Pharmacokinetics of cyclophosphamide and its metabolites in bone marrow transplantation patients*

Pharmacokinetics of cyclophosphamide and its metabolites in bone marrow transplantation patients

Objectives: To characterize the pharmacokinetics of cyclophosphamide and 5 of its metabolites in bone marrow transplant patients and to identify the mechanism of the increase in 4-hydroxycyclophosphamide area under the plasma concentration-time curve (AUC) from day 1 to day 2 of cyclophosphamide administration. Metboa%: Cyclophosphamide was administered by intravenous infusion (60 mg/kg over 1 hour, once a day) for 2 consecutive days to 18 patients. Cyclophosphamide and 4-hydroxycyclophosphamide concentration-time data on day 1 and day 2 were fitted to a model to estimate 4-hydroxycyclophosphde formation (CLf) and elimination (CL,) clearances. Erythrocyte aldehyde dehydrogenase-1 activity was measured ex vivo just before the frost cyclophosphamide infusion was started (0 hours) and 24 hours after the second cyclophosphamide in&ion (48 hours). Results: From day 1 to day 2, the AUC of cyclophosphamide, deschloroethyl cyclophosphamide and phosphoramide mustard decreased 24.8%, 51%, and 29.4% (P < .02), the AUC of 4-hydroxycyclophosphamide and carboxyethylphosphoramide mustard increased 54.7% and 25% (P < .Ol), whereas the AUC of phosphoramide mustard was not significantly changed (P > .3). The CLf of 4-hydroxycyclophosphamide increased 60% (P < .OOl), its CL,,, decreased 27.7% (P c .OOl), and the fraction of cyclophosphamide dose converted to 4-hydroxycyclophosphamide increased 16% (P c .OOl) from day 1 to day 2. The activity of patient erythrocyte aldehyde dehydrogenase-1 decreased 23.3% (P < .02) from 0 hours to 48 hours. Conclusions: The AUC of 4-hydroxycyclophosphamide increased from day 1 to day 2 as a result of increased formation and decreased elimination clearances of 4-hydroxycyclophosphamide. Aldehyde dehydrogenase- 1 activity appears to decline as a consequence of cyclophosphamide administration. (Clin Pharmacol Ther 1998;64:289-301.)

Song Ren, BS, Thomas F. Kalhorn, BS, George B. McDonald, MD, Claudio Anasetti, MD, Frederick R Appelbaum, MD, and John T. Slattery, PhD Seattle, Wash.

Cyclophosphamide is one of the most frequently used alkylating agents in the treatment of malignancy and in preparative regimens for bone marrow transplantation. l It is a pro-drug that must be oxidized to 4-hydroxycyclophosphamide, a reaction primarily catalyzed by CYP2C9 and CYP3A4 in human liver.2-4 4-Hydroxy-

From the Department of Pharmaceutics, University of Washington, and the Fred Hutchinson Cancer Research Center.

Supported in part by grants CA 18029 and GM 32165 from the National Institutes of Health (Bethesda, Md.).

Received for publication March 23, 1998; accepted June 24, 1998. Reprint requests: John T. Slattery, PhD, Fred Hutchinson Cancer

Research Center, Thomas Building, D2-100, 1100 Fairview Ave. N, PO Box 19024, Seattle, WA 98109-1024. E-mail:

[email protected] Copyright 0 1998 by Mosby, Inc. 0009-9236/98/$5.00 + 0 13/I/92753

cyclophosphamide is a circulating metabolite that enters cells and decomposes (through its tautomer aldophosphamide) to phosphoramide mustard and acrolein.’ Phosphoramide mustard is a bifunctional alkylator of DNA, the ultimate cytotoxic metabolite of cyclophosphamide.5-7 Alternatively, 4-hydroxycyclophosphamide is detoxified to 4-ketocyclophosphamide by cytochrome P450 and carboxyethylphosphoramide mustard by aldehyde dehydrogenase- 1.1 Cyclophosphamide is also oxidized to deschloroethyl cyclophosphamide, although this reaction accounts for little of cyclophosphamide disposition in humans. L4 The remaining route of elimination of cyclophosphamide is renal, which accounts for approximately 30% of its total clearance.’ A partial metabolic scheme is shown in Figure 1.

In bone marrow transplantation, cyclophosphamide is usually administered once a day by intravenous infu-

289

290 Ren et al. CLINICAL PHARMACOLOGY & THERAPEUTICS

SElTEhtBER 1998

Jmimcyclophosphamide

+GSH

0

-GSH

@fM

0s

4-GScy

M= - N(CH+H#)2

,-Cl

/ A +

P450 Deschl&ylCY

(DCW cl H

o-M

cz, ./’

Chloroecetaldehyde (CA)

Cyclophosphamide c-v

P450 [O]

4-hydrox$Y VW

4-K&&y (TcarboxyethylPM + FEPM)

Acrolein

I-

GSH F’hosphommide mustard (PM)

Figure 1. Partial metabolic scheme of cyclophosphamide.

sion for 2 to 4 consecutive days primarily to facilitate cyclophosphamide converted to 4-hydroxycyclophos- engraftment of donor cells, although an antileukemic phamide (eg, induction of cyclophosphamide metabo- effect may also be obtained. There are several reports lism) or a decrease in 4-hydroxycyclophosphamide of a significant decrease in cyclophosphamide elimina- elimination clearance or both. However, the fraction of tion half-life after repeated doses.*-12 One study showed cyclophosphamide converted to 4-hydroxycyclophos- that the area under the plasma concentration-time curve phamide versus other primary metabolites was deter- (AUC) of 4-hydroxycyclophosphamide (measured as mined to be about 0.95 at therapeutic concentrations of acrolein) increased by 73% from day 1 to day 2.12 It cyclophosphamide in human liver microsomal incuba- has been suggested that such changes are caused by the tions in vitro.4 Because the renal clearance of induction of cyclophosphamide metabolism.11,12 The cyclophosphamide is approximately 30% of its total AUC of 4-hydroxycyclophosphamide is determined by clearance, it is apparent that the fraction of a dose of the fraction of the dose of cyclophosphamide converted cyclophosphamide eliminated through the formation of to 4-hydroxycyclophosphamide and the elimination 4-hydroxycyclophosphamide is about 60% to 70%.’ clearance of 4-hydroxycyclophosphamide. The increase Because the maximum possible value of the fraction of in the AUC of 4-hydroxycyclophosphamide could be the dose of cyclophosphamide converted to 4-hydroxy- the result of an increase in the fraction of the dose of cyclophosphamide is 1, the increase in the AUC of 4-

CLINICAL PHARMACOLOGY & THERAPEUTICS VOLUME 64, NUMBER 3 Ren et al. 291

hydroxycyclophosphamide cannot exceed 30% to 40% if 4-hydroxycyclophosphamide elimination clearance is unchanged. An increase of 73% was observed,12 therefore it appears that the elimination clearance of 4- hydroxycyclophosphamide decreases from day 1 to day 2 of cyclophosphamide administration.

The high-dose cyclophosphamide used in transplant preparative regimens is associated with substantial and not entirely predictable toxicity.1 In a previous study,‘3 we found considerable variability in the metabolism of cyclophosphamide. If efforts to possibly modify the dose and scheduling of cyclophosphamide based on the individual patient pharmacokinetics are to be success- ful, a detailed understanding of the pharmacokinetics of the drug and its metabolites in the setting of bone marrow transplantation is needed. The objectives of this investigation were to characterize the pharmacokinet- its of cyclophosphamide and 5 of its metabolites in bone marrow transplantation patients on day 1 and day 2 of cyclophosphamide infusion to identify the mechanism of the increase in the AUC of 4-hydroxycyclophosphamide from day 1 to day 2.

PATIENTS AND METHODS Material. Cyclophosphamide, indole-3-acetaldehyde,

indole-3-acetic acid, and nicotinamide adenine dinu- cleotide (NAD) were purchased from Sigma Chemical Company (St. Louis, MO.). p-Nitrophenylhydrazine was purchased from Fluka Chemical (Ronkonkoma, N.Y.). Trifluoroacetic anhydride was purchased from Aldrich Chemical (Milwaukee, Wis.). Deschloroethyl cyclophosphamide, carboxyethylphosphoramide mustard, and phosphoramide mustard were gifts from ASTA Medica AG (Frankfurt, Germany). 4-Hydroperoxycyclophos- phamide, 4-ketocyclophosphamide, and tetradeuteurocarboxyethylphosphoramide mustard were prepared in our laboratory according to published methods.‘4 Dia- zomethane and 2-bis(N-propyl)amido-1,3,2-oxazaphos- phoridin-P-oxide’s were also prepared in our laboratory according to standard methods.

Patients. Eighteen patients scheduled to receive unrelated donor bone marrow transplants were studied under a protocol approved by the Institutional Review Board of the Fred Hutchinson Cancer Research Center (Seattle, Wash.). Patients received l-hour intravenous infusions of 60 mg/kg cyclophosphamide daily for 2 days, followed by 12 to 13.5 Gy total body irradiation. Diphenhydramine, droperidol, ondansetron, and lorazepam were given for nausea and vomiting as needed during cyclophosphamide infusion. Flucona- zole was administered prophylactically to all patients. No additional drugs were routinely administered.

Sampling procedure. Blood samples were obtained from a central venous access Hickman catheter just before the cyclophosphamide infusion was begun, at % and 1 hour after the beginning of the infusion, and at 1, 3,6,8, and 24 hours after the end of the infusion on both day 1 and day 2 of cyclophosphamide treatment. For the analysis of 4-hydroxycyclophosphamide, 1 mL blood was immediately placed in 1 mL derivatizing solution (p-nitrophenylhydrazine-citrate buffer [pH 3.5]),13 sealed, inverted 3 to 6 times and centrifuged at 10,OOOg for 1 minute at the patient’s bedside. The supernatant was transferred to a clean tube, sealed, stored in a -20°C cooler for transportation to the laboratory and frozen at -70°C until analysis. For the analysis of cyclophosphamide and its other metabolites, 1 mL blood was centrifuged at 10,OOOg for 1 minute at the bedside. Plasma was transferred to a clean tube, sealed, stored in a -20°C cooler for transportation to the laboratory, and frozen at -70°C until analysis.

Urine was collected for the measurement of cyclophosphamide renal clearance and deschloroetbyl cyclophosphamide formation clearance on both days of cyclophosphamide treatment between 1 and 3 hours after the end of the infusion, or from just before the infusion was begun to 24 hours after the end of the infusion, depending on clinical constraints. The total volume of urine was recorded and an aliquot was stored at -70°C until analysis.

Erythrocytes were collected for the determination of aldehyde dehydrogenase- 1 activity just before the day 1 infusion was begun (0 hours) and 24 hours after the end of the day 2 infusion (48 hours). Ten milliliters of venous blood was obtained and centrifuged. The plasma and buffy coat were removed and the erythrocytes were transferred to a clean tube and stored at -70°C until analysis.

Analysis of plasma and urine. For the analysis of cyclophosphamide, deschloroetbyl cyclophosphamide, and 4-ketocyclophosphamide, 500 pL plasma was extracted, derivatized, and assayed by a gas chromato- graph equipped with a nitrogen-phosphorus detector.* For the analysis of carboxyethylphosphoramide mustard and phosphoramide mustard, 500 pL of plasma was extracted and assayed by LC/MS.* For the analysis of 4-hydroxycyclophosphamide, 500 pL of derivatized plasma was assayed as described previously.13

For the analysis of cyclophosphamide and deschloroethyl cyclophosphamide in urine, 100 pL urine was extracted, derivatized, and assayed by a gas chromato- graph equipped with a nitrogen-phosphorus detector.*

*Kalhom TF, Ren S, Howald WN, Lawrence RF, Slattery JT. Analy- sis of cyclophosphamide and 5 metabolites from human plasma using LC-MS and GC-NPD. Submitted for publication.

292 Ren et al. CLINICAL PH.4Bh4ACOLoGY & THElWl’EUTIC.9

SEI’IEMBER 1598

Form&on of carboxyethylphosphoramide mustard from 4-hydroxycyclophosphamide and indole3-acetic acid from indole-3-acetaldehyde in human liver cytosolic incubations. Human livers were obtained from the human liver bank in the Departments of Phar- maceutics and Medicinal Chemistry at the University of Washington (Seattle, Wash.). Liver was homogenized in 100 mmol/L potassium phosphate buffer (pH 7.4), centrifuged at lO,OOOg, 4”C, for 30 minutes. The supernatant was filtered through 6-ply surgical gauze and centrifuged at lOO,OOOg, 4°C for 60 minutes. The resulting supernatant cytosolic fraction was stored at -70°C until use. Protein concentration was determined with Bio-Rad (Oakland, Calif.) protein assay reagent, with bovine serum albumin as the standard.l6

To evaluate the formation of carboxyethylphosphoramide mustard from 4-hydroxycyclophosphamide, 4- hydroperoxycyclophosphamide (5 mmol/L) was reduced to 4-hydroxycyclophosphamide by adding sodium thiosulfate (25 mmol/L) and allowing the mixture to stand on ice for 1 hour immediately before incubation. Carboxyethylphosphoramide mustard formation was evaluated in an incubation system containing 2.5 to 120 pmol/L 4-hydroxycyclophosphamide, 1 mg/mL cytosolic protein, 0.5 rnmol/L NAD, and 100 mmol/L potassium phosphate buffer (pH 7.4) in a total volume of 0.5 mL. The reaction mixture was preincubated at 37°C for 2 minutes before the reaction was started by the addition of NAD. The incubation was stopped after 5 minutes by the addition of 6 mL methylene chloride, followed by the addition of 15 pL internal standard, tetradeuteurocarboxyethylphosphoramide mustard (final concentration: 15 pmol/L), and 0.43 mL of 500 rnmol/L ammonium acetate buffer (pH 3.0). The mixture was sealed, shaken for 5 minutes and centrifuged for 10 minutes at 4°C. The supernatant (0.5 mL) was transferred to a clean tube, to which 0.5 mL acetonitrile was added. The sample was sealed, vortexed, and centrifuged for 5 minutes. Ten microliters of the supernatant was injected onto the LC/MS system used for the plasma carboxyethylphosphoramide mustard assay.

To evaluate the formation of indole-3-acetic acid from indole-3-acetaldehyde, indole-3-acetaldehyde (0.1 to 50 pmoVL), human liver cytosol(O.02 mg cytosolic protein/ml), and potassium phosphate buffer (100 mmol/L, pH 7.4) were preincubated at 37°C for 2 minutes before the reaction was started by the addition of NAD (0.5 mmol5). The incubation was stopped after 5 minutes by the addition of 50 yL of 1N sodium hydroxide and 100 pL of 10% zinc sulfate. The sample was sealed, vortexed, left on ice for 5 minutes, and centrifuged for 5 minutes. Five microliters of the super-

natant was injected into a Hewlett-Packard 1050 series HPLC equipped with a Rainin Microsorb Cl8 column and a fluorescence detector (excitation, 278 nm; emis- sion, 359 nm) to measure indole-3-acetic acid. The mobile phase was 27% acetonitrile and 73% of 25 mmol/L ammonium acetate buffer (pH 4.0), delivered at a rate of 0.9 mL/min. The retention time of indole- 3-acetic acid was 3.7 minutes and the run time was 5 minutes. The concentration of indole-3-acetic acid was quantified by the peak height.

The inhibition mechanism of aldehyde dehydrogenase-1 by indole-3-acetaldehyde was determined in an incubation system that contained indole-3-acetaldehyde (0, 0.4, 1.6, and 3 pmol/L), 4-hydroxycyclophosphamide (3.3, 6.6, and 30 pmol/L), cytosolic protein (1 mg/mL), and NAD (0.5 mmol/L). The reaction mixture was preincubated at 37°C for 2 minutes before the reaction was started by the addition of NAD. The incubation was stopped after 5 minutes by the addition of 6 mL methylene chloride and carboxyethylphosphoramide mustard was measured as described above.

Determination of erythrocyte aldehyde dehydrogenase-l activity. Erythrocyte aldehyde dehydrogenase- 1 was used as a surrogate marker for liver aldehyde dehydrogenase- 1 activity to assess the effect of cyclophosphamide administration on patient aldehyde dehydrogenase- 1 activity. l7 Hemolysate was prepared by diluting 0.2 mL of patient erythrocytes in 0.8 mL hemolyzing solution (2.7 mmol/L sodium ethylenediaminete- traacetic acid and 0.005% (voUvo1) 2-mercaptoethanol in water), freezing at -70°C for 30 minutes, and thaw- ing at room temperature. The formation of indole-3- acetic acid from indole-3-acetaldehyde was used to access erythrocyte aldehyde dehydrogenase-1 activity. Indole-3-acetaldehyde (40 pmol/L), hemolysate (2 pL red blood cells/n&), and potassium phosphate buffer (100 mmol/L, pH 7.4) were preincubated at 37°C for 2 minutes before the reaction was started by the addition of NAD (0.5 mmol/L). The incubation was stopped after 5 minutes by the addition of 50 pL of 1N sodium hydroxide and 100 lrL of 10% zinc sulfate. Indole-3- acetic acid was analyzed by the method described above.

Data analysis. The AUC values (from time zero to infinity) of cyclophosphamide and its metabolites were determined by noncompartmental analysis with use of the trapezoidal rule. Because the elimination rates of cyclophosphamide and its metabolites were faster on day 2 than on day 1, the mean of the elimination rate constants of day 1 and day 2 was used to extrapolate the AUC of cyclophosphamide and its metabolites from the last sampling time point on day 1. If there was residual cyclophosphamide or its metabolites present in the

CLINICAL P -COLOGY & THERAPEUTICS VOLUME 64, NUMBER 3 Ren et al. 293

sample just before the infusion of cyclophosphamide on day 2, then this residual AUC (calculated as the ratio of the concentration at the start of day 2 infusion to the mean of the elimination rate constants of day 1 and day 2) was subtracted from the AUC observed on day 2. The total clearance of cyclophosphamide was calculated as the ratio of the dose of cyclophosphamide to its AUC. The renal clearance of cyclophosphamide and formation clearance of deschloroethyl cyclophosphamide were calculated as the ratio of the amount of cyclophosphamide or deschloroethyl cyclophosphamide, respectively, excreted into the urine divided by the AUC of cyclophosphamide in plasma over that same period of time. The fraction of cyclophosphamide dose excreted unchanged in the urine was calculated as the ratio of cyclophosphamide renal clearance to total clearance. The elimination half-life of cyclophosphamide was calculated as the reciprocal of the slope of the last 3 log concentration-time points on day 1 and day 2. The apparent volume of distribution of cyclophosphamide was calculated as the product of its total clearance and elimination half-life divided by ln2.

To access the change of the formation clearance of 4-hydroxycyclophosphamide from cyclophosphamide and the elimination clearance of 4-hydroxycyclophosphamide from day 1 to day 2 of cyclophosphamide administration, the concentration-time profiles of cyclophosphamide and 4-hydroxycyclophosphamide on day 1 and day 2 were fitted separately by the fol- lowing model:

dCCY - = wo - Wf,“CY + CL,1 . C,y)Ng,cy dt

dCHcy - (CL,,,, -- dt ’ ’ ‘CY - cLm,HCY. CHCY)NP,HCY

in which Ko is the rate of infusion (Kc = 0 when t > infusion time), CLf,nCY is formation clearance of 4- hydroxycyclophosphamide from cyclophosphamide, CL, is the clearance of cyclophosphamide by pathways other than the formation of 4-hydroxycyclophosphamide, CLm,nCY is the elimination clearance of 4- hydroxycyclophosphamide, VP,~~ and Vp,ncu are the apparent volumes of distribution of cyclophosphamide and 4-hydroxycyclophosphamide, and Ccu and C,,, are the plasma concentrations of cyclophosphamide and 4-hydroxycyclophosphamide at time t. The estimated values of cyclophosphamide CL, (the mean value of the sum of cyclophosphamide renal clearance and deschloroethyl cyclophosphamide formation clearance) and the VP of cyclophosphamide from noncompartmen-

3 ‘n HCY

d

0.11 0 10 20 30 40 50

Time (hr)

Figure 2. Representative plasma concentration-time profiles of cyclophosphamide (CY), 4-hydroxycyclophos- phamdie (HCY), and deschloroethyl cyclophosphamide (DCCY; upper panel), and 4-ketocyclophosphamide (KetoCY), carboxyethylphosphoramide mustard (CEPM) and phosphoramide mustard (PM; lower panel) in 1 patient, unique patient number (UPN) 11196.The first cyclophosphamide infusion was started at 0 hours and the second at 23.3 hours.

tal analysis were used in the model to reduce the number of parameters for which values were estimated by curve fitting. The values of 4-hydroxycyclophosphamide formation clearance, elimination clearance, and apparent volume of distribution were estimated from concentration-time data of cyclophosphamide and 4-hydroxycyclophosphamide with use of a weighted least-squares procedure with the WinNonlin program (Scientific Consulting, Inc., Gary, N.C.). A weight of l/C was used in the iterative fitting process. The fraction of cyclophosphamide dose converted to 4-hydroxycyclophosphamide was calculated as the ratio 4-


SEPTEMBER 1998

Table I. The AUC of cyclophosphamide and its metabolites on day 1 and day 2 of cyclophosphamide infusion Cyclophos- 4-Hydroxy- Deschloroethyl Phosphoramide Carboxyethyl 4-Ketocyclophos- phamide cyclophosphamide cyclophosphamide mustard phosphoramide phumide

(PoVL. h) (PoUL * W (PoVL. 4 (woVZ. h) (woVL. h) (mom ’ h)

UPN Day I* Day 2* Day If Day2f Day18 Day2$ Day I$ Day2$ Day111 Day211 Day II Day2f

10484 3559 4930 61.9 179 414 156 269 231 109 120 130 71.3 10858 4050 3743 74.3 79.9 584 455 231 192 99.9 92.7 88.4 72.1 11196 4329 2556 68.1 132 430 161 329 308 173 178 194 111 11664 3851 3002 94.6 174 1345 159 756 1010 196 322 456 91.9 11672 3600 3113 ND ND 235 165 261 375 51.9 88.4 66.8 66.2 11779 2881 1899 56.8 65.2 329 269 394 262 240 262 83.7 61.3 11787 3970 3217 83.4 134 273 168 NDNDNDNDNTIND 11795 4240 2964 68.7 89.2 709 266 876 831 218 193 125 89.4 11812 4060 2624 61.7 86.5 183 68.2 353 431 140 198 174 58.6 11894 3444 2493 88.8 93.1 221 132 419 468 282 333 156 105 11927 3822 3074 117 134 654 467 314 335 187 201 199 174 11949 3747 2319 81.4 99.5 442 134 479 485 59.2 64.2 209 80.1 11995 4960 3417 ND ND 536 92.8 ND ND ND ND ND ND 12041 4327 2347 66.1 75.5 179 111 348 398 159 209 120 74.9 12100 4878 2848 79.3 83.2 449 82.8 ND ND ND ND 227 101 12245 3928 2295 61.5 66.7 423 69.4 489 476 226 231 197 100 12363 4446 3210 51.5 78.9 416 256 534 430 186 227 123 151 9407 2164 1976 29.2 97.0 307 308 230 399 125 261 96.5 159

Mean 3903 2890 71.5 104 452 196 419 442 164 199 165 98.0

E(B) 660 16.9 712 24.6 27.8 19.9 35.7 34.3 271 60.0 120 61.3 187 44.7 216 48.9 66.3 40.5 40.6 80.8 92.0 55.6 35.5 36.2

UPN, Unique patient number; ND, not determined. CV, coefficient of variation. *P = .00005; VP = ,001; SF’ = ,001; gP = ,389; /P = .OlO; nP = ,013. Comparisons of means were made with the paired two-tailed f test, with P < ,027 considered to

be statistically significant.

hydroxycyclophosphamide formation clearance to cyclophosphamide total clearance.

The maximal velocity (V,,) and Michaelis constant (K,) values of carboxyethylphosphoramide mustard formation from 4-hydroxycyclophosphamide and indole-3-acetic acid formation from indole-3-acetaldehyde in incubations of human liver cytosol, and the dissociation constant (Ki) of indole-3-acetaldehyde for aldehyde dehydrogenase-1 were estimated from the untransformed product formation rate and substrate concentration data using the WinNonlin program.

Data are reported as mean values f SD. Comparisons of mean values were made with the paired 2-tailed t test, with P < .05 considered to be statistically significant.

RESULTS Concentration-time profiles for cyclophosphamide, 4-

hydroxycyclophosphamide, deschloroethyl cyclophosphamide, 4-ketocyclophosphamide, carboxyethylphosphoramide mustard, and phosphoramide mustard in a representative patient are shown in Figure 2. The paral- lel concentration-time profiles of cyclophosphamide, 4- hydroxycyclophosphamide, carboxyethylphosphoramide mustard, and phosphoramide mustard indicate that the

kinetics of these metabolites are formation rate-limited. Deschloroethyl cyclophosphamide and 4-ketocyclophosphamide show elimination rate-limited kinetics.

The AUC values for cyclophosphamide and its metabolites are listed in Table I. The AUC of cyclophosphamide, deschloroethyl cyclophosphamide, and 4- ketocyclophosphamide decreased 24.8% (P < .OOOl), 51% (P < .OOl), and 29.4% (P c .02) from day 1 to day 2, respectively. The AUC of 4-hydroxycyclophosphamide and carboxyethylphosphoramide mustard increased 54.7% (P < .002) and 25% (P < .Ol) from day 1 to day 2, respectively.* The AUC of phosphoramide mustard on day 1 was not significantly different from day 2 (P > .3). The relationship between the AUC of cyclophosphamide and 4-hydroxycyclophosphamide is shown in Figure 3. There was no significant correlation between the AUC of cyclophosphamide and 4-hydroxycyclophosphamide on either day of cyclophosphamide infusion.

Cyclophosphamide pharmacokinetic parameters are listed in Table II. The total clearance of cyclophos-

*These differences are calculated from individual patient data, not the mean data shown in Table I.


200

150 I

Day 1

100

50 1 5: l

4 Oo 1000 2000 3000 4000 5000 6000 I

Day 2

100

50

l

l *

l l * l t l ++f* l

l

0 I I 0 1000 2000 3000 4000 5000 6000

AUCcr (pM.hr) Time (hr)

HCY I

30

Figure 3. Relationship between the area under the plasma Figure 4. Observed and predicted plasma concentration- concentration-time curve (AUC) of cyclophosphamide time profiles of cyclophosphamide (CY) and 4-hydroxy- (AU(&) and 4-hydorxycyclophosphamide (AUCHCY) in 16 cyclophosphamide (HCY) by the model (described in the patients on day 1 (r2 = 0.130; P = .170; upper panel) and day Methods section) in a representative patient (UPN 11196) on 2 (r2 = 0.343; P = .071; lower panel) of cyclophosphamide day 1 (upper panel) and day 2 (lower panel) of cyclophos- infusion. phamide infusion.

phamide increased from 0.0570 f 0.0128 L/h/kg (3.96 f 1.13 L/h) on day 1 to 0.0783 f 0.0176 L/h/kg (5.44 f 1.70 L/h) on day 2 (P < .OOOl). These values are similar to literature values: 0.0851 L/h/kg,‘* 5.76 L/h,‘9 and 4.72 L/h.*O The elimination half-life of cyclophosphamide decreased from 6.77 f 1.27 hours on day 1 to 4.51 rt 0.99 hours on day 2 (P < .OOOl), similar to 5.2 hours13 and 4.8 hours21 found previously. The apparent volume of distribution of cyclophosphamide was 0.5 11 f 0.09 L/kg (35.2 f 8.91 L) and 0.496 zt 0.096 L/kg (34.5 f 10.2 L) on day 1 and 2, respectively (P > .5), which were consistent with 45 Ltg and 0.49 L/kg*0 reported previously. Urine was collected in 5 patients to confirm the reported renal clearance of cyclophosphamide.t9,*0 Cyclophosphamide renal clearance was 0.913 f 0.291 L/h and 1.04 f 0.427 L/h on day 1 and 2, respectively (P > .6) compared with previous reports of 1.74 L/h19 and 1.37 L/h.*0 The fraction of cyclophosphamide dose excreted unchanged in urine and the formation clearance of deschloroethyl cyclophosphamide were also calculated in these patients. The fraction of cyclophosphamide

Day 1 1000 -

loo-\ CY

IO -

Day 2

CY

dose excreted unchanged in urine was 0.257 f 0.100 and 0.194 f 0.062 on days 1 and 2, respectively (P > .3). The formation clearance of deschloroethyl cyclophosphamide was 0.172 f 0.109 L/h on day 1 and 0.255 f 0.031 L/h on day 2 (P > .2), very close to 0.252 L/h reported previously.*0 Thus the fraction of the cyclophosphamide dose eliminated as deschloroethyl cyclophosphamide (estimated from its recovery in urine) was 4.3% on day 1 and 4.7% on day 2.

The pharmacokinetic mechanism of the increase in the AUC of 4-hydroxycyclophosphamide from day 1 to day 2 was sought by fitting cyclophosphamide and 4-hydroxycyclophosphamide concentration-time data on day 1 and day 2 separately to the model described in the methods section to estimate the values of 4-hydroxycyclophosphamide formation and elimination clearances on the 2 days of cyclophosphamide administration. Observed and predicted plasma concentration-time profiles of cyclophosphamide and 4-hydroxycyclophosphamide for a representative patient are shown in Figure 4. Table III shows the results of this analysis for all patients studied.


SElTEMBER 1998

Table II. Pharmacokinetic parameters of cyclophosphamide on day 1 and day 2 of cyclophosphamide infusion

CL ~uwks) CL ww vp ww VP W) ti: Ch) UPN AIBW (kg) Day I* Day 2* Day If Day 2t Day I$ Day 2# Day l§ Day 3 Day 1” Day 2*

10484 51 0.0604 0.0436 3.08 2.22 0.561 0.265 28.6 13.5 6.44 4.21 10858 68 0.0531 0.0574 3.61 3.90 0.714 0.593 48.5 40.3 9.32 7.15 11196 86 0.0497 0.0841 4.27 7.23 0.450 0.483 38.7 41.5 6.28 3.98 11664 67 0.0558 0.0716 3.72 4.78 0.447 0.642 29.8 42.8 5.55 6.22 11672 68 0.0597 0.0691 4.06 4.70 0.511 0.424 34.7 28.8 5.93 4.25 11779 64 0.0746 0.1132 4.77 7.24 0.688 0.579 44.1 37.0 6.39 3.54 11787 47 0.0542 0.0668 2.54 3.13 0.466 0.422 21.9 19.8 5.97 4.38 11795 66 0.0507 0.0725 3.37 4.81 0.482 0.537 32.0 35.6 6.59 5.13 11812 90 0.0530 0.0819 4.77 7.37 0.542 0.573 48.8 51.5 7.10 4.84 11894 81 0.0624 0.0862 5.05 6.98 0.588 0.564 47.6 45.7 6.53 4.53 11927 62 0.0562 0.0699 3.47 4.31 0.390 0.399 24.1 24.6 4.80 3.96 11949 72 0.0574 0.0927 4.13 6.67 0.532 0.544 38.3 39.1 6.42 4.06 11995 78 0.0433 0.0629 3.38 4.91 0.432 0.454 33.7 35.4 6.90 5.00 12041 57 0.0497 0.0916 2.85 5.26 0.409 0.466 23.5 26.7 5.70 3.53 12100 84 0.0441 0.0755 3.70 6.34 0.487 0.49 1 40.9 27.7 7.66 4.51 12245 73 0.0547 0.0937 3.99 6.84 0.593 0.634 43.3 46.3 7.51 4.69 12363 60 0.0484 0.0670 2.89 4.00 0.465 0.429 27.8 25.6 6.67 4.44 9407 74 0.0994 0.1088 7.37 8.07 0.440 0.428 32.6 31.8 10.0 2.73

Mean 68.5 0.0570 0.0783 3.96 5.44 0.511 0.496 35.2 34.5 6.77 4.51 SD 11.5 0.0128 0.0176 1.13 1.70 0.090 0.096 8.91 10.2 1.27 0.99 cv (%) 16.8 22.4 22.5 28.6 31.2 17.7 19.3 25.3 29.6 18.7 22.0

ABW, Adjusted ideal body weight. CL, Total clearance, VP apparent volume of distribution; tqelimination half-life of cyclophosphamide. *P = .OOOOl; tP = .00003; $P = ,533; $P = .654. Comparisons of means were made with the paired two-tailed t test, with P < .05 considered to be statistically

significant.

Table III. The estimated values of CL, CL,, VP and f, of 4-hydroxycyclophosphamide from the model fitting of concentration-time profiles of cyclophosphamide and 4-hydroxycyclophosphamide on day 1 and day 2 of cyclophosphamide infusion

CLf ww ct, (W VP 69 fm UPN DayI* Dq2* %of+l Dqlf Day3 %ofdayl DayI$ Day2$ %ofdayl DayI* Day2* %of&yl

9407 4.99 6.79 136 230 177 77.1 101 112 111 0.677 0.841 124 10484 2.20 2.05 93.2 171 101 59.1 86.8 101 117 0.714 0.922 129 10858 2.75 3.05 111 143 126 88.2 66.4 92.7 140 0.762 0.781 103 11196 3.34 6.24 187 241 130 53.9 147 94.8 64.4 0.781 0.863 110 11664 3.07 4.66 152 217 117 53.9 91.5 80.1 87.5 0.825 0.976 118 11779 3.57 6.68 187 383 205 53.6 54.5 59.7 110 0.748 0.922 123 11787 1.50 2.24 149 89.5 60.5 67.7 189 140 74.1 0.590 0.715 121 11795 2.45 4.11 168 177 149 84.0 89.8 103 114 0.728 0.854 117 11812 3.95 6.68 169 353 213 60.4 160 136 85.5 0.828 0.906 109 11894 4.27 6.06 142 197 127 64.3 97.8 83.3 85.2 0.845 0.868 103 11927 2.68 3.72 139 103 103 100 57.6 59.8 104 0.773 0.863 112 11949 3.18 5.56 175 201 141 69.8 101 61.2 60.7 0.769 0.833 108 12041 1.99 4.52 227 216 152 70.6 120 102 85.1 0.698 0.860 123 12245 2.98 5.95 200 167 215 129 110 50.0 45.5 0.746 0.870 117 12363 1.93 3.15 163 210 113 53.6 89.7 84.9 94.7 0.668 0.788 118 Mean 2.99 4.76 160 207 142 72.3 104 90.7 91.9 0.743 0.857 116

0.94 g (%) 31.6

1.65 34.1 78.8 44.5 21.0 37.3 26.8 24.7 0.068 0.064 7.98 34.7 21.4 38.2 31.4 29.0 35.8 29.5 26.9 9.11 7.43 6.89

CLf, Formation clearance; CL,, elimination clearance; VP apparent volume of distribution of 4-hydroxycyclophosphamide ; f,, fraction of the dose of cyclophosphamide converted to 4-hydmxycyclophosphamide.

*P = .OLXXMl; tP = .OOl; $P = ,075. Comparisons of means were made with the paired, tw&aikd f test, with P < .05 considered statistically significant.


Table Iv. The estimated values of V,,, &, and V,,& of carboxyethylphosphoramide mustard and indole-3- acetic acid formation from 4-hydroxycyclophosphamide and indole-3-acetaldehyde in human liver cytosolic incubations

Substrate V,, (nmollmin/mg protein) K,,, (WOW VAm (mL/min/nzg protein)

4-Hydroxycyclophosphamide HLC 118 0.36 6.78 HLC 127 0.72 11.6 HLC 132 0.44 6.79 HLC 133 1.24 23.2 HLC 142 1.65 20.6 HLC 144 1.88 8.52 Mean 1.05 12.9 SD 0.64 7.21

Indole-3-acetaldehyde HLC 118 13.0 1.24 HLC 127 31.7 2.60 HLC 132 11.5 0.83 I-KC 133 31.8 3.52 HLC 142 45.6 3.21 HLC 144 48.6 1.10 Mean 30.4 2.08 SD 15.7 1.17

V max, Maximal velocity; K,,,, Michaelis constant: V&K,,,, intrinsic clearance; HLC, human liver cytosol.

0.053 0.062 0.065 0.054 0.080 0.221 0.089 0.065

10.5 12.2 13.8 9.04

14.2 44.1 17.3 13.3

4-Hydroxycyclophosphamide formation clearance increased by 60% (P < .OOl) and its elimination clearance decreased by 27.7% (P < .OOl) from day 1 to day 2. The apparent volume of distribution of 4-hydroxycyclophosphamide was not different between day 1 and day 2 (P > .07). The fraction of the dose of cyclophosphamide converted to 4-hydroxycyclophosphamide increased 16% (P < .OOl) from day 1 to day 2.

There are 2 potential reasons for a decrease in 4- hydroxycyclophosphamide elimination clearance: (1) a saturation of aldehyde dehydrogenase- 1 attributable to a K, in the clinical concentration range or (2) a decrease in aldehyde dehydrogenase-1 activity by a slowly-reversible or irreversible mechanism. The value of K, of carboxyethylphosphoramide mustard formation from 4-hydroxycyclophosphamide was determined in cytosol prepared from human livers. The respective estimated values of V,, and Km of carboxyethylphosphoramide mustard formation were 1.05 + 0.64 nmol/min/mg protein and 12.9 f 7.21 pmol/L (Table IV). Indole-3-acetaldehyde was evaluated for use as an ex vivo probe substrate for carboxyethylphosphoramide mustard formation activity by aldehyde dehydrogenase- 1 by determining the kinetics of conversion of indole- 3-acetaldehyde to indole-3-acetic acid and the correlation between the V,, for indole-3-acetic acid and carboxyethylphosphoramide mustard formation in human liver cytosol. Indole-3-acetaldehyde was converted to indole-3-acetic acid in human liver cytosolic incuba-

tions, with a V,, of 30.4 f 15.7 nmol/min/mg protein and a K, of 2.08 * 1.17 l.tmol/L (Table IV). The V,, of indole-3-acetic acid formation (measured at 40 pmol/L indole-3-acetaldehyde) correlated very well with the V,,, of carboxyethylphosphoramide mustard formation (measured at 80 pmol/L 4-hydroxycyclophosphamide) as shown in Figure 5 (r2 = 0.863; P < .OOOl). In addition, indole-3-acetaldehyde compet- itively inhibited the formation of carboxyethylphosphoramide mustard in human liver cytosol, with a Ki of 5.92 f 3.86 l.tmol/L (n = 6). A representative Dixon plot of indole-3-acetaldehyde inhibiting carboxyethylphosphoramide mustard formation from 4-hydroxycyclophosphamide in human liver cytosol- 118 is shown in Figure 6. Use of indole-3-acetaldehyde as the probe substrate for aldehyde dehydrogenase- 1 provided the advantages of a simple assay and avoidance of the error that would be produced by subtracting the concentration of carboxyethylphosphoramide mustard present in the patient sample from the concentration at the end of an incubation to which 4-hydroxycyclophosphamide had been added exogenously.

The possibility of a decrease in 4-hydroxycyclophosphamide elimination clearance was accessed ex vivo by measuring patient erythrocyte aldehyde dehydrogenase-1 activity, before the first infusion of cyclophosphamide and 48 hours later (24 hours after the start of the second cyclophosphamide infusion). The aldehyde dehydrogenase-1 activity, as measured by V,,, of

298 Ren et al. CLINICAL PHARMA COLOGY &THERAPEUTICS

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HCY

(IM

0 I 0

I I 1 0.5 1 1.5 2

V,, of CEPM formation (nmol/min/mg protein)

Figure 5. Correlation between indole-3-acetic acid (IAA) and carboxyethylphosphoramide mustard (CEPM) formation maximal velocity (V,,,) in human liver cytosol (r2 = .863; P < .oool).

indole-3-acetic acid formation in erythrocytes obtained from 9 patients, decreased 23.3% (P < .02) from cells obtained just before the initial infusion of cyclophosphamide to 48 hours after the start of the first cyclophosphamide infusion (Table V).

DISCUSSION The major findings of this study with regard to the

effect of repeated administration of cyclophosphamide on exposure to cyclophosphamide and its metabolites were (1) that the AUC of cyclophosphamide, deschloroethyl cyclophosphamide, and 4-ketocyclophosphamide decreased from day 1 to day 2 of cyclophosphamide administration, (2) that the AUC of 4-hydroxycyclophosphamide and carboxyethylphosphoramide mustard increased from day 1 to day 2, and (3) that the AUC of phosphoramide mustard was not significantly different between day 1 and day 2. The major findings that provide mechanistic insight into the change of pharmacokinetics in cyclophosphamide and its metabolites were (1) that the total clearance of cyclophosphamide increased from day 1 to day 2, (2) that the elimination clearance of 4-hydroxycyclophosphamide decreased from day 1 to day 2, (3) that the aldehyde dehydrogenase-1 in vitro K, was within the clinical 4-hydroxycyclophosphamide concentration range, and (4) that 2 days of cyclophosphamide administration decreased patient erythrocyte aldehyde dehydrogenase- 1 activity.

In agreement with previous reports,s-12 we observed a significant decrease in the AUC of cyclophosphamide

16

14

12

6.6

-6 -4 -2 0 2 4

IAL (Pm

Figure 6. A representative Dixon plot of indole-3-acetaldehyde (IAL) inhibiting carboxyethylphosphoramide mustard (CEPM) formation from 4-hydroxycyclophosphamide (HCY) in human liver cytosol-118. The estimated dissociation constant (KJ for indole-3-acetaldehyde was 1.77 pmol/L in this liver.

from day 1 to day 2. Because deschloroethyl cyclophosphamide formation clearance and cyclophosphamide renal clearance were not significantly different between day 1 and day 2, the most likely explanation for the change in the AUC of cyclophosphamide is that the formation clearance of 4-hydroxycyclophosphamide was increased. Although it is impossible to measure 4- hydroxycyclophosphamide formation clearance directly in vivo, model-based analysis of simultaneous fitting to cyclophosphamide and 4-hydroxycyclophosphamide concentration-time data suggested that it-increased 60% (P < .OOl) from day 1 to day 2. Cyclophosphamide has been shown to induce CYP2C9 and CYP3A4 in primary human hepatocyte culture,** the isozymes that account for the formation of 4-hydroxycyclophosphamide in human liver microsomes.4

The AUC of deschloroethyl cyclophosphamide decreased significantly from day 1 to day 2. This AUC is determined by the fraction of cyclophosphamide dose converted to deschloroethyl cyclophosphamide, which is the ratio of its formation clearance to cyclophosphamide total clearance, and the elimination clearance of deschloroethyl cyclophosphamide. Because deschloroethyl cyclophosphamide formation clearance was unchanged and cyclophosphamide total clearance increased from day 1 to day 2, the fraction of cyclophosphamide dose converted to deschloroethyl cyclophosphamide would decrease from day 1 to day 2, as would the AUC of deschloroethyl cyclophosphamide.

CLINICAL PHARMACOLOGY &THERAPEUTICS VOLUME 64, NUMBER 3 Ren et al. 299

We found previously that CYP2C9 is the major enzyme responsible for 4-hydroxycyclophosphamide formation, but the enzyme responsible for deschloroethyl cyclophosphamide formation could not be identified because of its very low in vitro formation rate at therapeutic concentrations of cyclophosphamide.4 Because deschloroethyl cyclophosphamide formation clearance was unchanged and 4-hydroxycyclophosphamide formation clearance was increased (see below) from day 1 to day 2, these data indicate that the formation of deschloroethyl cyclophosphamide and 4-hydroxycyclophosphamide are dominated by different enzymes; that is, the enzyme responsible for deschloroethyl cyclophosphamide formation at therapeutic concentrations of cyclophosphamide is probably not CYP2C9.

The AUC of 4-hydroxycyclophosphamide increased 54.7% (P < .002) from day 1 to day 2 of cyclophosphamide administration. This AUC is determined by the fraction of cyclophosphamide dose converted to 4- hydroxycyclophosphamide and the elimination clearance of 4-hydroxycyclophosphamide. The increase in the AUC of 4-hydroxycyclophosphamide could be attributable to an increase in the fraction of cyclophosphamide dose converted to 4-hydroxycyclophosphamide or a decrease in 4-hydroxycyclophosphamide elimination clearance or both. The renal clearance of cyclophosphamide was 25.7% and 19.4% of its total clearance, respectively, on days 1 and 2, and the total clearance of cyclophosphamide increased about 30% from day 1 to day 2. Thus, together these data indicate that the AUC of 4-hydroxycyclophosphamide cannot increase more than 20% if its elimination clearance is unchanged. Because an increase of 54.7% was observed, it appears that its elimination clearance decreased from day 1 to day 2.

The formation of carboxyethylphosphoramide mustard has been suggested to be the major pathway of 4- hydroxycyclophosphamide elimination. Carboxy- ethylphosphoramide mustard formation is catalyzed by aldehyde dehydrogenase- 1, presumably in the liver, the location of most of the body’s aldehyde dehydrogenase- 1 .23 The decrease in 4-hydroxycyclophosphamide elimination clearance could be caused by a K,,, for aldehyde dehydrogenase-1 that is in the clinical 4-hydroxycyclophosphamide concentration range, coupled with a higher 4-hydroxycyclophosphamide concentration on day 2 than on day 1 caused by autoinduction of 4-hydroxycyclophosphamide formation clearance increasing the fraction of cyclophosphamide dose converted to 4-hydroxycyclophosphamide, or a decrease in the vmax of aldehyde dehydrogenase- 1. Pharmacoki- netic calculations with use of the V,,, and K, of 4-

Table V. The formation rate of indole-3-acetic acid from indole-3-acetaldehyde in patient erythrocyte preparations at 0 and 48 hours of cyclophosphamide infusion

Formation rate of indole9-acetic acid (nmoUmi@L RBC)

Patient No. 0 Hours* 48 Hours*

Percentage of 0 hours

26 0.109 0.083 75.8 27 0.101 0.126 124 28 0.087 0.053 61.2 29 0.045 0.050 111 30 0.064 0.040 61.5 31 0.105 0.075 70.8 32 0.137 0.087 63.5 33 0.112 0.077 68.1 34 0.117 0.062 53.3 Mean 0.098 0.072 76.7 SD 0.028 0.026 24.4 CV% 28.8 35.2 31.9

RBC, Red blood cells. *I’ = ,017. Comparisons of means were made with the paired two-tailed

t test, with P < .05 considered to be statistically significant.

hydroxycyclophosphamide for aldehyde dehydrogenase- 1, the concentrations of 4-hydroxycyclophosphamide, and the increase in the fraction of cyclophosphamide dose converted to 4-hydroxycyclophosphamide observed in this study, indicated that the capacity-limited formation of carboxyethylphosphoramide mustard from 4-hydroxycyclophosphamide and the increase in the fraction of cyclophosphamide dose converted to 4- hydroxycyclophosphamide would account for only about 30% of the increase in the AUC of 4-hydroxycyclophosphamide. Pharmacokinetic considerations therefore suggest that the apparent decrease in 4-hydroxycyclophosphamide elimination clearance was in part caused by a decrease in the V,, of aldehyde dehydrogenase- 1. We fitted cyclophosphamide and 4-hydroxycyclophosphamide concentration-time data on day 1 and day 2 separately with our model and estimated the values of the fraction of cyclophosphamide dose converted to 4-hydroxycyclophosphamide and 4-hydroxycyclophosphamide elimination clearance. The fraction of cyclophosphamide dose converted to 4-hydroxycyclophosphamide increased 16% (P < .OOl) and 4- hydroxycyclophosphamide elimination clearance decreased 27.7% (P < .OOl> from day 1 to day 2.

Erythrocyte aldehyde dehydrogenase- 1 has been shown to be identical to liver aldehyde dehydrogenase- 1 in peptide sequence. 24 Furthermore, a strong correlation between erythrocyte and hepatic aldehyde dehy-

300 Ren et al. CLINICAL PHAIWACOLOGY & THEIL4PEUTIC.9

SEPTEMBER 1998

drogenase-1 activity has been reported for healthy humans.17 We measured aldehyde dehydrogenase-1 activity with use of indole-3-acetaldehyde as the probe substrate in patient erythrocytes on day 1 and day 2 (at 0 and 48 hours after the start of the first cyclophosphamide infusion). Erythrocyte aldehyde dehydrogenase-1 activity decreased 23.3% (P < .02) from 0 to 48 hours. The extent of the decrease in patient erythrocyte aldehyde dehydrogenase- 1 activity was comparable to that in 4-hydroxycyclophosphamide elimination clearance in vivo (27.7%).

Preincubation of human liver cytosol with 5 pmol/L 4-hydroxycyclophosphamide for 10 minutes in vitro caused about 65% inhibition of aldehyde dehydrogenase-1 activity as measured by indole-3-acetic acid formation rate compared with 25% inhibition without preincubation (data not shown). This suggested that aldehyde dehydrogenase-1 could be inhibited by the degradation product(s) of 4-hydroxycyclophosphamide.

We did not detect any correlation between the AUC of cyclophosphamide and 4-hydroxycyclophosphamide on either day of cyclophosphamide infusion, although both positiveta and negative21 correlations have been reported. Our findings are expected on pharmacokinetic grounds because the fraction of cyclophosphamide dose converted to 4hydroxycyclophosphamide was 0.743 f 0.068 on day 1 and 0.857 -+ 0.064 on day 2, both close to 1, which indicated that the AUC of 4hydroxycyclophospharnide should be relatively independent of the AUC of cyclophosphamide. In a previous report,‘3 we found a significant negative correlation between the AUC of cyclophosphamide and 4-hydroxycyclophosphamide in 7 patients receiving cyclophosphamide and TBI only if 1 patient (UPN 8807) was omitted. It appears that that patient may not, in fact, be an outlier, and that no such correlation should be expected. The in vivo fraction of cyclophosphamide dose converted to 4-hydroxycyclophosphamide determined herein is consistent with our previous determination of the fraction of cyclophosphamide dose converted to 4-hydroxycyclophosphamide relative to other primary metabolites (0.95 1 f 0.024) in human liver microsornal incubations.4

Although the formation clearance of carboxyethylphosphoramide mustard decreased from day 1 to day 2, which contributed to the decrease in the fraction of the amount of carboxyethylphosphoramide mustard formed from 4-hydroxycyclophosphamide, the AUC of carboxyethylphosphoramide mustard increased from day 1 to day 2. This could be quantitatively explained by the fact that the fraction of cyclophosphamide dose converted to 4-hydroxycyclophosphamide increased from day 1 to day 2 and this contributed to the increase in the AUC of carboxyethylphosphoramide mustard.

The AUC of phosphoramide mustard is determined by the amount of cyclophosphamide converted to 4- hydroxycyclophosphamide, the amount of 4-hydroxycyclophosphamide converted to phosphoramide mustard, and the elimination clearance of phosphoramide mustard. Although the formation fraction of carboxyethylphosphoramide mustard from 4-hydroxycyclophosphamide decreased from day 1 to day 2, the AUC of phosphoramide mustard was not significantly different between day 1 and day 2. The 2 possible explanations are (1) the formation clearance of phosphoramide mustard decreased or (2) the elimination clearance of phosphoramide mustard increased from day 1 to day 2. Our data gave no insight into which mechanism is operative. Although phosphoramide mustard is the ultimate cytotoxic species of cyclophosphamide, circulating phosphoramide mustard does not contribute to cytotoxicity because it is largely ionized at physiological pH and does not enter cells.1

The AUC of 4-ketocyclophosphamide decreased significantly from day 1 to day 2. As with phosphoramide mustard, this could be explained by a decrease in 4- ketocyclophosphamide formation from 4-hydroxycyclophosphamide or an increase in 4-ketocyclophosphamide elimination from day 1 to day 2. The data do not permit speculation as to specific cause.

In summary, we studied the pharmacokinetics of cyclophosphamide and its metabolites in bone marrow transplantation patients on day 1 and day 2 of cyclophosphamide infusion. The AUC of cyclophosphamide decreased from day 1 to day 2 as a result of an increase in its metabolic clearance. The AUC of 4-hydroxycyclophosphamide increased from day 1 to day 2 as a result of increased formation and decreased elimination clearances. No correlation between the AUC of cyclophosphamide and 4-hydroxycyclophosphamide was detected on either day of cyclophosphamide administration. Alde- hyde dehydrogenase-1 activity appears to decline as a consequence of cyclophosphamide administration. It is important to elucidate the mechanism of the decline of aldehyde dehydrogenase-1 activity in future studies.

We appreciate the technical assistance of Michelle Bouvier, MSN, Ami Batchelder, Bruce Comeau, Scott McDonald, and Steven Ellis, and the support of the medical and nursing staff of the Fred Hutchin- son Cancer Research Center and Swedish Medical Center.

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Pharmacokinetics of cyclophosphamide and its metabolites in bone marrow transplantation patients*