Metabolism of viprostol — A synthetic vasolidator PGE2 analog

PROSTAGLANDINS

METABOLISM

OF VIPROSTOL

- A SYNTHETIC ANALOG

VASODILATOR

G. Nicolau, '* D.B. Cosulich ', A. Tonelli ,*,’ KS. Pruzinsky:' and D. 61um

PGE2

S.il.Chen,I,

Depdrtmerits of Pharn:acodyn$i,:ics,' dt?d Cher,lictilPrcccss Medical Research Division, Research and Development, American Cyanamid Company, Pearl River, New York.

Abstract (I) Metabolic studies were done with 14C-Viprostol administered by various routes (I.V., oral and topical) to Total radioactivity and six animal species and to man. metabolic profiles were analyzed in plasma, tissues and excreta. The main metabolites were isolated and identified by capillary GC/MS. Plasma and urinary metabolic profiles were qualitatively similar across species, with two major metabolic reactions being predominant: rapid hydrolysis to the pharmacologically active free acid (II) and oxidation of the alpha-chain to dinor and tetranor acids (III, IV). In the monkey and man, reduction of the 9.keto group lead to PGF2 type metabolites (VI-VIII). In the rat, omega oxidation of the beta-chain occurred as well, resulting in the formation of dicarboxylic acids (V). Introduction Viprostol is a potent vasodilator with prolonged antihypertensive activity when administered transdermally to man and laboratory animals (l-3). Its effect is produced primarily by a direct relaxant action on arteriolar smooth muscle (4). Because of its peripheral vasodilator properties and its persistence in the skin following topical administration (5). viprostol is considered potentially active in disease states that could benefit from increased blood flow. * Reprint requests to: Gabriela Nicolau, Ph.D., Department of Pharmacodynamics, Medical Research Division, American Cyanamid Company, Pearl River, NY 10965

APRIL 1986VOL. 31 NO. 4

811

PROSTAGLANDINS

The compound (I), synthesized under the code name CL 115,347 is a methyl ester that differs from the natural PGE2 mainly in the beta-chain in which the metabolically unstable CI50H, group was replaced with a tertiary OH group at CI6, containing also a vinyl group. This paper summarizes the metabolism studies done with 14C-viprostol. Data on deposition and metabolism in the skin First-pass metabolism through will be reported separately. human skin was estimated to be extensive (6). I.

Methods _I__ A.

Compounds -_

1) 14C-labeled viprostol was synthesized as described was at the C5 position. previously (1). The radiolabel Specific activity was 19 uCi/mg. The structure is shown in Figure 1.

a

“1,c=c, /”

A”,

.*-\ HO

Figure

812

1:

H

,c=c<

CH,(cH,)*c00cH, H

CH=CH*

CH, ’ CHz (cH,)) CH, t H

Structure of viprostol: methyl (+)-(11,5Z(and 5E),13E, 16R(and 16s).169ethenyl-ll,l%-dihydroxy9-oxaprosta-5,13-dien-1-oate

APRIL 1986 VOL. 3 1 NO. 4

PROSTAGLANDINS

Figure 2:

Synthesis of reference compounds; schematic representation

APRIL 1986 VOL. 3 1 NO. 4

813

PROSTAGLANDINS

2) Reference compounds were synthesized as outlined in Figure 2 by stereospecific total synthesis starting from furan. Reaction of furan with monomethyl succinate catalyzed by aluminum chloride followed by reduction with sodium borohydride gave alpha-hydroxy furan 1. Rearrangement of 1 under acidic conditions and protection3s the trimethyl-silyT (TMS) ester or ether, gave the key cyclopentenoid intermediate 2 (45% overall yield from furan). Conjugate addition of th> mixed organocuprate of vinylstannate (1)3 followed by deprotection under midly acidic conditions gavZ the PGE type reference compound 4 in 60% yield. Reduction of 4 with L-Selectride (lithium trizec-butylborohydride) gave Exclusively the PGF2-alpha reference compound 5. The cis relationship between the 5 alpha-OH group and tFie4 alphapropionic acid side chain allowed for slow formation of cislactone 7. Reduction of cyclopentanone 4 with sodium borohy dride in-ethanol produced the PGF2-betaFeference compound 6 as a crystalline solid which did not form a trans-lactone. B.

Dosing Material and Animals

Intravenous dosing solutions were prepargd in 5-10% ethanol/saline. For oral drug administration, C-viprostol was dissolved in neobee. Topical formulations were prepared in a petrolatum base ointment containing: petrolatum 72X, isopropyl adipate 20% and paraffin 8%. Drug concentrations in the various formulations were made up as needed to obtain the desired doses. A summary of the species, dose levels (mg/kg) and routes of administration is shown in Table I. In a human study, y male volunteers were administered topically 2 me Se; ",'f"W C-viprostol in petrolatum formulation on a 10 cm area. C.

Sample Collection and Handling Blood was collected at various time intervals after dosing, serum or plasma se arated by centrffugation and stored frozen at -20' to -408C until analyzed. All handling of samples was performed at low temperatures and in most cases the samples were treated with acetone or methanol immediately after collection to stop potential (in vitro) enzymatic activity. Urine was collected in containers surrounded by dry ice and kept frozen until analyzed.

814

APRIL 1986 VOL. 3 1 NO. 4

PROSTAGLANDINS

Route

species

Dose

@WW

Intrsvenous

0.1 0.005 1.0

Rat

29

Yi 132 3

Dog

1.0

3

Rabbit

0.1 1.0

3

Monkey

0.05 0.025 0.1 1.0

: 7

Mouse

2.0

Guinea

Pig

Man

Table I:

Topical

Oral

3

6

9 40

9

44

1.5

10

2.0’

4

ADIIE - Absorption, distribbution, metabolism and excretion studies with C-viprostol (number of animals or subjects) *

Total dose/subject

Expired CO was collected from rats placed in Itec,ially designed metabo ?.ism cages. In the clinical study, CO2 was monitored in the expired air at selected timepoints with a CO trapping solution consisting of 1M hyamine hydroxide (2 Although ml ‘i , absolute ethanol (2 ml) and thymolphtalein. radioactivity was observed intermittently, extrapolation to quantifiable values was not possible. as well as silica from thin layer Plasma, urine and CO chromatography (TLC) plaY es were counted directly in 3a70B scintillation cocktail (Research Products Int., Mt. Prospect, IL.), by using Beckman LS 8000 and LS 9000 Scintillation Spectrometers.

APRIL 1986 VOL. 31 NO. 4

815

PROSTAGLANDINS D.

Determination of Metabolic Profiles

Metabolic profiles in plasma and urine were analyzed by TLC and high pressure liquid chromatography (HPLC). The samples were extracted at neutral and acidic pH with ethyl acetate. The extraction efficiencies from plasma and urine More than 90% of the were > 90% and 50-75%, respectively. radioactivity left in the aqueous phase of the urine samples was extracted with ethanol after saturating the aqueous phase with K2CO . Sample extraction was also performed by using CIB Sep-Pa% cartridges (Waters Assoc., Milford, MA.). The organic phases (ethyl acetate, ethanol) containing the extracted radioactive compounds were evaporated under nitrogen, the dry residues redissolved in small volumes (20100 ul)of methanol or ethyl acetate and platedIjn Brinkman C-viprostol Silicp4Gel GF or Fluka RP12 plates, alongside and C-CL 115,129 (the free acid, II) as reference standards. The Silica Gel plates were developed in solvent systems containing chloroform:methanol:acetic acid at ratios of 90:5:5 or 90:10:5. The reversed phase (RP) plates were developed with H20:CH3CN, 65:35. Radioactivity was measured directly on the plates by using a Berthold Model LB 282 TLC linear analyzer (Dana Scientific Co., Philadelphia, PA). Autoradiograms were obtained by exposing the whole plates to Kodak Medical Si? 5 X-Ray film. Metabolic profiles by HPLC were obtained by using Waters HPLC systems with reversed phase columns Cl8 ODS2 and ODS3 (Whatman Inc., Clifton, NJ) with MeOH/H20 and CH3CN/H 0 as mobile phases and UV detection at 214 nm. Effluent frac 2. ions were collected and counted for radioactivity. E.

Isolation of Metabolites

Larger samples (up to 100 mL urine) were extracted as described above and the dry residues applied in ethyl acetate to preparative (0.5 mm) TLC plates. The radioactive areas of interest were removed, extracted and replated individually for a second or (as needed) third TLC purification. HPLC was used, in some cases, as an additional purification step. Reaction with diazomethane followed by replating of the reaction product was used for the preliminary identification of carboxylic acids.

816

APRIL 1986 VOL. 31 NO. 4

PROSTAGLANDINS

F.

Derivatization for&C/MS --_

Pentafluorobenzyl esters and TMS (trimethylsilyl) derivatives for GC/MS analysis were prepared as described of one of the main urinary earlier (7). Derivatization metabolites found in all species is shown as an example in Figure 3.

6H

I40

OH

Ii0

1V M.W.=324

M.W.=664

467---

I 68TFA

c (Piperidine) OTMS M.W.=648

Figure 3:

M.W.=720

Schematic representation of reactions used to derivatize metabolite IV for TX/MS analysis and of relevant fragments.

Esterification of the CODH group was done with TMS derivatives of the OH groups pentafluorobenzyl bromide. were prepared with BSTFA (bis-trimethylsilyltrifluoroacetamide) in pyridine. Enolization of the C=O group followed by TNS derivatization occured when piperidine was used as a reaction medium (8).

APRIL 1986 VOL. 3 1 NO. 4

817

PROSTAGLANDINS

G.

Fletabolite Identification by tll? _~I_-~Each urinary metabolite (25-1000 ng) was examined at each derivatization stage (Figure 3)by GC/MS on a Finnigan 4021T quadrupole mass spectrometer by electron impact (EI) and negative ion chemical ionization (NICI), with methane gas at 0.4 torr. flass spectral parameters were: sources: temperature, 220° (EI) and 270' (NICI); manifold temperature, 95-1000; transfer lines' temperatures, 285-300'; EV 50 (EI), 44 (NICI), GC parameters: 15M DB5 capillary column (J&W Scientific Co.) programmed in the splitless mode from 70-300' at lO'/min. for samples dissolved in acetonitrile (after esterification and the first silylation step and from 180300' at lO'/min. for the final derivative in tetradecene. Eletabolic material in the silyl derivatives was identified in the GC profiles by the characteristic base peak ion, m/z 185+, produced by cleavage at the 15-16 bond. In the rat, (V) was observed by the strong m/z 395t due again to cleavage of the 15,16 bond. The molecular weight data were derived from the strong carboxylate anions generated in NICI (El-CH C6F ), by careful comparison of the GC retention times for tie E? and NICI runs. The metabolites were distinguished from endogenous materials and could be identified as lites from the appearance of the isotopic clusters. ;;;aj! C-metabolites enhanced the +2 from 14C-viprostol isotopes 12-15s (see below). For metabolite IV, synthetic IV was prepared and derivatized. By GC/blS it was identical by retention time and mass spectral data with the metabolite derivatives of IV from both monkey and human urine. Ketabolite III was not isolated; it is the result of the first beta-oxidation leading to IV. Identification of the PGF-type metabolites resulted from comparison of the data after the first and second silylation steps; PGF-type material remained unchanged, whereas PGE-type samples increased the observed ions by 72 amu (TNS) due to silylation of the enol form of the ketone. The metabolite VIII derivative was identical by GC/MS to synthetic F-betaVIII and different from synthetic F-alpha-VIII by GC/MS retention time (done by several coinjections of mixtures of rfftabolite and synthetic F-alpha and F-betIa4-VIII). Since C-viprostol had been administered, the C enhanced +2 isotope of the carboxylate anion in NICI was used to track the metabolite elution in the mixture with the synthetic materials.

818

APRIL 1986 VOL. 3 1 NO. 4

PROSTAGLANDINS The mass spectra of synthetic F-alpha and F-beta-VIII were identical. Metabolite VII was also identified using the above methodologies. The plasma metabolite (II) was the species which was monitored in plasma (7) and was identified by GC/MS from its GC retention time , EI spectrum and NICI m/t 593- (MCH2CcF5)* Because of the low levels present, metabolite VI in human and monkey plasma was identified without isolation. By examination with NICI and multiple ion detection (IlID)at each silylation stage, there was no difference between the two stages in the amount of 595. (M-CH2C6H5) present. That it was not a plasma artifact from (II) was demonstrated by its absence in normal plasma and in plasma spiked with (II) and stored for 4 months at -4O'C. That it was formed from (I) metabolically was shown by MID scans to develop the isotope clusters: human plasma samples after administration of "cold" viprostol gave a normal isotopic patt rn, whereas, monkey plasma after topical administration of $4 C-viprostol (12.5% enrichment at C-5) showed a 12-15X enrichment of the +2 isotope of 595.. II.

Results --A.

Metabolic Profiles __-~--_-

Plasma metabolic profiles indicate that viprostol was rapidly and extensively metabolized in all species. Hydrolysis of the methyl ester group in the alpha-chain to the free carboxylic acid II (CL 115,129) was the first rapid - metabolic reaction. This reaction also occurred even in vitro, catalyzed by plasma esterases. The free acid is pharmacologically active (3). It is the compound for which the analytical methodology has been developed for pharmacokinetic studies (7). Table II shows the TLC metabolic profiles in rat plasma (expressed as percent of extracted radioactivity) at 5 min., 45 min. and 2 hours following intravenous dosing and at 2 hours following topical dosing in petrolatum. At 5 minutes after intravenous dosing, only about 12% of the radioactivity was found as unchanged I and more than 25% was the free acid II, as confirmed by GC/MS of isolated material. By 45 minutes, most of the radioactivity (ca 35%) was found in a more polar compound, subsequently identified as the tetranor acid (IV).

APRIL 1986 VOL. 3 1 NO. 4

819

PROSTAGLANDINS

At least five more radioactive compounds were seen, most being more polar than I and II. Significant free acid (II) (about 31% of radioactivity) was seen at 2 hours following topical dosing as compared to only 5% following I.V. dosiny. This confirms previous findings on the slow transdermal release of active drug following topical administration of viprostol (5).

TLC Metabolic Profile of 14C=VIPROSTOL in Rat Plasma (Oh of Radioactivity Plated) Area Number

Reference Compounds

: 3

Intravenous 5 min.

45 min.


<1 10

15

;:

Topical 2 hrs.
11 5 26

2 hr.
5 12 20

2 6 : 9 10

11 12 13 14 15

Table II:

820

10 CL 115,129

26

CL 115,347

12

Std.degrad. Front

11
1 6 4
5 17 -

31 1 1

6
2
Rat plasma samples were extracted (>90% recovery) and analyzed as described in the Methods Section. to the tetranor acid Area No. 4 corresponds The compound in area No. 8 has metabolite (IV). the same chromatographic properties as the PGF2 system: Solvent metabolite (VI). chloroform:methanol:acetic acid 90:5:5. Reference compounds: CL 115.347 = viprostol (I), CL 115,129 not = free acid (II). Background radioactivity, visible as discrete spots on the plate, accounted for 8-18X of that originally plated.

APRIL 1986 VOL. 31 NO. 4

PROSTAGLANDINS

A

tc)

figure

in: (A) 4: TLC metabolic profiles of 14C-viprostol monkey urine (O-24 hour) following oral administration of 1 mg/kg and (B) human urine (80 12 hour sample) following topical administration Solvent of 2 mg (total dose) in petrolatum. acid 90:5:5 systems: chloroform:methanol:acetic (A) and 90:10:5 (B). Areas marked (a), (b) and (c) were used as sources for the isolation of the respective metabolites VIII, IV, and VI.

APRIL 1986 VOL. 31 NO. 4

821

PROSTAGLANDINS

Renal excretion was predominant, especially in the larger species. Radioactivity in urine accounted for 66, 54 and 80% of dose by four days after I.V. dosing to the dog, monkey and rabbit, respectively. In the mouse and rat, biliary excretion was predominant, but >32% of dose was still recovered in the urine. Therefore, urine was used as source for the isolation of metabolites. Urinary metabolic profiles were qualitatively similar in most species studied regardless of route of administration. There was no evidence of unchanged I or the main early plasma metabolite II. TLC metabolic profiles in monkey urine following oral administration and in human urine following topical dosing are shown in Figure 4. The corresponding HPLC urinary metabolic profiles also indicated the presence of several metabolites, more polar than I or II.

Figure 5:

822

TLC urinary metabolic profiles of 14C-viprostol in the rabbit following topical and intravenous dosing and in the rat after intravenous dosing. Dose: 1 mg/kg, collection interval O-24 hours. Reference standards: I = viprostol, II = free acid (see Figure 6). Solvent system as in Figure 4 (A)

APRIL 1986 VOL. 31 NO. 4

PROSTAGLANDINS

Rabbit urine contained a significant amount of a metabolite not seen in other species as well as a compound having the same Rf as authentic II. The autoradiogram of the plate is shown in Figure 5. B. _e Metabolite

Identification --uI_

The main metabolites from rat, monkey and man were isolated and characterized by capillary GC/MS. The high potency (low dose) of viprostol necessitated processing large amounts of biological fluid for metabolite isolation. The small amount of sample isolated coupled with the relatively large amount of accompanying endogenous materials even after numerous purification steps precluded characterization by IR and NMR which require larger sample sizes and/or purer preparations. However, capillary GC/IlS spectral data from both EI and NICI allowed structural postulates to be made. In addition, the two-step silylation procedures used, enabled classification of metabolites as either E- or Fprostaglandins. The validity of the characterization methods used was verified by the finding that the GC/MS properties of synthetic IV and VIII were identical with their respective metabolites. Mass spectral data are shown in Table III. In general, the EI spectra were weak because of the small amounts of sample available, but structural data for the beta-chain were provided from the dominant cleavage at 15, 16 bonds and occassionally at 16, 17 (loss of butyl). The NI spectra exhibited intense carboxylate anions, used for molecular weight assignments. Mass spectral fragmentations were largely derived from TMS and TMSOH losses. The structures postulated for the metabolites viprostol and a suggested metabolic pathway are shown Figure 6.

APRIL 1986VOL. 31 NO. 4

of in

823

774

648

720

858

930

776

776

740

740

722

722

IV (1s)

IV (25)

v (1s)

v (251

VI (IS)

VI l2s.I

VII (1s)

VII (2s)

VIII (1s.)

VIII

495

-567

-541

-541 469

469

495

(ND Data 1

595

-567

(MID Data)

595

-749

677

451

451

477

477

659

-587

449

539

503

-431

-377 467

521

467

-593

521

379

379

405

405

-377

431

361

361

569

497

287

341 431

479

307

720

774

707

707

705

Done

665

665

Not Done

Not

630

684

612

615

1s = after 1 silylation step; 2s =-after silylation steps ** 185+ = (CH2CH=C(OTP?S)C4Hg); 395+ = (CH2CH=C(OTMS)C$t6COOCti2C6F6)+ Underlined ions are the base peaks

(251

702

(1s)

I1

II (25)

575

575

573

HI fragments of (M - CH2C6F5) I-. m/z) El Fragments (+. m/z) ________________c__~__c_________________~~~~~~~~~~~~~~~~~-~~~~--~~~~-“--~~-~--~----“~--~~~~~~~-~~~~~-~~~~-~~~~~~Derivattve -72 -72 -15 -57 Metabol i te Mol. Wt. 0 -72 -90 -90 -2x90 -2x90 -3x90 n+ -15 -57 -90 -90 -90

Table III: Mass Spectral Fragments of Viprostol Metabolites (Roman numerals correspond to those of the structures shown in Fig.6)

-185

-185

-185

-185

395

395

-185

-185

-185

-185

l*

181

181

181

181

-181

-181

181

181

181

181

CH2C6F5

PROSTAGLANDINS

Figure 6:

Chemical structures of metabolftes and suggested metabolic pathway for viprostol. Metabolites IIV: PGE series, Metabolites VI-VIII: PGF series

APRIL 1986 VOL. 3 1 NO. 4

825

PROSTAGLANDJNS

The main plasma metabolite found at early times following intravenous dosing and throughout a plateauing time of up to 8 hours following topical dosing was identified in all species as the acid II. (It was monitored by GC/MS in the viprostol absorption - phramacokinetic studies.) The dinor and tetranor acids III and IV, resulted form one and two beta oxidation steps of the alpha-chain, respectively. Aminor pathway seemed to be an additi#al oxidative decarboxilation CO2. It accounted for about at C with the production of 5% o+ the administered radioactivity in the rat ans was detected below measurable levels in man. In the rat, omega oxidation occured as well, to form a dicarboxylic metabolite (V) not found in larger species. A significant metabolic pathway in man and the monkey lead to the formation of PGF type metabolites: F2 viprostol (VI) was detected in the plasma of man and monkey and to a The reduction of the C-9 keto lesser extent in the rat. group was sterospecific in the tetranor acid (VIII) from both monkey and human urine, since synthetic F-beta-VIII was identical with the VIII metabolite and synthetic F-alpha-VIII was different. The dinor metabolite (VII) was noted in monkey urine. Our data indicate viprostol undergoes extensive metabolism, similar in some respects to the known such as the beta oxidation biodegradation of endogenous PGE P' evertheless, significantly reactions in the alpha-chain. greater metabolic stability was achieved by changing the metabolically unstable C150H group to a tertiary OH at C16. This change apparently also protected the 13-14 double bond since the metabolites were excreted in urine without reduction in the beta-chain The greater metabolic stability of viprostol correlates with its prolongued pharmacological activity as compared to that of the natural PGE2.

Acknowledgements We wish to thank A. Lanzilotti for the synthesis of 14Cviprostol, W. McWilliams for valuable technical assistance and C. Gesner for the prepration of the manuscript.

826

APRIL 1986 VOL. 31 NO. 4

PROSTAGLANDINS References

1.

Birnbaum, J.E., P. Cervoni, P.S. Chan, S.M. Chen, M.B. Floyd, C.V. Grudzinskas, M.J. Weiss and F. Dessy: Prostaglandins and congeners. (16RS)-(+)-15-deoxy-16-hydroxy-16-vinyl-prostaglandin E2, an orally and transdermally active hypotensiveagent of prolonged duration., J. Med. Chem., 25:492, 1982.

2.

Chan, P.S., P. Cervoni, M.A. Ronsberg, R.C. Accomando, G.J. Quirk, P.A. Scully, L.M. Lipchuck: Antihypertensive activity of dl-15deoxy-16-1-hydroxy-16-(a//B)-vinyl prostaglandin E2 methyl ester (CL 115,347), a new orally and transdermally long-acting antihypertensive agent., J. Pharm. Exp. Ther., 226:726, 1983.

3.

Birnbaum, J.E., P.S. Chan, P. Cervoni, F. Dessy, L. van Humbeeck: Cutaneous erythema and blood pressure lowering effects of topically applied 16-vinyl prostaglandins., Prostaglandings, 23(2):185, 1982.

4.

Nicolau, G., V.K. Batra, S. Gordon, A. Tonelli, W. McWilliams: Transdermal absorption of HDV-PGE2 Prostaglandin in the rat. Vth International Conference on Prostaglandins, Abstracts, p. 506, 1982.

5.

Lai, F.M., T.K. Tanikella, P. Cervoni: The vasorelaxanteffectof (CL (+)-15-deoxy-16-hydroxy-16(a/8)-vinyl-prostaglandin E 115,129) and its methyl ester (CL 115,347) on the isolated $ uctus arteriosus preparation., Life Sciences, 34:1861, 1984.

6.

Yacobi, A., R.A. Baughman, D.B. Cosulich, G. Nicolau: Method for determination of first-pass metabolism in human skin., J. Pharm. Sci., 73(10):1499, 1985.

7.

Cosulich, D.B., N.A. Perkinson, V.K. Batra: Capillary gas chromatographic-mass spectrometric analysis of 15-deoxy-16hydroxy-16-vinylprostaglandin E2., J. Pharm. Sci., 74(1):76, 1985.

8.

Rosello, J., J. Tusell, E. Gelpi: Profiles of prostaglandins A,B,E, and F (Series 1 and 2) obtained by gas chromatography with multiple ion detection., J. Chromat., 130:65, 1977. Editor: J. Pike

Received: 9-3-85

APRIL 1986 VOL. 3 1 NO. 4

Accepted: 3-14-86

827