Pharmaceutical Research, Vol. 19, No. 8, August 2002 (© 2002)
Research Paper
effects (1), it is now known to possess significant and unique
immunomodulatory and anti-inflammatory activities. Numer-
ous reports have appeared on the successful use of thalido-
mide in the treatment of type 2 lepra (erythema nodosum
leprosum) reactions (2,3), chronic graft-versus-host disease
Chemical Stabilities and Biological
Activities of Thalidomide and Its
N-Alkyl Analogs
(4), discoid lupus erythematosus (5), aphthous ulcers in pa-
tients with AIDS (6), and rheumatoid arthritis (7). Its effec-
tiveness has been attributed to its ability to inhibit the syn-
thesis of tumor necrosis factor-alpha (TNF-␣). Studies have
shown that thalidomide is a powerful inhibitor of TNF-␣ pro-
duced by monocytes in vitro (8,9).
1
,6,7
2
Colleen Goosen,
Timothy J. Laing,
1
3,4
Jeanetta du Plessis, Theunis C. Goosen,
Tharaknath B. Rao, and Gordon L. Flynn
5
6
Oral therapy of thalidomide has proven to be effective in
rheumatoid arthritis but is associated with unacceptable side
effects (7). There is good reason to believe that thalidomide
Received February 8, 2002; accepted May 1, 2002
Purpose. To determine whether the N-alkyl analogs of the thalido- acts on TNF-␣ expression at the local tissue level (8,10–12).
mide are active and stable, their stabilities in buffer and their abilities
Therefore, if the delivery of the drug can be targeted, via
to inhibit tumor necrosis factor alpha (TNF-␣) in vitro in human
percutaneous delivery, it should be feasible to treat localized
peripheral blood mononuclear cell cultures were investigated.
inflammation selectively. However, thalidomide itself is too
Methods. TNF-␣ concentrations were determined with the aid of
polar and too high melting to achieve this end. Therefore,
ELISA kits. Chemical stabilities of the compounds were determined
several N-alkyl analogs of thalidomide that do not possess its
marked polarity and high melting properties were prepared.
We have shown that these N-alkyl analogs of thalidomide are
in three phosphate buffer solutions (pH 6, 6.4, and 7.4) at 25 and 32°C
by high-pressure liquid chromatography, and half-lives were calcu-
lated.
Results. The addition of N-alkyl groups to the glutarimide ring of the delivered percutaneously far easier than thalidomide itself
thalidomide molecule had little effect on the ability such compounds (13). However, these improvements would be little avail if the
have to inhibit TNF-␣ production. There was no statistical difference analogs were not themselves active compounds. Therefore, in
between the activity of thalidomide and its N-alkyl analogs at a 95%
this study, the TNF-␣ inhibitory effects of thalidomide and its
confidence level. Like thalidomide, the N-alkyl analogs in this series
N-alkyl analogs were assessed in cell culture. Peripheral
inhibit an average of 60% of the TNF-␣ synthesis in lipopolysaccha-
blood mononuclear cells (PBMCs) were stimulated with lipo-
ride-stimulated peripheral blood mononuclear cell cultures. Thalido-
polysaccharide (LPS), which induces TNF-␣ production. Tha-
mide and its N-alkyl analogs are hydrolyzed at very similar rates, with
lidomide was used as a control.
half-lives ranging from 25 to 35 h at 32°C at pH 6.4 and an average
rate constant of 2.35 × 10 /h.
−
2
Conclusions. Alkylating thalidomide had little effect on its ability to MATERIALS AND METHODS
inhibit the production of TNF-␣ in these cell cultures. All of the
compounds tested seem to have some, perhaps comparable, thera-
peutic potential.
Synthesis Method
The racemic form of thalidomide and its N-alkyl analogs
KEY WORDS: thalidomide; N-alkyl analogs; biological activity; tu-
mor necrosis factor alpha; chemical stability; half-life.
were synthesized according to literature methods but with
some modifications in the purification procedures. Detailed
synthetic procedures have been presented in our previous
study (14). Briefly, thalidomide was prepared by condensing
N-phthaloyl-DL-glutamic anhydride with urea. Similarly, N-
methyl, N-propyl, and N-pentyl thalidomide were synthesized
by condensing N-phthaloyl-DL-glutamic anhydride with meth-
yl amide, propyl amine, and amyl amine, respectively. Iden-
tification and levels of purity >96% were assured through
element analysis, electron impact mass spectrometry, nuclear
magnetic resonance spectroscopy, high-pressure liquid chro-
matography (HPLC), and by the sharpness of melting points.
INTRODUCTION
Thalidomide (␣-N-phthaloyl-glutamic-acid-imide) was
widely prescribed as a sedative in the late 1950s but was with-
drawn from the market in 1961 because of its teratogenic
effects. Thalidomide has a relatively simple chemical archi-
tecture (Fig. 1). In addition to its sedative and teratogenic
1
Department of Pharmaceutics, School of Pharmacy, Potchefstroom
University for Christian Higher Education, Potchefstroom, 2520,
Republic of South Africa.
Department of Pharmacology, School of Pharmacy, Potchefstroom
3
Stability Determination
University for Christian Higher Education, Potchefstroom, 2520,
Republic of South Africa.
Department of RheumatologyUniversity of Michigan, Ann Arbor,
The chemical stabilities of thalidomide and its N-alkyl
analogs were investigated. Methanolic stock solutions of each
compound (160 g/mL) were prepared. A series of dilutions
of each compound in study into phosphate buffer solutions of
pH 6, 6.4, and 7.4 was made to give final drug concentrations
of 2 g/mL containing less than 1% methanol. To obtain the
two lower pH values, the 0.1 M phosphate buffer (pH 7.4) was
adjusted to pH 6 and 6.4 with ortho-phosphoric acid. The
2
Michigan 48109.
4
Department of Pharmacology, University of Michigan, Ann Arbor,
Michigan 48109.
5
Department of Internal Medicine, University of Michigan, Ann Ar-
bor, Michigan 48109.
College of Pharmacy, University of Michigan, Ann Arbor, Michigan
6
4
8109.
7
0
To whom correspondence should be addressed. (e-mail: fmscg@ various solutions were incubated at 25 and 32°C for 24 h, with
puknet.puk.ac.za)
samples being taken at 1, 2, 4, 6, 8, and 24 h. The chemical
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1232