first-pass metabolism of midazolam by the human intestine*

Pharmacoltinetics and pharmacodynamics of the monoamine oxidase B inhibitor mofegiline assessed during a phase I dose tolerance trial

The safety, pharmacokinetics, and pharmacodynamics of single oral doses of up to 48 mg and daily (for 28 days) doses of up to 24 mg mofegiline were investigated in healthy male volunteers. Plasma pharmacokinetics indicated rapid absorption and elimination: time to reach maximum concentration occurred at about 1 hour; half-life ranged from 1 to 3 hours. Maximal plasma concentration and area under the plasma concentration-time curve increased and oral clearance decreased disproportionately with dose. Mofegiline rapidly and markedly inhibited platelet monoamine oxidase B (MAOB) activity, which returned to baseline within 14 days. Urinary excretion of phenylethylamine increased proportionately with doses up to 24 mg. No changes in urinary elimination of catecholamines, blood pressure, heart rate, or ECG were observed. A classic maximum tolerated dose was not achieved in these studies. How- ever, the 48 mg single dose and the 24 mg multiple daily dose far exceeded the dose (1 mg) that was associated with >90% platelet MAOB inhibition. (CLIN P HARMACOL THER 1995;58:342-53.)

Maxine Stoltz, PhD, Donald Reynolds, PhD, Linda Elkins, PhD, Daniel Salazar, PhD, and Scott Weir, PharmD, PhD IGmus City, MO., and PGaceton, 2V.J

Mofegiline is currently undergoing clinical evalua- tion for the treatment of Parkinson’s disease. The drug is a selective, irreversible inhibitor of the B isozyme of monoamine oxidase (MAOB). The two recognized monoamine oxidase isozymes, MAOA and MAOB, are outer mitochondrial membrane proteins that cata- lyze the breakdown of biogenic amines.’ In humans, MAOA is predominantly localized in aminergic neu- rons and is the major isozyme in the gut and placenta. MAOB is primarily distributed in glial cells and liver and is the exclusive isozyme in platelets. The isozymes have different but overlapping specificities for various biogenic amines, including dopamine, se- rotonin, norepinephrine, and tyramine.’ Inhibitors of MAOA carry the risk of hypertensive crisis in con- junction with ingestion of tyramine-containing foods

From Marion Merrell Dow, Inc., Kansas City, and Bristol Myers- Squibb, Princeton.

Received for publication Dec. 29, 1994; accepted April 21, 1995. Reprint requests: Maxine L. Stoltz, PhD, U.S. Clinical Pharmacoki-

netics Department, Marion Merrell Dow, Inc., PO Box 9627, Kansas City, MO 64134.

Copyright 0 1995 by Mosby-Year Book, Inc. 0009-9236/95/$5.00 + 0 13/l/65758

342

(the “cheese” effect) and are not widely used clinically. An inhibitor of MAOB, such as mofegiline, may offer new therapeutic applications because it avoids the “cheese” effect.

The dopamine-sparing and antioxidative properties of mofegiline are being directed toward the treatment of Parkinson’s disease. The only MAOB inhibitor currently on the market is L-deprenyl (selegiline, El- depryl). Results of the Deprenyl And Tocopherol Anti- oxidative Therapy Of Parkinsonism (DATATOP) study3 suggested that MAOB inhibition delays the progression of Parkinson’s disease and the need to ini- tiate levodopa therapy. Preclinical studies have shown that mofegiline has greater potency and greater selec- tivity for MAOB than L-deprenyl.4 Mofegiline has other advantages over L-deprenyl in that it does not appear to be metabolized to sympathomimetic amines and is devoid of amphetamine-like activity.5 See structure for comparison of the molecular structures of mofegiline and deprenyl. Mofegiline is taken up into rat brain tissue after single oral doses and inhibits brain MAOB activity in a dose-dependent mamrer.4

The studies were performed to investigate the clinical pharmacology of mofegiline in healthy male vol-

CLINICAL PHAFMACOLOGY & THERAPEUTICS VOLUME 58, NUMBER 3 Stoltz et al. 343

CH3 I CECH

MDL 72,974 L-Deprenyl

Molecular structure comparison of mofegiline (MDL 72,974) and r-deprenyl.

unteers after administration of single and multiple oral solution doses of the drug. The objectives of the study were to identify the maximum tolerated dose after single- and multiple-dose regimens, to characterize the safety profile of the drug over a range of doses, and to determine the preliminary pharmacokinetics and pharmacodynamics of mofegiline.

METHODS Subjects and study design. These studies were con-

ducted with use of single-blind (sponsor unblinded), placebo-controlled, randomized, parallel-group escalat- ing-dose designs with a placebo lead-in. A total of 40 healthy male subjects participated in the study; 20 participated in a single-dose study, and the remaining 20 were enrolled in a multiple-dose trial. Thirty-six subjects were white, two were black, and two were His- panic. Of the 40, five were smokers. The mean (?SD) age, height, and weight of the volunteers were 30 + 7 years, 179 t 8 cm, and 76 i: 9 kg, respectively.

Doses were prepared from a stock solution of mofegiline (lot No. RJ9101) that contained 100 mg mofegiline, 500 mg benzyl alcohol, 775 mg citric acid mono- hydrate, and 1793 mg sodium phosphate dibasic per 100 ml purified water. The matching placebo oral solution (lot No. RJ9102) contained all the above ingre- dients except mofegiline. Prestudy hematology, serum and urine chemistry, blood pressure and heart rate measurements, and 12-lead electrocardiography (ECG) were performed as screening criteria for subject inclu- sion in the study.

All subjects received an oral dose of placebo on day 1. On day 2 for the single-dose study or on days 2 through 29 for the multiple-dose study, mofegiline or placebo were administered as oral solution doses. Dif- ferent groups of four subjects each (three receiving active drug and one receiving placebo) were used at each dose level. Each dose was evaluated for safety and tolerance to mofegiline before subsequent escala- tion.

Safety assessments. The safety of mofegiline was assessed with use of serial three-positional heart rate and systolic and diastolic blood pressure measurements, ECG readings, clinical laboratory measurements, neurobehavioral testing, and reported adverse events. Clinical laboratory, neurobehavior, and adverse event reports are not discussed in detail in this article. Times of other safety assessments in relation to dose administration are shown in Table I.

Plasma sampling for mofegiline concentrations. As shown in Table I, serial blood samples were col- lected for determination of plasma mofegiline concentrations on day 2 of the single- and multiple-dose studies and on day 29 of the multiple-dose study. Trough samples were also taken at various times during multiple-dose treatment. A method to quantitate mofegiline concentrations in human plasma was validated over the concentration range of 100 to 10,000 pg/ml with use of a 1 ml sample volume. Sample cleanup was achieved by solid-phase extraction with use of cyan0 solid-phase cartridges. Drug and its deuterated form (internal standard) were isolated from plasma, purified through use of a selective wash step, and eluted with acetonitrile/l,2N hydrochloric acid (95 : 5). The solution was evaporated to dryness and the ana- lytes were derivatized with pentafluoropropionic anhy- dride in methylene chloride (60” C for 30 minutes). The derivatized sample was evaporated, reconstituted in a smaller volume, and injected into a gas chromato- graph coupled to a mass spectrometer. A DB-17 column (30 m X 0.25 mm internal diameter, 0.15 pm film; Hewlett-Packard Co., Wilmington, Del.) was used to separate drug and internal standard from other components. Positive ion chemical ionization was used with 5% ammonia in methane as the reagent gas. The MNH+ ions for drug (m/z = 361) and internal standard (mlz = 365) were quantified with use of selective ion monitoring.

Validation was completed by assaying two replicates of seven calibration standards and five to seven

344 Stoltz et al. CLINICAL P HARMACOLOGY & THERAPEUTICS

SEPTEMBER 1995

Table I. Times of safety assessments and pharmacokinetic samples in relation to dose administration during single- and multiple-dose phase I studies of mofegiline

Safety Single-dose Multiple-dose measure study study

Dosing Lead-in Active drug

Blood pressure/heart rate

12-Lead ECG

Plasma pharmacokinetic samples

Platelet-rich plasma samples

24-Hr urine samples: PEA and catecholamines

Day 1 Day 2 Day 1: 0, 1, 3, 6, 9, 12 hr Day 2: 0, 1, 3, 6, 9, 12, 24, 48 hr

Day 1: 0 hr Day 2: 0, 4, 12, 24, 48 hr

Day 2: 0, 0.33, 0.67, 1, 1.5, 2, 3, 4, 5, 6, 8 hr

Day 1: 0 hr Day 2: 0, 6, 12, 24, 48, 96, 168,

336 hr

Days 1-2, 2-3, 3-4

Day 1 Days 2-29 Day 1: 0, 1, 3, 6, 9, 12 hr Day 2: 0, 1, 3, 6, 9, 12 hr Days 3-28: 0 hr Day 29: 0, 1, 3, 6, 9, 12, 24, 48 hr Day 1: 0 hr Day 2: 0, 4, 12 Days 3, 4, 7, 10, 17, 24: 0 hr Day 29: 0, 48 hr Day 2: 0, 0.33, 0.67, 1, 1.5, 2, 3, 4, 5,

6, 8 hr Days 10, 17, 24: 0 hr Day 29: 0, 0.33, 0.67, 1, 1.5, 2, 4, 6,

9, 12 hr Day 1: 0 hr Day 2: 0, 6, 12 hr Days 3, 10, 17, 24: 0 hr Day 29: 0, 6, 12, 24, 48, 96, 168,

336 hr Days 1-2, 2-3, 10-11, 17-18, 24-25,

29-30

PEA, Phenylethylamine.

replicates of eight quality control samples on 3 separate days. The line of best fit for calibration standards was calculated by weighted (l/x) quadratic least- squares regression based on peak area ratios. Peak area ratios were proportional to the amount of mofegiline added to plasma over the concentration range from 100 to 10,000 pg/ml. Calibration curve precision (percent relative standard definition) ranged from 3.1% to 10% over 3 days, with accuracies (percent relative error) of -0.7% to 1.0%. Intraday assay precision was 1.4% to 15%, and intraday assay accuracy ranged from -18% to 9.0%. Interday assay precision ranged from 4.8% to 13%, with interday accuracies of -11% to 1.3%.

Plasma pharmacokinetic analysis. Plasma mofegiline concentration versus time data were analyzed model independently. The area under the plasma concentration-time curve to the last measurable time point (AUC[n]) was calculated by the trapezoidal rule and extrapolated to infinity (AUC[m]) by dividing the last concentration by the log-linear terminal rate con- stant. Maximum plasma mofegiline concentrations (C,,,) and time to C,, (f,) were observed from the raw data. The terminal elimination rate constants and half-lives (tl,J were determined by curve-stripping

methods. The apparent oral clearance was calculated by dividing the mofegiline dose by the corresponding AUC[m] for each volunteer.

Blood sampling for platelet MAOB activity. Blood samples were withdrawn for analysis of platelet MAOB activity serially, as shown in Table I. Platelet- rich plasma was separated by centrifugation at 300g for 10 minutes. A Coulter Counter (Coulter Corp., Iw- ing, Texas) was used for platelet count determination. Phenylethylamine labeled with carbon 14 was incu- bated with a fixed amount of platelet-rich plasma at 37” C for 30 minutes. The hydrolytic product was then extracted with an organic solvent and the extract was centrifuged. The radioactivity in the organic layer was determined by means of liquid scintillation counting. MAOB activity was calculated by division of the specific activity of 14C-phenylethylamine by the product of extraction recovery and counts per minute (cpm) to yield units of nanomoles of phenylethylamine hydrolyzed per hour per milliliter of platelet-rich plasma. Data were finally expressed in absolute platelet counts.

Precision of the assay was established from three runs on 3 separate days. Three levels of bovine MAO in buffer were prepared as buffer quality control samples. These served as external enzyme controls for

CLINICAL PHABMACOLOGY & THERAPEUTICS VOLUlME 58, XUMBER 3 Stoltz et al. 345

0

-I- 4mg U 12mg 1 1 + 24mg

-O- 48mg

0 1 2 3 4 5 6 7 8

Time After Dosing (hr)

Fig. 1. Mean 5 SD plasma mofegiline (MDL 72,974) concentration-time profiles after administration of single oral solution doses of mofegiline to healthy male volunteers.

the incubation condition and hydrolytic product extraction. Two levels of platelet-rich plasma quality control samples were prepared from a single pool: the high level had the inherent MAOB activity; the low level was prepared by spiking the platelet-rich plasma with 1 rig/ml mofegiline. These samples served as ma- trix controls with and without the inhibitor (mofegiline) and were tested for storage stability. Precision of the platelet-rich plasma preparation was shown by centrifugation and Coulter Counter reproducibility. Freeze/thaw stability was also established.

Platelet MAOB activity was calculated for indi- vidual subjects at each time point measured. We estimated percentage of MAOB inhibition by averaging enzyme activity on day 1 (placebo lead-in) and day 2 (before dosing) and using this value as baseline. The following equation was then applied:

[lo0 - (Activity at time t/activity at baseline)] 100

Urine sampling and assay for phenylethylamine concentrations. Phenylethylamine concentrations were determined in serial urine samples as shown in Table I. Urine was also analyzed for creatinine and for catecholamine and metabolites. After addition of an internal standard (phenylpropylamine), the urine was made alkaline and extracted with cyclohexane. The solvent was separated from the aqueous phase and backex- tracted with 1 mmol/L hydrochloric acid. Phenylethyl- amine was quantified with a reversed-phase HPLC technique involving precolumn derivatization with o-phthaldialdehyde and fluorescence detection. A single set of calibration standards at or near the beginning of each curve defined a standard curve from which six replicates of quality control samples at three concentrations were determined. Five consecutive standard curves, assayed over a period of 10 days, were used to determine the interday variation. Calibration curve precision (percent coefficient of variation) ranged

346 Stoltz et al. CLINICAL PHARMA COLOGY & THERAPEUTICS

SEPTEMBER 1995

0 2 4 6 8

Time After One Dose (hr)

10 12

15000

I T

/i]

loooO-

5ooo-

0-Y 0 2 4 6 8 10 12

Time After 28 Doses (hr)

Fig. 2. Mean i: SD plasma mofegiline (MDL 72,974) concentration-time profiles after administration of one or multiple oral solution doses of mofegiline to healthy male volunteers.

Table II. Pharmacokinetic parameters estimated from plasma drug concentrations of healthy male subjects after single or 28 daily oral doses of mofegiline

Parameter 4 mg (n = 6) Single-dose study

12 mg (n = 6) 24 mg (n = 6) 48 mg (n = 3)

:m..(w (&ml)

A?C[n] (pg/ml

0.78 562 2 ‘- 0.10 280 2,874 0.92 2 k 0.30 1,357 9,836 0.92 It -c 4,182 0.30 27,202 1.17 ? +- 0.29 10,967

. hr) 610 -c 376 4,181 ? 1,930 14,854 ? 8,670 103,741 ? 52,192 AUC[@JI (pghl h-1 ND 4,343 + 1,962 15,064 r 8,628 105,170 rf: 52,685 CL,,, wd ND 3,719 t 1,848 2,048 2 1,158 557 2 313 b/* (W ND 1.00 + 0.18 1.18 I: 0.42 3.02 -c 0.57

Data are mean values t SD. C nlax, Maximum plasma concentrations; f,, time to reach C,,,; AUC[n], area under the plasma concentration-time curve to the last measurable time point;

AUC[m], AUC extrapolated to infinity; CL oral, apparent oral clearance; tl/z, half-life; ND, not determined because of scant data during the elimination phase.

CLINICAL PHARMA COLOGY & THERAPEUTICS VOLUME 58, NUMBER 3 Stoltz et al. 347

from 0.6% to 5.7%, with accuracy (percent relative error) of -2.1% to 2.3%. Interday assay precision was 5.1% to 7.8%, with accuracy ranging from -0.7% to 3.4%. Intraday assay precision ranged from 2.8% to 3.3%, with accuracy of -1.5% to 2.3%. The linear range was 5 to 100 rig/ml, with a 5 rig/ml limit of quantitation.

Urine assay for catecholamine and catecholamine metabolite concentrations. It was anticipated that if mofegiline caused significant MAOA inhibition, changes in peripheral metabolism and hence the urinary elimination of certain catecholamines and catecholamine metabolites would be detectable. Dopamine is metabolized by MAO to dopamine oxidation products or methylated by catechol-0-methyltransferase to yield 3-methoxytyramine. Therefore, increases in do- parnine and 3-methoxytyramine urinary excretion were monitored in this study. Similarly, increases in norepinephrine and epinephrine were monitored because they also are oxidized by MAO or methylated by COMT to normetanephrine and metanephrine, respectively. Normetanephrine and metanephrine are further metabolized by MAO to the major excretory products, 3-methoxy-4-hydroxyphenylethylene glycol (MHPG) and 3-methoxy-4-hydroxymandelic acid (VMA); only MHPG was monitored in this study. De- creased urinary elimination of MHPG and increases in 3-methoxytyramine, normetanephrine, and metanephrine have been shown to be sensitive markers for peripheral MAOA inhibition6-’

Total 24-hour urine collections (Table I) were used for determination of catecholamine and catecholamine metabolite concentrations. For analysis of metanephrines (metanephrine, normetanephrine, and 3-methoxytyramine), pH-adjusted (1.0 to 3.0) urine specimens were diluted with ammonium pentaborate buffer and an internal standard (4-0-methyldopamine) was added. After elution from a cation exchange column,

Multiple-dose study 4 mg (n = 3) 12 mg (n = 3) 24 mg (n = 3)

768 -c 5 5,989 2 4,065 14,018 i- 6,607 0.84 +- 0.23 0.89 t 0.19 1.17 -+ 0.29

1,440 ? 284 18,726 2 15,058 54,037 -+ 31,774 ND 18,857 2 15,107 54,359 i 32,040 ND 1,118 t 1,018 537 t 245 ND 2.64 -c 0.48 2.79 + 0.25

the eluate was applied to an anion exchange column. Metanephrines were finally eluted with ammonium acetate buffer then injected onto a cation exchange HPLC column and monitored by electrochemical detection. Quantification was achieved by comparison of metanephrine/intemal standard peak height ratios in the unknown to that of a urine standard.

For the catecholamines (epinephrine, norepinephrine, and dopamine), pH-adjusted (1.0 to 3.0) urine specimens were diluted with ammonium acetate buffer and an internal standard (4-0-methyldopamine) was added. After pH adjustment to 6.0 to 7.0, the sample was applied to a column that contained Bio-Rex 70 (Bio-Rad Laboratories, Hercules, Calif.) which re- tained the compounds of interest. Ammonium pentaborate buffer was used to elute the compounds from the column. The eluate was injected onto a cation exchange HPLC column and monitored by electrochemical detection. Quantification was achieved by comparison of catecholamine/intemal standard peak height ratios in the unknown to that of a urine standard.

For both metanephrines and catecholamines, two levels of assayed control samples were used with each run. Unknowns, urine standards, and control samples were processed simultaneously. After initial standard- ization, a standard and one control level were re- checked after every eight unknowns, alternating control levels. The lower limit of detection for all metanephrines and catecholamines was 2 p,g/L urine.

Non-pH-adjusted urine was used for MHPG assay. Conjugated MHPG was hydrolyzed with enzymes specific for sulfate and glucuronide. The degree of hy- drolysis was monitored with labeled MHPG sulfate and MHPG glucuronide. An ethyl acetate extract of the hydrolysate was further purified and quantified by HPLC with electrochemical detection. Calculation of the MHPG was by comparison to external standards. The linear range of the assay was 0.25 to 4 mg/L for each fraction, with an expected uncertainty (SD) of 10%. Two levels of control samples and at least one standard were included in the analysis of each batch of unknowns. Urinary concentrations of phenylethylamine and the various catecholamines and catecholamine metabolites were expressed as micrograms excreted per day normalized to grams of creatinine excreted per day.

Additional pharmacodynamic assessments. Serial three-positional heart rate and blood pressure measurements determined on days 1 (placebo lead-in), 2 (first active dose), and 29 (last active dose) were compared within each parameter by calculation of the daily area under the effect curve (AUEC) with the trapezoidal

348 Stoltz et al. CLINICAL PHARMA COLOGY & THERAPEUTICS

SEPTEMBER 1995

120000 -

-o- AUC(x1)

lOoooO- + AUC(x28)

c 80000- c ?.I E

j 6WOO-

e 0’

zrn

26000-

OL 0 12 24 36 40

Dose (mg)

Fig. 3. Values for area under the plasma concentration-time curve to the last measurable time point (ALJC[n]) plotted as a function of dose of mofegiline when administered to healthy male volunteers.

Table III. Mean (SD) percent platelet MAOB inhibition after single oral doses of mofegiline in healthy male volunteers

Day(s) after dosing Placebo (n = 5) I mg (n = 3) 4 rng (n = 3) 12 mg (n = 3) 24 mg (n = 3) 48 mg (n = 3)

‘/i - 1.6(49.2) 87.9(4.3) 97.7(0.8) 97.6(1.7) 97.6(4.2) 100(0.0) 1% 4.6(34.5) 72.4(30.6) 95.7(1.3) 96.5(2.0) 97.1(3.0) 100(0.0) 1 - 13.2(33.7) 68.7(9.2) 90.0(3.8) 93.0(1.9) 92.4(4.6) 98.8(0.8) 2 -16.3(43.4) 53.4(4.1) 78.3(7.5) 84.2(2.8) 80.7(6.4) 91.3(4.2) 4 -12.5(29.3) 0.1(8.6) 49.6(15.0) 58.3(3.4) 56.4(24.1) 73.9(11.8) 7 -15.3(32.6) -19.9(17.4) -4.3(23.8) 17.5(1.8) 36.9(9.4) 15.4(38.5)

14 -17.1(37.4) -3.9(24.2) 1.4(45.6) -64.3(13.4) -91.4(80.8) -18.7(51.2)

MAOB, Monoamine oxidase B.

rule. Maximum and minimum effect (E,, and E,,, respectively) were determined from observed raw values. The average effect (E,,) was calculated from the quotient: AUEC/r, in which r is 24 hours. The percent daily fluctuation was calculated with use of the following formula:

[OLx - Emd(Eavg)l 100

RESULTS Dose tolerance. Single oral doses of up to 48 mg

mofegiline were extremely well tolerated. No serious adverse events were reported in subjects receiving multiple doses of mofegiline: three reports of hypertension were judged by the investigator to be related to treatment; all three occurred in the lowest dose group (0.1 mg). Six events (back pain, chest pain, stiff

CLINICAL. PHARMA COLOGY & THERAPEUTICS VOLUME 5X. NUMBER 3 Stoltz et al. 349

Platelet MAOB Inhibition

Fig. 4. Percent platelet monoamine oxidase B (MAOB) inhibition during and after administl of multiple oral solution doses of mofegiline to healthy male volunteers.

-ation

neck, and leg pain) in five subjects (three in the 24 mg group; two in the placebo group) were judged to be related to treatment. No changes in heart rate were clinically significant. Any noted ECG change was transient and within normal limits. No clinical chemistry, hematology, or urinalysis value which fell out of normal range was considered to be significant.

Plasma pharmacokinetics. Most plasma samples obtained from subjects receiving doses of 1 mg mofegiline, and all samples obtained from subjects receiving 0.1 mg, were not analyzed for mofegiline after it was found that four consecutive samples (0.33, 0.67, 1, and 1.5 hours) from two subjects receiving 1 mg doses had concentrations below the quantitation limit of 100 pg/ml. In addition, only certain pharmacokinetic parameters could be estimated for subjects receiving 4 mg doses because only three to five consecutive plasma samples contained quantifiable drug concentrations. Plasma concentration versus time curves are illustrated in Fig. 1 (single-dose profiles) and Fig. 2 (single- versus multiple-dose profiles).

As summarized in Table II, absorption of mofegiline was rapid; t,,, occurred, on the average, between 0.8 and 1.2 hours after treatment. Disappearance of the drug from plasma was also rapid (t,/, ranging from 0.7 to 3 hours) and monophasic. The plasma elimina-

tion tl/, after single doses of 12 and 24 mg was -1 hour; the tl/, was slightly prolonged and ranged from 2.6 to 3.0 hours after single 48 mg doses and multiple doses of 12 and 24 mg. Over the single-dose range of 4 to 48 mg and the multiple-dose range of 4 to 24 mg, C max and AUC values increased disproportionately, whereas apparent oral clearance decreased with increasing dose. The nonlinearity of the kinetics is further illustrated in Fig. 3, where AUC is plotted against dose of mofegiline administered.

MAOB inhibition. Table III shows the percentage of inhibition of platelet MAOB after single oral doses of mofegiline. Enzyme activity was markedly reduced as early as 6 hours (l/4 day) after treatment, that is, 90% inhibited at the lowest dose tested (1 mg) and greater than 95% at the higher doses. MAOB activity appeared to approach baseline levels by day 4 in subjects treated at the 1 mg level, by day 7 after 4 mg doses, and by day 14 after administration of higher doses. The tl,* of MAOB activity recovery was 0.4 days after the 1 mg dose and then ranged from 2.8 to 4.6 days after the 4, 12, 24, or 48 mg doses.

Fig. 4 illustrates the inhibition of platelet MAOB activity during and after daily multiple-dose treatment with mofegiline. Enzyme activity was inhibited >30% at 6 hours after the first 0.1 mg dose and >90% at

350 Stoltz et al. CLINICAL l’Hz%MA COLOGY & THEBAPEUTICS

SEPTEMBER 1995

Linear Regression xl Dose y = 39.7 +0.006x cork coeff. q 0.995

I I I I I I 2oooo 40000 60000 60000 100000 120909

Area Under the Plasma MDL 72,974 Concentration Time Curve

Fig. 5. Relationship between total mofegiline (MDL 72,974) exposure and phenylethylamine urinary excretion after single oral doses of mofegiline to healthy male volunteers. PEA, Phenylethyl- amine.

Table IV. Mean phenylethylamine urinary excretion (in micrograms per gram of creatinine) after single oral doses of mofegiline in healthy male volunteers

Day of study* Placebo (n = 5) I mg (n = 3) 4 mg (n = 3) 12 mg (n = 3) 24 mg (n = 3) 48 mg (n = 3)

1 to 2 <LLQ <LLQ <LLQ <LLQ <LLQ <LLQ 2 to 3 <LLQ <LLQ 24 88 114 589 3 to 4 1 <LLQ <LLQ 5 7 99

LLQ. Lower limit of quantitation of the assay (5 rig/ml urine). *Active dose administered on day 2.

doses of 1,4, 12, and 24 mg. Steady-state enzyme inhibition of approximately 80% was achieved by the eighth day of treatment at 0.1 mg. After treatment at 1 mg, steady-state inhibition was in the range of 85%; higher doses caused nearly complete (195%) inhibition. After the last dose, MAOB activity slowly returned, ap- proaching baseline levels by day 41 (2-week washout: approximate platelet turnover rate). The tl/, values of MAOB recovery were 4.0,4.2,2.0,4.2, and 4.0 days, respectively, for the 0.1, 1,4, 12, and 24 mg daily doses.

Phenylethylamine urinary excretion data support the peripheral inhibition of MAOB activity after treatment with mofegiline. Phenylethylamine is a specific sub-

strate for MAOB; normal urinary concentration is 7.4 2 2 pg/gm creatinine.’ As shown in Table IV, within the constraints of the small sample size, phenylethylamine elimination increased proportionately to dose after administration of single oral doses of 4, 12, and 24 mg mofegiline but disproportionately after the single 48 mg dose. There was also a slower rate of re- turn to baseline phenylethylamine levels after the 48 mg dose. As pointed out earlier, estimated plasma pharmacokinetic parameters were also disproportion- ate to dose. As shown in Fig. 5, when plasma AUC values are plotted against cumulative phenylethylamine urinary excretion data, a correlation coeffi-

CLINICAL P HARMACOLOGY & THERAPEUTICS VOLUME 58, NUMBER 3 Stoltz et al. 351

q Phcebo

q 0.1 mg

0 1 mg

44

E 12mg

4 24mg

17to18 Day of Study

24 to 25 29 to 30

Fig. 6. Phenylethylamine (PEA) urinary excretion during and after administration of multiple oral solution doses of mofegiline to healthy male volunteers.

cient of >0.99 supports the direct relationship of MAOB inhibition to mofegiline exposure.

Phenylethylamine urinary excretion data after multiple oral doses of the drug are illustrated in Fig. 6. For the mofegiline dose levels tested, phenylethylamine elimination increased proportionately to dose. In addition, phenylethylamine excretion reached a steady state at all dose levels, suggesting that loss of specific MAOB inhibition did not occur with multiple- dose treatment of up to 24 mg daily for 28 days.

Catecholamine and catecholamine metabolite excretion. No clinically significant change or pattern of change was detected in the urinary concentrations of the catecholamines dopamine, epinephrine, and norepinephrine when compared across dose in relation to baseline and placebo group concentrations. For dopamine, baseline values ranged from 142 to 211 kg/gm creatinine. After 28 daily doses of mofegiline, dopamine values ranged from 165 to 218 kg/gm creatinine; normal values range from 106 to 256 p,g/gm creatinine. For norepinephrine and epinephrine, no differ- ences between baseline and end-study values could be detected, and all values over the course of the study remained within normal range. Treatment with mofegiline also produced no detectable changes in urinary elimination of MHPG, 3-methoxytyramine, normetanephrine, or metanephrine, all thought to be sensitive

markers for peripheral MAOA inhibition. Based on the development objectives for mofegiline, the lack of MAOA inhibition is a desirable result from a safety standpoint.

Dynamic effects on blood pressure and heart rate. Oral doses of mofegiline had no effect on hemody- namics as assessed from serial blood pressure and heart rate measurements and subsequent pharmacodynamic analyses. For example, over the range of multiple daily doses tested (0.1 to 24 mg), the mean values for AUEC for immediate standing systolic blood pressure ranged from 2495 to 2898 mm Hg . hr after one dose compared with 2452 to 2698 mm Hg . hr on day 29 after 28 daily doses. The same pattern was found for diastolic blood pressure and heart rate with all three-positional data sets. No placebo-versus- active-drug effects or single-versus-multiple-dose- treatment effects could be established.

DISCUSSION The results of this study show that single oral doses

of mofegiline of up to 48 mg and multiple (28 consecutive daily) oral doses of up to 24 mg are very well tolerated in healthy male volunteers. No increase in adverse events with increasing dose was observed, and no pattern of effect on any vital sign or laboratory parameter was shown.

352 Stoltz et al.

Mofegiline was shown to be a potent inhibitor of platelet MAOB at all doses tested. Enzyme activity was nearly completely inhibited within 6 hours (earli- est sampling time) of treatment with single or multiple doses of 1 mg or higher. At the 0.1 mg multiple dose level, time to peak MAOB inhibition was slower (18 days) and the degree of inhibition was less (-80% maximal) than at the higher doses. Activity returned toward baseline levels more slowly as the dose was increased but generally returned to baseline levels after a 1Cday washout period, which is the approximate platelet turnover rate.

It was noted that after single doses of 12 and 24 mg (Table III) and multiple doses of 1 and 4 mg (Fig. 4), percentage of platelet MAOB inhibition appeared to rebound toward the end of the sampling period. It is not known whether this was a true biological phenom- enon or was the result of normal laboratory variability. It is possible that at the higher dose levels (48 mg single and 12 and 24 mg multiple), sampling for platelet MAOB inhibition was terminated before the rebound effect occurred. Platelet MAOB inhibition will continue to be used as a peripheral marker for drug activity, allowing further investigation into this finding.

In further support of the peripheral MAOB inhibition observed from platelet enzyme activity, phenylethylamine urinary excretion reflected a dose-related increase in elimination of this MAOB-specific sub- strate as a function of increasing dose. After single doses of 4, 12, and 24 mg, these increases were generally proportional to dose. However, after a single dose of 48 mg the increase was at least twofold higher than levels anticipated from those after single doses of 24 mg. The apparent increase in availability and urinary excretion of this pressor amine was not accompanied by any identifiable changes in safety-related parameters, that is, blood pressure or heart rate. There were also no changes seen in catecholamine metabolism after treatment with mofegiline. If hemodynamic effects had occurred, these potentially would have been accompanied by increases in norepinephrine urinary excretion; norepinephrine has been shown to be a sensitive indicator of change in sympathetic tone.” Orally administered mofegiline also caused no clinically significant changes in cardiac conduction as assessed from pharmacodynamic analyses of ECG parameters (data not shown). Furthermore, during multiple-dose treatment, steady-state phenylethylamine elimination was achieved by day 10. The lack of further increases suggests that mofegiline, given during a multiple-dose regimen of up to 24 mg daily, remains a specific inhibitor of MAOB.

CLINICAL PHARMA COLOGY & THERAPEUTICS SEPTEMBER 1995

Plasma concentrations of mofegiline and resulting pharmacokinetic parameters after single and multiple oral doses of the drug showed large intersubject variability. The drug has a relatively short plasma tl/, (ranging from 1 to 3 hours), suggestive of rapid and extensive elimination. The drug has been shown to be extensively metabolized in rats, dogs, and monkeys.4 Only small percentages of an initial dose are detected in urine as unchanged compound and as many as eight (monkey) to 15 (rat) different metabolites have been found. Further, data generated during this study sug- gest nonlinear pharmacokinetics, that is, AUC and C values increased and apparent oral clearance de- crF;ed disproportionately with an increase in dose. In addition, further decreases in clearance were seen after multiple doses compared with single-dose regimens. A definitive dose-proportionality study is currently in progress to address the apparent nonlinear pharmacokinetics.

A classic maximum tolerated dose was not achieved in this study. This was based on the lack of adverse events associated with single- and multiple-dose treatment with mofegiline. However, the highest single dose (48 mg) and multiple dose (24 mg) tested were 48- and 24-fold greater than the dose that produced >90% platelet MAOB inhibition (1 mg). A dose of 12 mg is the anticipated therapeutic dose of mofegiline for the treatment of Parkinson’s disease.

We acknowledge Harris Laboratories, Lincoln, Neb for the conduct of the clinical portion of this study and, in particular, Jean Lee, PhD, and Gaylin Nickell, PhD, of Harris for the analysis of platelet MAOB and urinary phenylethylamine and for coordination of the analyses of catecholamine and metanephrine urine concentrations. We also acknowledge Kelly Reith, MS, and Larry Eden, MS, of Marion Mer- rell Dow for the analysis of mofegiline in plasma samples.

References Murphy DL, Donnelly CH. Monoamine oxidase in man: enzyme characteristics in human platelets, plasma and other human tissues. Adv Biochem Psychopharmacol 1974;12:72-86. Fowler CJ, O’Carroll A, Tipton KF. Deamination of dopamine by monoamine oxidase A and B in rat and in man. In: Tipton KP, Dostert P, Strolin-Benedetti M, eds. Monoamine oxidase and disease. London: Academic Press, 1984:393-402. Parkinson Study Group. Effect of deprenyl on the progression of disability in early Parkinson’s disease. N Engl J Med 1989;321:1364-71. Investigational drug brochure for MDL 72,974A; ver- sion 3. Kansas City, Missouri: Marion Merrell Dow, October 1991.

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5. MDL 72,974A. Drugs Future 1991;16:428-31. 6. Linnoila M, Karoum F, Potter WZ. Effect of low-dose

clorgyline on 24-hour urinary monoamine excretion in patients with rapidly cycling bipolar affective disorder. Arch Gen Psychiatry 1982;39:513-6.

7. Murphy DL, Brand E, Goldman T, et al. Platelet and plasma amine oxidase inhibition and urinary amine excretion changes during phenelzine treatment. J Nerv Ment Dis 1977;164:129-34.

8. Sunderland T, Mueller EA, Cohen RM, Jimerson DC, Pickar D, Murphy DL. Tyramine pressor sensitivity changes during deprenyl treatment. Psychopharmacol- ogy 1985;86:432-7.

9. Lentner C, ed. Geigy scientific tables; vol 1. West Cald- well, New Jersey: Ciba-Geigy Corporation, 1981:70.

10. Goldstein DS, McCarty R, Polinsky RJ, Kopin JJ. Rela- tionship between plasma norepinephrine and sympathetic neural activity. Hypertension 1983;5:552-9.

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first-pass metabolism of midazolam by the human intestine*

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