Pure Enantiomers of 2-Aryipropionic Acids: Tools in Pain Research and Improved Drugs in Rheumatology Kay

Brune,

The

MD,

mode

Geisslinger,

Gerd

of action

PhD,

MD,

and

S. Menzel-Soglowek,

PhD

drugs in pain is widely referred to as inhibition of acid, however, at low doses, is an analgesic but not a This “enigma” may be resolved by recent findings employing 2-arylpropionic acids. Pure enantiomers of these chiral drugs show a different pharmacodynamic and pharmacokinetic profile. Using pure enantiomers of flurbiprofen, ibuprofen, and ketoprofen, we could show that (1) R-enantiomers of these drugs are inverted to S-enantiomers to a different degree in different species, including humans, (2) the pharmacokinetic parameters of both pure enantioniers differ in a drugand a speciesspecific manner, and (3) both enantiomers exert differential analgesic effects. It appears particularly interesting that R-flurbiprofen, for instance, which is not or only to a small extent inverted in humans and rats, is practically devoid of prostaglandin synthesis inhibition in vitro. Consequently, in line with current thinking, R-flurbiprofen is not prostaglandin synthesis. potent anti-inflammatory

toxic

of aspirinlike Salicylic agent.

to the gastrointestinal

tract

concepts, however, of pain and nociception. R-enantiomers of 2-arylpropionic

this

current models

of peripheral

prostaglandin

and

shows

synthesis

inhibition

M

ore than one hundred years ago pure salicylic acid or its sodium salt were first employed in the treatment of rheumatic diseases. They had, however, to be given in quantities of more than 10 g/ day.1’2 Within the last decades, several double-blind controlled studies could show that salicylic acid at low doses yielded an analgesic effect comparable to low doses of aspirin.3 Moreover, other antipyretic analgesics as phenazone or paracetamol do not interfere with prostaglandin synthesis at analgesic doses.4’5 Also the analgesic effectiveness of many

nonsteroidal

anti-inflammatory

drugs

(NSAID5)

in

current use do not correlate very well with their potency as prostaglandin synthesis inhibitors.6 These findings conflict to some extend with the concept of VANE,7 that all aspects of the mode of action of anti-

From the Department of Experimental and Clinical Pharmacology and Toxicology, University of Erlangen-Nuernberg, Universitaetsstr, Erlangen, Germany. Supported in part by a grant from the Deutsche Forschungsgemeinschaft Brune, Toxicology,

0-8520

944

(SFB

353,

Al).

Address

for reprints:

and Clinical Pharmacology University of Erlangen-Nuernberg, Universitaetsstr. Erlangen, Germany.

Department

of

Experimental

#{149} J dIn Pharmacol

1992;32:944-952

no anti-inflammatory

effects.

enantiomer does exert analgesic It is concluded that R-flurbiprofen acids may exert novel analgesic

Kay and

22,

in inflamed

In contrast

to

activity in different and, possibly, other effects independently

tissue.

pyretic analgesics depend on the inhibition of prostaglandin synthesis. Some of the arguments came invalid about 20 years ago by our finding the acidic function of the NSAIDs, in contrast to nonacidic drugs such as phenazone, propyphena-

zone,

and

paracetamol,

does

induce

drug

the bethat the

accumula-

tion in inflamed tissue and, by that, may explain the superior anti-inflammatory activities of these compounds.8 Still, the salicylic acid/aspirin enigma remains unresolved. We have now tackled this problem again, taking advantage of the availability of pure enantiomers of 2-arylpropionic acids (for review,9-11) the development of easy-to-handle and reliable stereoselective assays for both enantiomers in tissues and body fluids,128 and the possibility of measuring both drug and eicosanoid19 concentrations in body fluids and tissues at the same time. We here report that, in contrast to current thinking, part of the analgesic activity of NSAIDs may not be related to peripheral prostaglandin synthesis inhibition in inflamed tissue. This observation may explain why salicylic acid is analgesic at low doses despite lack of prostaglandin synthesis inhibition. It also may suggest the selective use of pure R-enan-

PURE

tiomers

of 2-arylpropionic

verted, contrasted reactions

THE THE

ENANTIOMERS

acids,

which

in pain conditions with little to those with considerable when pure S-enantiomers

PROBLEM OF DEVELOPMENT

are

OF

not

inflammation inflammatory are indicated.

2-ARYLPROPIONIC

One difficulty encountered in comparing the results on the effects of enantiomers from different research groups is sometimes the absence of data concerning the optical purities of the enantiomers used.9 The optical purity of the enantiomers we have used exceeded 98.5%, with the exception of tiaprofenic acid (95%), in every case as determined by high-performance liquid chromatography and optical rotation. The enantiomers of flurbiprofen, ibuprofen, ketoprofen, and tiaprofenic acid were kindly supplied by PAZ Arzneimittelentwicklungsgesellschaft mbH (Frankfurt/Main, Germany,) Pharmatrans Sanaq Ltd. (Basel, Switzerland), Rhone Poulenc (Paris, France), and Albert-Roussel (Wiesbaden, Germany), respectively.

inas

OPTICAL PURITY AND OF CHIRAL ASSAYS

Within the last years, several assays for the stereoselective determination of R- and S-enantiomers of 2arylpropionic acids in body fluids and tissues have been developed. For the separation of chiral compounds, three methods are currently used: (1) the formation of diastereomeric derivatives20-22; (2) the use of a chiral mobile phase23; and (3) the use of chemically bonded chiral stationary phases.24-26

Most

published

stereoselective

assays

are

based

on THE INVERSION IS SPECIES AND

the formation of diastereomeric derivatives. We developed stereospecific high-performance liquid chro-

matographic

methods

procedure

for

the

without

determination

any

derivatization

and

quantification

of flurbiprofen,15 ibuprofen,14’16 ketoprofen,14 fenoprofen,14 using chiral stationary phases.

of therapeutic

doses

in biologic

fluids

and

OF R- TO SUBSTANCE

tis-

15.2729

R.ibuprofenR-ketoprofen nn

S-ENANTIOMERS SPECIFIC

With the aid of stereoselective high-performance liquid chromatography assays and the availability of pure R- and S-enantiomers of flurbiprofen, ibuprofen, and ketoprofen, we could show that different cell types3#{176}of a given species were able to invert Renantiomers provided that in this species, the general ability of inversion exists (Figure 1). The extent of inversion, however, appears to vary depending on the tissue investigated.31 Moreover, a systematic in-

and They

are easy to handle, reliable, and accurate, but not inexpensive. Also, the quantification limits were low enough for measuring the concentrations of enantiomers of 2-arylpropionic acids after the administra-

tion sues.

ACIDS

A-flurblproten

I

man in-vItro

I

rat ratl

Ii

dog guinea pig

0

2

4

60

2

4

60

2

4

6

AUD (S) / AUD (R)

Figure 1. B- to S-inversion of different 2-arylpropionic acids in different species. The amount of inversion was calculated as the ratio AI]DS/AUDR after administration of pure R-ibuprofen (A), R-ketoprofen (B) and R-jlurbiprofen (C) to different species (values are means, +SD, n = 3). The human subjects were dosed orally with 400 mg, 25 mg, and 50 mg of R-ibuprofen, B-ketoprofen, and B-flurbiprofen, respectively. The animals were treated with an intravenous dose of 10mg/kg bw of the individual B-enantiomers. The concentrations of the drugs in tissue culture was 20 g/ml. The degree of inversion was assessed after 120 hours. AUD5: area under the S-enantiomer data points. AUD5: area under the R-enantiomer data paints. Man in vitro: human hepatoma cell line (Hep G2, for detail30). Rat in vitro: hepatoma cell line

of the

rat

(H4IIE).

CHIRAL SYMPOSIUM

945

BRUNE

ET

AL

TABLE I Inhibition of Release of 6-keto-PGF1 in Gastric Mucosa, Jejunal Tissue, and Lung of the Rat and Inhibition Release of PGF2a in Rat Brain and of TXB2 in Whole Rat Blood After Administration of Various Doses of the Flurbiprofen Enantiomers41 (%)

Inhibition 6.keto.PGF1, Gastric Dose (mg/kg bw)

Mucosa

R

Jejunal

S

1 5

12±5 44±3

80±3 86±2

25

39±7

95±5

R

0±

20±4 3±25

Lung S

0

animals

PHARMACODYNAMIC OF ENANTIOMERS 2-ARYLPROPIONIC

and

S

42±2

15±9

73±3

68±7

90±2 99±0

91±2

67±9

94±1

in humans.

Pharmacol

1992;32:944-952

Whole

R

S

Blood

R

0±0

53±3

23±

48±5 /

90±2 /

79±2 93±

S

16

98±0 97±0 98±0

0

richment and (marginal) inversion of the R-enantiomer in the different tissues. When R- and S-enantiomers were employed in the standard models of pain and inflammation research, only the S-enantiomers proved to be active anti-inflammatory compounds causing a dose-dependent inhibition of different forms of paw inflammation42 (Figure 2). Surprisingly, however, R-flurbiprofen, being practically devoid of anti-inflammatory activity, proved to be an effective analgesic agent42 (Figure 3). This analgesic effect was equally visible after pinching either the inflamed or the noninflamed

EFFECTS OF SOME ACIDS

The enantiomers of flurbiprofen, ibuprofen, and ketoprofen blocked prostaglandin synthesis in macrophages differently (for methodologic details, see references 4 and 37). The R-enantiomers are always 100 to 1000 times less potent than the S-enantiomers. These data are in line with results published previously using other in vitro prostaglandin synthesis inhibition models.9’38#{176} It is possible that this small effect of the R-enantiomers is related to impurities of S-enantiomers in the approximately 98.5 to 99% pure R-enantiomers. Interestingly, despite a basic lack of anti-inflammatory activity of R-flurbiprofen in the rat, a considerable inhibition of prostaglandin synthesis ex vivo does occur in the gastric mucosa, in the blood plasma, in the lung, and in the central nervous system, but not in the mucosa of the small intestine41 (Table 1). It is difficult to explain this differential effects by the small amount of S-enantiomer co-administered as impurity. It appears more plausible to assume a quantitatively different degree of en-

946 #{149} J CIIn

Brain

R

vestigation of humans and available laboratory animals confirmed and extended the observations of other groups that the R-enantiomers of 2-arylpropionic acids, as for example, flurbiprofen3234 and ketoprofen,35’36 are not or only marginally inverted in some species and actively transformed in others (Figure 1). R-ibuprofen, by contrast, was inverted to its optical antipode in every species tested.9 These observations did allow for the pharmacodynamic re-investigation of the activity of 2-arylpropionic acids not only in isolated cell systems but also in experimental

TXB2

PGF,

Tissue

of

Inhibitionof edema

(%)

Treatment

-s--

0,01

0,1 Dose

(mg/kg)

1 p.o.

-B’--

$

-

Rac

10

Figure 2. The anti-inflammatory effect of R-, S-and racemic flurbiprofen as dened in the carrageenan edema of the rat paw (for details42) was related to their potency as PG synthesis inhibitors in vitro. Inhibition was calculatedfrom the means ofn = 6maleSprague-Dawley rats per dose as compared with controls (n = 6). *p