Method for pretreatment and method for analysis of lenalidomide in biological sample

Abstract

The present invention addresses the problem of providing a novel method for the pretreatment of a biological sample containing lenalidomide enantiomer and thereby establishing a simple and accurate method for the quantitative analysis of lenalidomide enantiomer. In the present invention, the racemization and decomposition of lenalidomide enantiomer in a biological sample can be prevented by the deproteinization under acidic conditions of the biological sample containing lenalidomide enantiomer, and the lenalidomide enantiomer can be simply and accurately quantitatively analyzed by subjecting to HPLC the biological sample that has been pretreated in such a way.

Claims

1. A method for quantifying enantiomers of lenalidomide in a biological sample, comprising pretreating a biological sample containing enantiomers of lenalidomide by deproteinizing the biological sample under acidic conditions to minimize racemization of the enantiomers of lenalidomide relative to a method where a biological sample containing enantiomers of lenalidomide is not deproteinized under acidic conditions; and separating and quantifying the enantiomers of lenalidomide by high-performance liquid chromatography of the biological sample containing enantiomers of lenalidomide that has been pretreated.

2. The method according to claim 1, wherein the acidic conditions are pH 5 or lower.

3. The method according to claim 1, wherein an acid selected from among perchloric acid, trichloroacetic acid, trifluoroacetic acid, metaphosphoric acid, hydrochloric acid, succinic acid and maleic acid is added.

4. The method according to claim 1, wherein the biological sample is blood, serum, blood plasma, urine, saliva, breast milk, spinal fluid, semen, tissue or microsomes.

5. The method according to claim 1, wherein the enantiomer of lenalidomide is the S-form.

6. The method according to claim 1, wherein the enantiomer of lenalidomide is the R-form.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a set of graphs showing the stability of lenalidomide stored at room temperature and at 50 C. for 24 hours, in different solvents. The solid black bars represent enantiomer 1, and the diagonally shaded bars represent enantiomer 2.

(2) FIG. 2 shows separation of lenalidomide enantiomers using CHIRALPAK (chiral chromatography column) IC (4.6 mm i.d.250 mm) as the optical resolution column, with ethyl acetate as the mobile phase.

(3) FIG. 3 shows purification of lenalidomide enantiomers with CHIRALPAK (chiral chromatography column) IC (20 mm i.d.250 mm) as the optical resolution column, and with ethyl acetate as the mobile phase. The enantiomer eluted in fraction 1 was designated as LL1, and the enantiomer eluted in fraction 2 was designated as LL2.

(4) FIG. 4 is a set of chromatograms of purified (fractionated) enantiomers in fraction 1 (A-1) and fraction 2 (B-1). CHIRALPAK (chiral chromatography column) IA was used for the analysis.

(5) FIG. 5 is a chromatogram showing S-thalidomide (a) and R-thalidomide (b).

(6) FIG. 6 shows time-dependent changes in the peak area of (S*)-LL and (R*)-LL after adding aqueous buffer at room temperature.

(7) FIG. 7 shows time-dependent changes in enantiomer excess in aqueous buffers (pH 4 to 9) at room temperature.

(8) FIG. 8 is a plot of log.sub.10(Enantiomer Excess) with respect to time, at room temperature.

(9) FIG. 9 is a pair of chromatograms of (a) LL enantiomer in water (control) and (b) LL enantiomer in human serum. Sample: (a) 0.1 mg/ml racemic LL in water, (b) 0.1 mg/ml racemic LL in human serum, (c) LL-free human serum. HclO.sub.4 was added immediately after dissolving the LL in serum or water. HPLC conditions: Column: CHIRALPAK (chiral chromatography column) IA (4.6 mm i.d.250 cm)+Security guard C8 (3.0 mm i.d.4 mm), flow rate: 0.75 mL/min, injection rate: 2 L.

(10) FIG. 10 is a set of chromatograms of (a) 0.01 mg/mL LL in Na-phosphorate buffer (pH 7.4), (b) 0.01 mg/mL LL in serum, and (c) LL-free blank serum.

(11) FIG. 11 is a chromatogram for human serum at 37 C., after adding 10 g/mL of (S*)-LL.

(12) FIG. 12 shows the racemization half-lives of LL and TD enantiomers in Na-phosphorate buffer (pH 7.4, diagonal-shaded bars) and human serum (solid black bars). The bar graphs represent the average values for 3 tests, and the error bars represent SD. The significant differences were **p<0.01 and ***p<0.001.

(13) FIG. 13 shows a scheme for preparing pure enantiomers of lenalidomide based on the results for stability and enantiomer separation of lenalidomide.

(14) FIG. 14 shows a scheme for elucidating the racemization half-life of lenalidomide and its pH dependence.

(15) FIG. 15(a) shows a conventional method of measuring thalidomide or lenalidomide in blood (serum and blood plasma) or urine using HPLC or measuring enantiomers of TD in biological samples.

(16) FIG. 15(b) shows an analysis method for the pretreatment method for lenalidomide based on the knowledge of enantiomer stability under acidic conditions demonstrated in Example 5.

(17) FIG. 16 shows a scheme in which the obtained pure enantiomers were used to evaluate the racemization rate of lenalidomide under biological buffer conditions (pH 7.4, 37 C.) and in human serum (37 C.).

DETAILED DESCRIPTION OF THE INVENTION

(18) Lenalidomide (LL) is a derivative of thalidomide (TD) which is known as a therapeutic agent for multiple myeloma, and it is widely used as an effective immunomodulating drug for different malignant blood diseases such as multiple myeloma. It has the chemical structure represented by the following structural formula, having an asymmetric center on a dioxopiperidine ring, similar to thalidomide.

(19) ##STR00001##

(20) When used in combination with dexamethasone, lenalidomide has a high success rate of 60% for recurrent intractable multiple myeloma, and it is receiving approval throughout the world, having been approved in about 50 countries so far, and has become a blockbuster drug with sales exceeding 400 billion yen. It was approved in 2010 in Japan, but with the condition of compliance with administrative procedures by medical personnel, patients and family, from the viewpoint of ensuring safety, as well as the requirement to investigate the merit of its use in all cases. Lenalidomide has a greater drug effect than thalidomide and fewer side-effects such as sleepiness and numbness, but also, like thalidomide, carries the risk of teratogenicity, and therefore its use is contraindicated for pregnancy. Because lenalidomide is a derivative of thalidomide, it is also expected to racemize rapidly in the blood in the same manner as thalidomide. It is therefore sold as an R-form:S-form=50:50 racemic mixture, and it is currently difficult to obtain pure enantiomers of lenalidomide. Furthermore, since analysis conditions for controlling racemization and accomplishing stable pretreatment and separation have not been established, virtually no data exist regarding the pharmacokinetic properties and racemization rate of pure enantiomers of lenalidomide.

(21) The present inventors have found that it is possible to separate and purify pure enantiomers of lenalidomide with controlled decomposition and racemization, by using an organic solvent selected from the group consisting of aprotic solvents, secondary alcohols and mixtures thereof as the mobile phase, for optical resolution by chromatography. According to one mode of the invention, therefore, there is provided a method for separating and purifying lenalidomide enantiomers, wherein a sample containing an enantiomeric mixture of lenalidomide is supplied for chromatography, and an organic solvent selected from the group consisting of aprotic solvents, secondary alcohols and mixtures thereof is used as the mobile phase for stable optical resolution of each enantiomer of lenalidomide from an enantiomeric mixture of lenalidomide.

(22) In the chromatographic method, a sample containing an enantiomeric mixture in a stationary phase supporting a compound with asymmetric discriminatory power (a chiral discriminator) is supplied together with the organic solvent as the mobile phase, adsorbing each enantiomer and utilizing their difference in retention time for optical resolution of each enantiomer. It is generally carried out using a high-performance liquid chromatography (HPLC) apparatus comprising an optical resolution column.

(23) An aprotic solvent to be used as the mobile phase is not particularly restricted so long as it allows the object of the invention to be achieved, and examples include esters such as acetonitrile and ethyl acetate, ketones such as acetone, ethers such as diethyl ether and diisopropyl ether, and combinations of the foregoing. Preferred are acetonitrile, ethyl acetate and combinations thereof.

(24) A secondary alcohol to be used as the mobile phase is not particularly restricted so long as it allows the object of the invention to be achieved, and examples include isopropanol, 2-butanol, cyclopentanol and cyclohexanol, and combinations of the foregoing. Isopropanol is preferred among these.

(25) The optical resolution column to be used for the method of the invention is not particularly restricted so long as it allows the object of the invention to be achieved, and it may be a normal-phase column or reversed-phase column, or a separation mode column or a multimode column comprising a combination of these. A polysaccharide derivative chiral column will typically be used. A polysaccharide derivative chiral column is a column in which a polysaccharide derivative as the chiral discriminator is immobilized on a support. The polysaccharide derivative supported on the polysaccharide derivative chiral column may be an amylose derivative or cellulose derivative, for example. According to the invention, it is preferred to use CHIRALPAK (chiral chromatography column) IA or CHIRALPAK (chiral chromatography column) IC.

(26) The optical resolution method of the invention allows pure enantiomers of lenalidomide to be separated and purified from an enantiomeric mixture (such as a racemic mixture) of lenalidomide, without decomposition or racemization.

(27) When pure enantiomers of lenalidomide in a biological sample are to be separated and purified, the macromolecules such as proteins that can lower the low-molecular separation efficiency are generally removed beforehand. Pretreatment of thalidomide has conventionally been carried out by extracting a thalidomide-containing biological sample with a hydrophobic organic solvent (such as an n-hexane/ethyl acetate mixture), either once or several times, drying the obtained organic solvent layer, and then redissolving it in an organic solvent such as dioxane. Lenalidomide, however, has higher polarity than thalidomide and lower solubility in the organic solvent layer, such that the conventional method has had poor efficiency and has been inadequate. In order to solve this problem, the present inventors conducted much diligent research on the effect of pH on racemization of lenalidomide enantiomers, and as a result have found that enantiomers of lenalidomide are highly stable under acidic conditions. According to another mode of the invention, therefore, there is provided a method for pretreatment of a biological sample containing enantiomers of lenalidomide, wherein the biological sample is deproteinized under acidic conditions.

(28) Such acidic conditions are typically pH 5 or lower, preferably in the range of pH 2 to pH 5, and most preferably in the range of pH 4 to pH 5.

(29) The acid to be added to the sample to produce the acidic conditions is not particularly restricted so long as it allows the object of the invention to be achieved, and may be perchloric acid, trichloroacetic acid, trifluoroacetic acid, metaphosphoric acid or hydrochloric acid, or a dicarboxylic acid such as succinic acid or maleic acid.

(30) The biological sample is not particularly restricted so long as it contains lenalidomide, and examples include blood, serum, blood plasma, urine, saliva, breast milk, sweat, spinal fluid, semen, tissue and microsomes.

(31) By supplying a pretreated biological sample containing lenalidomide enantiomers by chromatography, and preferably high-performance liquid chromatography (HPLC), it is possible to conveniently and efficiently separate and quantify enantiomers of lenalidomide in the biological sample.

(32) Moreover, by keeping the fractionated lenalidomide enantiomers in an aqueous buffer under acidic conditions, it is possible to store the lenalidomide enantiomers. Such acidic conditions are typically pH 5 or lower, and preferably in the range of pH 2 to pH 5. The aqueous buffer is not particularly restricted so long as it allows the acidic conditions to be maintained, and examples include citrate buffer, phosphate buffer, acetate buffer, glycine-hydrochloride buffer, MES-HEPES buffer, Tris buffer and borate buffer.

EXAMPLES

Example 1. Stability of Lenalidomide in Different Solvents

(33) A racemic mixture of 0.5 mg/mL lenalidomide in methanol, ethanol, isopropanol, acetonitrile or ethyl acetate, as the sample (product of Selleck Chemicals, US) was incubated at room temperature or at 50 C. for 24 hours, and the following HPLC conditions were employed for quantification of the lenalidomide enantiomers, for evaluation of the stability of lenalidomide in the different solvents.

(34) HPLC conditions

(35) Apparatus: Nanospace SI-2 Series (Shiseido Corp.)

(36) Column: CHIRALPAK (chiral chromatography column) IA (4.6250 cm, 5 m, product of Dicel), RT

(37) Mobile phase: EtOH (100%)

(38) Flow rate: 1.0 mL/min

(39) Injection volume: 5 L, Detection: UV 230 nm.

(40) The peak areas for the enantiomers before and after incubation are shown in FIG. 1 (the initially observed peaks are enantiomer 1, and the next observed peaks are enantiomer 2). Lenalidomide was highly unstable in methanol and ethanol, but no decomposition was observed in acetonitrile and ethyl acetate. Lenalidomide was also stable even in isopropanol. Considering these results, it is thought that decomposition of lenalidomide is accelerated by the weak basicity of the alcohol that attacks the -carbon atom of the carbonyl group. This does not contradict the results indicating stability of lenalidomide in acetonitrile and ethyl acetate, which are aprotic solvents.

Example 2. Separation of Lenalidomide Enantiomers Using Different Solvents

(41) In order to elucidate the racemization rate of enantiomers of lenalidomide it is very important to separate and quantify the pure enantiomers of lenalidomide. As demonstrated in Example 1, lenalidomide in an alcohol (especially methanol and ethanol) or water (>pH 7) solvent is unstable, and therefore a highly stable aprotic solvent should be used as the mobile phase or sample solvent.

(42) Separation of enantiomers of lenalidomide (LL) and thalidomide (TD) was tested using the different organic solvents listed in Table 1 as the mobile phase, under the following HPLC conditions. The samples used were 0.5 mg/mL LL (Wako Pure Chemical Industries, Ltd.) and TD (Sigma-Aldrich Japan, KK.) dissolved in acetonitrile.

(43) HPLC conditions

(44) Apparatus: Nanospace SI-2 Series (Shiseido Corp.)

(45) Column: CHIRALPAK (chiral chromatography column) IC (4.6250 cm, 5 m, product of Dicel), RT

(46) Mobile phase: Solvent listed in Table 1

(47) Flow rate: 1.0 mL/min

(48) Injection volume: 5 L

(49) Detection: UV 230 nm or 254 nm.

(50) The mobile phase used was an aprotic solvent comprising acetonitrile (ACN), ethyl acetate (EtOAc), tetrahydrofuran (THF) and t-butyl methyl ether (BME). Isopropanol (IPA) is a secondary alcohol, and it was also tested because it has excellent stability, as shown in FIG. 1. The enantiomer separating power was evaluated using the separation factor (RS), defined by the following formula.
RS=2(T.sub.2T.sub.1)/(W.sub.1+W.sub.2)

(51) In the formula, numeral 1 and numeral 2 refer to enantiomer 1 and enantiomer 2, respectively (peak 1 eluting before peak 2), T represents the retention time, and W represents the peak width. Table 1 shows the separation of LL enantiomers when using different solvents as the mobile phase.

(52) TABLE-US-00001 TABLE 1 Enantiomer separation using CHIRALPAK (chiral chromatography colunm) IC with different inert mobile phases Method Mobile phase composition (%) Separation No. ACN EtOAc THF IPA tBuOMe LL TD 01 100 7.26 2.51 02 100 12.51 0.59 03 100 2.93 NS 04 75 25 6.33 1.08 05 75 25 2.05 NS 06 75 25 4.78 NS 07 50 50 6.40 NS 08 50 50 NS NS 09 50 50 4.51 NS 10 50 50 4.90 NS 11 50 50 NS NS 12 50 25 25 4.04 NS 13 50 50 1.53 NS 14 50 25 25 2.45 1.43 15 25 75 7.62 1.84 16 75 25 8.24 1.35 17 75 25 10.11 NS 18 100 NS NS NS: No separation

(53) Among these mobile phases, the most effective ones for separation of LL enantiomers was ethyl acetate, the highest separation being obtained when using ethyl acetate as the mobile phase (Table 1, No. 02) (RS=12.51). FIG. 2 shows that LL enantiomers were satisfactorily separated when using ethyl acetate as the mobile phase. Moreover, using a mobile phase with a fixed amount of ethyl acetate mixed exhibited more excellent separation than when no ethyl acetate was present or the amount was very low (for example, No. 3, 10, 11 and 17). These results demonstrated that ethyl acetate is a highly superior solvent as the mobile phase in chromatography for LL enantiomer separation.

(54) Acetonitrile also exhibited satisfactory separation (Table 1, No. 01, RS=7.26). Since acetonitrile also exhibits excellent stability as shown in FIG. 1, it can serve as a satisfactory solvent for the mobile phase in chromatography for separation of LL enantiomers.

(55) For TD, on the other hand, sufficient separation was not observed when using any solvent as the mobile phase.

Example 3. Preparation of Pure Enantiomers of Lenalidomide

(56) A method for preparing pure enantiomers of lenalidomide was established, based on the results for stability and enantiomer separation of lenalidomide. Specifically, pure enantiomers of lenalidomide were prepared according to the scheme in FIG. 13 and the following HPLC conditions.

(57) HPLC conditions

(58) Apparatus: HPD PUMP and LAMBDA1010 (Bischoff Co.)

(59) Column: CHIRALPAK (chiral chromatography column) IC (2.0 mm i.d.250 cm, 5 m, product of Dicel), RT

(60) Mobile phase: Ethyl acetate

(61) Flow rate: 10 mL/min

(62) Injection volume: 2.3 L/mL LL racemic mixture in 5000 L of acetonitrile

(63) Detection: UV 254 nm.

(64) As shown in FIG. 3, the LL enantiomers were completely separated. After separating off the fractions and drying them, over 80 mg of the pure enantiomers was obtained from 180 mg of the racemic mixture. The fractions were dissolved in acetonitrile (1 or 0.1 mg/mL) and stored at 25 C. Based on the integral peak areas in the chromatograms shown in FIG. 4 (A-2, B-2), fraction 1 and fraction 2 recovered as lenalidomide enantiomer 1 (LL1) and lenalidomide enantiomer 2 (LL2) exhibited sufficient enantiomer purities of 99.02% and 99.96%, respectively.

Example 4. Inference of Absolute Configurations of LL1 and LL2 from Elution Order

(65) The absolute configurations of LL1 and LL2 can be reasonably inferred by comparing the order of elution of LL1 and LL2 with commercially available R-thalidomide and S-thalidomide (Sigma-Aldrich Japan, KK.). The order of elution was confirmed with the following HPLC conditions.

(66) HPLC conditions

(67) Apparatus: Nanospace SI-2 Series (Shiseido Corp.)

(68) Column: CHIRALPAK (chiral chromatography column) IA (4.6 mm i.d.250 cm, 5 m, product of Dicel), RT

(69) Mobile phase: 0.1% Formic acid in EtOH/H.sub.2O (95/5, v/v)

(70) Flow rate: 0.75 mL/min

(71) Injection volume: 5 L of 0.1 mg/mL (S)-TD, (R)-TD

(72) Detection: UV 230 nm.

(73) As clearly seen from FIG. 5, S-thalidomide eluted earlier than R-thalidomide. Considering that LL1 elutes earlier than LL2 under the same HPLC conditions, presumably the absolute configuration of LL1 corresponds to the S-enantiomer while the absolute configuration of LL2 corresponds to the R-enantiomer (hereunder, LL1 and

(74) LL2 will be defined as S*-LL and R*-LL).

Example 5. Dependence of Racemization Half-Life on pH

(75) In order to elucidate the racemization half-life of lenalidomide and its pH dependence, an experiment was conducted with the scheme in FIG. 14 and the following HPLC conditions.

(76) HPLC conditions

(77) Apparatus: Nanospace SI-2 Series (Shiseido Corp.)

(78) Column: CHIRALPAK (chiral chromatography column) IA (4.6 mm i.d.250 cm, 5 m, product of Dicel), RT

(79) Mobile phase: 0.1% Formic acid in EtOH/H.sub.2O (95/5, v/v)

(80) Flow rate: 0.75 mL/min

(81) Injection: 5 L,

(82) Detection: UV 230 nm.

(83) The variation in (S*)-LL at different pH is shown in FIG. 6. A small amount of (R*)-LL was produced with incubation at pH 6, while racemization was significantly accelerated at pH 7 and higher. Since the peak areas of (S*)- and (R*)-LL were reduced at pH 9 and 8, it was concluded that racemization and decomposition of LL takes place under these pH conditions.

(84) The enantiomeric excess rate (EE, [(SR)/(S+R)100], %) at each point is plotted in FIG. 7, and the log.sub.10(EE) vs. time plot is shown in FIG. 8. As reported for thalidomide, since racemization is thought to be a pseudo-first-order reaction, FIG. 8 shows an approximately ideal straight line.

(85) This linearity was used to calculate the time for EE=50 based on a straight line approximation (y=ax+b), to determine the racemization half-life. The stability of the pure enantiomer is very highly dependent on pH, the half-life being 10 times longer for every pH reduction of 1, and estimation of the half-life was no longer possible at pH 4. At pH 9, on the other hand, sudden racemization and decomposition were observed. These results clearly indicate that enantiomers of lenalidomide are stable under acidic conditions (<pH 4).

Example 6. Establishing Biological Sample Pretreatment Method

(86) Numerous reports already exist of measuring thalidomide or lenalidomide in blood (serum and blood plasma) or urine using HPLC. Measurement of enantiomers of TD in biological samples has been reported, and the racemization half-life has been determined both in vivo and in vitro (Eriksson T et al., Chirality., 1995, 7(1), p.44-52 and Knoche B et al., J. Chromatogr. A., 1994, 666, p.235-240). These measurements are all based on liquid-liquid extraction methods. The conventional method is shown as FIG. 15(a). Measurement is possible by this method because an aprotic solvent is essentially inert to racemization or decomposition. However, since the solubility of thalidomide or lenalidomide is not high in hydrophobic organic solvents, it is necessary to use a large amount of solvent and carry out repeated extraction.

(87) A very simple and efficient pretreatment method for lenalidomide was therefore established, based on the knowledge of enantiomer stability under acidic conditions demonstrated in Example 5. The analysis method of the invention is shown as FIG. 15(b). The acid used to suspend the reaction while simultaneously precipitating and removing the protein was HClO.sub.4.

(88) FIG. 9 shows chromatograms obtained using the method of FIG. 15(b). As shown in FIG. 9(b), lenalidomide enantiomers in serum can be satisfactorily separated and quantified without being affected by contaminating components such as proteins, and therefore the method can be applied to biological samples as well.

Example 7. Racemization of Lenalidomide Under Physiological Conditions

(89) The obtained pure enantiomers were used to evaluate the racemization rate of lenalidomide under biological buffer conditions (pH 7.4, 37 C.) and in human serum (37 C.), by the scheme in FIG. 16.

(90) HPLC conditions

(91) Apparatus: Nanospace SI-2 Series (Shiseido Corp.)

(92) Column: CHIRALPAK (chiral chromatography column) IA (4.6 mm i.d.250 cm, 5 m)+Security guard C8 (3.0 mm i.d.4 mm)

(93) Temperature: 40 C. Mobile phase: 0.1% Formic acid in EtOH/H.sub.2O (95/5, v/v)

(94) Flow rate: 0.75 mL/min

(95) Injection: 20 L

(96) Detection: UV 230 nm.

(97) As clearly seen from the chromatogram for (S*)-LL shown in FIG. 10 [0.01 mg/mL (a) in buffer, (b) in serum], the method of the invention has sufficient retention and separation and detection sensitivity (signal-to-noise ratio). The blank sample (c) derived from serum alone without LL contained no contaminants that inhibit measurement of LL in the method of the invention. The peak area for (S*)-LL in FIG. 10(b) was 91% of FIG. 10(a). This indicates an adequate recovery rate for pretreatment of lenalidomide in serum.

(98) FIG. 11 shows continuous change in the chromatogram for incubation of S*-LL. The LL enantiomers were separated and quantified to a sufficient level for detection of racemization product, without being inhibited by other substances in the sample.

(99) The racemization half-life was calculated by the same method described in Example 5. The racemization half-life in aqueous buffer was estimated from this, as follows: (S*)-LL, (R*)-LL, (S)-TD and (R)-TD=2721.3, 2671.1, 26610 and 29524 (min), respectively. These results demonstrated that at pH 7.4, LL enantiomers have a similar half-life as TD in aqueous buffer.

(100) The half-life in human serum was calculated in the same manner: (S*)-LL, (R*)-LL, (S)-TD and (R)-TD=1314.9, 1092.0, 21.81.0 and 32.925 (min), respectively. The results are shown in FIG. 12. Thalidomide in human serum has a half-life of only 8.2% (S-form) and 11.1% (R-form) compared to buffer, indicating that racemization of thalidomide is extremely accelerated in serum. It is believed that the accelerated racemization for these enantiomers is due to the biomolecules including proteins such as human albumin in serum.

(101) For LL in serum, the results showed that (S*)-LL has a racemization half-life of 6 times longer than (S)-TD, while (R*)-LL has a racemization half-life of 3 times longer than (R)-TD. These results clearly indicate that the LL enantiomers, and especially (S*)-LL, are much more stable compared to thalidomide. In contrast to TD, (S*)-LL was more stable than (R*)-LL. This difference in the half-lives of the S- and R-enantiomers should also be noted.