in twophases over a 15-month period.
Duringthe first phase, 23 patients were ran-domly assigned to
receive 60 mg pam-idronate, diluted in 500 mL of 0.9%saline, as an
intravenous infusion over aperiod of 4 or 24 hours. To ensure
ade-quate urine flow, a total of 2 L of intra-venous fluid was
given during the day ofpamidronate administration and the
fol-lowing day. Blood samples were notdrawn during this phase of
the study.When analysis disclosed that the amountof drug excreted
in urine was similar atboth infusion rates, we started the
secondphase of the study.
During the second phase, an additionalgroup of 14 patients was
given 60 mgpamidronate, diluted in 250 mL of 0.9%saline, over a
period of an hour withouthydration. Blood and urine samples
werecollected in this group of patients.
Among this group of patients, fourwere selected in whom the
1-hour infu-sion was repeated at 4- to 5-week inter-vals at a
maximum of four infusions.
Urine Collection and Blood Sampling
During the first part of the study, urinecollections were
performed at 4-hourintervals for 12 hours (three collections)and
then at 12-hour intervals for a further36 hours (three
collections). During thesecond part of the study, collection
ofblank samples and two collections ofurine were performed from 0
to 4 hoursand from 4 to 24 hours.
Table 1. Patient characteristics
No. of patientsMale/femaleMean age, y (range)Mean height, cm
(range)Mean weight, kg (range)Median creatinine clearance, mL/min
(range)No. of breast carcinomasNo. of prostatic carcinomasNo. of
bronchial carcinomasNo. of hypernephromasNo. of bone
metastases*
15
Pamidronate infusion60mg/4 h
122/10
67 (56-80)162 (135-175)
62 (34-85)65 (37-98)
1011
—
372
60 mg/24 h
110/11
63 (28-78)157(148-165)
60(52-71)66 (48-102)
11———
272
rates60 mg/ 1 h
143/11
62 (42-73)162(139-178)
65 (52-75)68 (35-110)
112
—1
194
•Evaluated on standard x rays.
Vol. 84, No. 10, May 20, 1992 REPORTS 789
-
Blood samples were obtained onlyduring the second part of the
study,before and then every 15 minutes for 2hours after the start
of the infusion andthen at 2.5, 3, 5, and 24 hours.
Assay of Pamidronate in Urine andPlasma
The assay of pamidronate in plasmaand urine was performed by
high-performance liquid chromatography withfluorescence detection,
after derivatiza-tion of pamidronate with fluorescamine(22,23). The
limits of quantitation were1.4 (jimol/L in plasma and 1.8 jjimol/L
inurine.
Clinical Assessment
In the first part of the study, serumcalcium, phosphate,
creatinine, andcreatinine clearance levels were assessedbefore
treatment.
Because of concerns about the pos-sibility of increased toxicity
of the morerapid infusion rate used during the sec-ond part of the
study, the followingparameters were measured in serumbefore
infusion and at 24 and 48 hoursafter infusion: urea, creatinine,
calcium,phosphate, albumin, bilirubin, alkalinephosphatase, and
transaminases. In addi-tion, the following parameters weremeasured
in urine: calcium, phosphate,creatinine, sodium, protein,
hydroxy-proline, and lysozyme. Routine urinal-yses were done for
the presence of bloodand protein.
In all patients, the number of bonemetastases was evaluated on
standardx rays and reported in three groups—those with fewer than
five metastases,those with between five and 15 meta-
stases, and those with more than 15 met-astases. In seven
patients, a moreaccurate determination of the number ofbone
metastases was obtained by bonescan. Clinical examinations for
toxicitywere performed daily during the infusionperiod and then 2
to 4 weeks later.
Pharmacokinetic Calculations
Noncompartmental techniques wereused in the pharmacokinetic
analysis(24). The total urinary excretion (TUE)at 0-24 hours was
the total amount ofpamidronate excreted in urine over 24hours. The
body retention (BR) at 0-24hours was estimated as follows: BR (%of
dose) = dose (100%) - TUE (% ofdose); fecal excretion was, in all
proba-bility, negligible as known from animalexperiments (17).
The area under the curve (AUC) fordrug concentration in plasma X
time at0-24 hours, expressed as (u,mol/L) xhours, was determined by
the linear tra-pezoidal rule from 0 to 24 hours. Cmax((i-mol/L) was
the maximal plasma con-centration, and Tmax (hour) was the timeat
which Cmax was reached. The apparentplasma distribution half-life
(tl/2 inhours) of pamidronate was calculatedfrom the slope of the
linear least-squaresregression line through the concentra-tion-time
points in the time interval from1 to about 2 hours. The total
plasmaclearance (CL, [L/hour]) was calculatedas the dose/AUC (0 -
), taking AUC(0-24 hours) as AUC (0 - oo) because all24-hour plasma
concentrations werebelow the limit of detection and taken aszero.
The renal clearance (CLr [L/hour])was calculated as the TUE (0-24
hour)/AUC (0-24 hour). The nonrenal clear-ance (CLnr [L/hour]) was
CL, - CLr.
No statistical comparison of the treat-ments was performed. This
study beingthe first pharmacokinetic trial with pami-dronate
disodium, no information on theintersubject variability was
available.The results of this trial showed a highinterpatient
variability in the urinaryexcretion, and, therefore, the number
ofpatients included in each group of thestudy was too low to
perform meaningfulstatistical tests with sufficient power.
Results
Pharmacokinetics in Urine
The TUE over 24 hours of eachpatient and the mean (±SD) values
areshown graphically in Fig. 1, according tothe three different
infusion rates. Themean TUE (0-24 hours) (±SD) was notsignificantly
different and was measuredat 31% ± 15.2%, 34.9% ± 13.9%, and41.0% ±
15.4% of the 60 mg of pami-dronate administered over periods of
1hour, 4 hours, and 24 hours, respec-tively. Considerable
between-patientvariability was noted.
To explain the wide disparity of TUE(0-24 hours) among patients,
TUE wascorrelated with creatinine clearance andwith tumor bone
involvement. A weakrelationship was found between cre-atinine
clearance and TUE (0-24 hours)by linear least-squares regression
anal-ysis (r = 0.42), but a stronger associa-tion was demonstrated
between thenumber of bone metastases and BR (0-24hours). The mean
(±SD) of the BR(0-24 hours) values in patients withfewer than five
bone metastases was50.6% ± 11.8% and increased withincreasing
number of bone metastases to76.4% ± 12.0% in patients with more
20 30
Time [h]
40
70
60
50
|60 mg in 4 h
20 30
Time [h]
20 30
Time [h]
Fig. 1. Total unnary excretion [TUE (0-24 hours)] of each
patient after an infusion of 60 mg of pamidronate disodium given
over 24-, 4-, and 1 -hour periods at aconstant infusion rate. Mean
values are presented as dash lines.
790 Journal of the National Cancer Institute
-
than 15 metastases (Fig. 2). These find-ings were confirmed in
the subgroup ofseven patients, where an accurate countof bone
involvement could be made bybone scintigraphy and where the
coeffi-cient of correlation was 0.82 (linearleast-squares
regression). No correlationwas found between serum and
urineparameters such as calcium, phosphate,alkaline phosphatase,
hydroxyproline,and TUE (0-24 hours).
Pharmacokinetics in Plasma
After the 1-hour infusion of 60 mg ofpamidronate disodium, the
mean (±SD)of the AUC for drug concentration inplasma x time at 0-24
hours was 22.0± 8 . 8 (itnol/L X hours; the mean Cmax(±SD) was 9.7
(xmol/L ± 3.2 in 14patients. The median value of Tmax was0.8 hour.
The mean (±SD) plasma con-centration times profile is shown in
Fig.3. The decline of pamidronate concentra-tions in plasma
followed a multiexponen-tial course. The distribution of
pami-dronate was rapid with a mean (±SD)apparent tV2 of 0.8 hour
(±0.3). Plasmaconcentrations of pamidronate were nearthe limit of
quantitation in most patientsat 3 hours and below the limit at 5
and24 hours. Therefore, no terminal tmcould be estimated for the
slow releaseof pamidronate from bone.
Two hours after the start of the infu-sion, a slight increase in
plasma con-centration occurred in the majority ofpatients, possibly
corresponding to arelease of pamidronate from an
unknowncompartment. The mean (±SD) CL, was10.7 L/hour (±3 .7 ) ,
and the mean
100-
8 0
6 0 -
4 0 -
2 0 -
o-
BR% of
N-
r11•
-
BR (0-24 hours) was 50% in patientswith few bone metastases and
75% inthose with numerous bone metastases.The relationship was
stronger in a subsetof patients in whom a more
accuratedetermination of the number of metasta-tic sites was
performed by countinglesions on bone scan.
The influence of renal function on uri-nary excretion of
pamidronate is un-known. In the population studied whichhad either
a normal or a slightly reducedcreatinine clearance, the renal
functionhad a weak effect on TUE (0-24 hours).However, in a study
in which patientswith osteodystrophy had a creatinineclearance of
only 5-35 mL/minute, TUE(0-24 hours) of Tc-99m-etidronate was11.4%
of the administered dose (25).The disparity in skeletal invasion
and inrenal function could explain the almostthreefold variability
in BR (0-24 hours)among patients in our study.
The plasma pharmacokinetics of pami-dronate at 60 mg/hour
followed a multi-exponential course and was characterizedby a short
distribution phase in plasma,followed by a rapid elimination of
un-changed drug in urine. The distributionof pamidronate was rapid
with a tm of0.8 hour. Clodronate was also removedpromptly from the
blood, with a tmbetween 1.8 hours and 2.3 hours inhealthy subjects
(16) and in patients withbone disease (18,20). The total
clearanceof pamidronate was 10.7 L/hour with aCLnr clearance of 7.5
L/hour. For clodro-nate, values varied between studies, witha total
clearance of 6.4 and 11.5 L/hour,possibly due to different methods
ofmeasurement, a factor which preventsany direct comparison with
our data.Plasma concentration increased slightly 2hours after the
start of the infusion. Pam-idronate may have been released fromthe
reticuloendothelial system, where itcould have accumulated
temporarily. Asimilar observation has been made inanimals but not
previously in humans. Inmice, pamidronate accumulated in spleenand
liver in contrast to clodronate andetidronate.
When pamidronate was infused at adose of 60 mg over a 1-hour
period at 4-to 5-week intervals, its pharmacokineticvalues remained
unchanged. With thisschedule and dose, the bone compart-ment was
not saturated. Similar conclu-sions were reached after five
dailyinfusions of clodronate, where no signifi-
cant variations were detectable betweendaily infusions (20).
Clinically, the tolerance of patients forall three infusion
rates was similar, withonly minimal toxicity. In particular,
norenal toxicity was observed after the1-hour infusion rate.
Previous studieshave shown a transient increase inplasma creatinine
(26) and rare cases ofrenal failure (27) after etidronate
andclodronate administration. The plasmacreatinine did not rise
within 48 hoursafter the infusion of pamidronate. Pro-teinuria
remained within normal range.The minimal elevation of
aspartateaminotransferase seen in one patient hadno clinical
significance and could berelated to a possible uptake of
pami-dronate into the reticuloendothelial sys-tem. A larger number
of patients,however, should be studied for toxicityat the fastest
rate of infusion beforesafety can be ensured.
On the basis of the pharmacokineticdata combined with the
clinical findings,we conclude that BR (0-24 hours) ofpamidronate
was dependent on theamount of bone metastases and was notinfluenced
by the rate of infusion. Thus,the 1-hour infusion rate can be
proposedas a kinetically relevant treatment sched-ule for patients
with malignant bonediseases.
References
(/) FLEISCH H: Bisphosphonates—history andexperimental basis.
Bone 8:23-28, 1987
(2) SHINODA H, ADAMEK G, FELIX R, ET AL:Structure-activity
relationships of variousbisphosphonates. Calcif Tissue Int
35:87-99,1983
(3) BOONEKAMP PM, VAN DER WEE P A L S L, VANWIJK-VAN LENNEP MML,
ET AL: Two modesof action of bisphosphonates on
osteolyticresorption of mineralized bone matrix. BoneMin 1:27-39,
1986
(4) FAST DK, FELIX R, DOWSE C, ET AL: Theeffects of
diphosphonates on the growth andglycolysis of connective-tissue
cells in cul-ture. Biochem J 172:97-107, 1978
(5) THIEBAUD D, JAEGER P, GOBELET C, ET AL: Asingle infusion of
bisphosphonate AHPrBP(APD) as treatment of Paget's disease ofbone.
Am J Med 85:207-212, 1988
(