- Original Articles
- Open Access
Selection of Novel Analogs of Thalidomide with Enhanced Tumor Necrosis Factor α Inhibitory Activity
© Molecular Medicine 1996
- Published: 1 July 1996
Tumor necrosis factor α (TNFα) is thought to mediate both protective and detrimental manifestations of the inflammatory response. Recently, thalidomide (α-n–phthalimidoglutarimide) was shown to partially inhibit monocyte TNFα production (by 50–70%) both in vivo and in vitro. More efficient inhibition of TNFα may, however, be necessary to rescue the host from more acute and extensive toxicities of TNFα-mediated inflammation.
Materials and Methods
Three structural analogues of thalidomide were selected for study based on increased activity against TNFα production. The parent drug and the analogs were tested in vitro in human peripheral blood mononuclear cell cultures for their effects on lipopolysaccharide (LPS) induced cytokine protein and mRNA production using ELISAs and Northern blot hybridization. The in vitro effects of the drugs were then confirmed in vivo in a mouse model of LPS induced lethality.
The new compounds (two esters and one amide) showed increased inhibition of TNFα production by LPS-stimulated human monocytes, relative to the parent drug thalidomide. The analogs and the parent drug enhanced the production of interleukin 10 (IL-10), but had little effect on IL-6 and IL-1β protein and mRNA production. When tested in vivo, the amide analog protected 80% of LPS-treated mice against death from endotoxin induced shock.
Analogs of thalidomide designed to better inhibit TNFα production in vitro have correspondingly greater efficacy in vivo. These finding may have therapeutic implication for the treatment of human diseases characterized by acute and extensive TNFα production such as tuberculous meningitis or toxic shock.
Thalidomide was initially used as a sedative without knowledge of its mechanism of action, its immunoregulatory properties, or its teratogenic activity (1). Recent studies have shown that, in addition to its other known effects, thalidomide is an inhibitor of tumor necrosis factor α (TNFα) production by monocytes in vitro (2,3). When leprosy patients with erythema nodosum leprosum (ENL) are treated with thalidomide, there is an inhibition of TNFα production in vivo as well as a delayed reduction in interferon-γ (IFNγ) serum levels (4). In patients with tuberculosis, thalidomide treatment lowers TNFα protein and TNFα mRNA production and also reduces the levels of interleukin 1β (IL-1β) mRNA in peripheral blood mononuclear cells (5). Thalidomide appears to inhibit TNFα production by enhancing the degradation of TNFα mRNA (3). However, thalidomide inhibits only 50–70% of the TNFα mRNA and TNFα protein produced by monocytes. This may be in part attributed to the instability of the drug in aqueous solution. Thalidomide undergoes rapid degradation at physiological pH (6) through the hydrolysis of the glutarimide ring to generate either a n-phthaloyl substituted glycinamide or a n-phthaloyl substituted γ-aminobutamide (7).
Although TNFα production is important in the host defense against infection, the accompanying toxicities associated with increasing levels of TNFα and other cytokines may lead to serious pathology in the host. For example in experimental models of endotoxin induced shock, TNFα production as well as the generation of other pro-inflammatory cytokines has been shown to be associated with the rapid onset of multiorgan failure and death. Reversal of shock is dependent on, among other interventions, complete inhibition of production of cytokines such as TNFα (8,9). Selective inhibitors of TNFα would therefore be useful in this situation. Since the efficiency of thalidomide in inhibition of TNFα production may depend on its stability in aqueous solution, we designed and synthesized a series of thalidomide analogs with improved activity and stability (10). We then selected three of the structural analogs which had superior TNFα inhibitory activity in preliminary experiments. These compounds contained the phthaloyl ring in which the glutarimide moiety was replaced. The resulting analogs, one amide and two esters, were evaluated for their ability to inhibit and or increase monocyte cytokine production. The analogs were also compared with thalidomide in respect to cytokine mRNA accumulation in lipopolysaccharide (LPS)-stimulated human monocytes in vitro and evaluated in vivo for efficacy in a mouse model of LPS-induced death.
Peripheral blood mononuclear cells (PBMC) from normal donors were obtained by Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, NJ, U.S.A.) density centrifugation (2). Monocytes were enriched by incubation of PBMC with neuraminidase-treated (Vibrio cholera neuraminidase; Calbiochem-Behring Corp., La Jolla, CA, U.S.A.) sheep erythrocytes (Cocalico Biologicals, Reamstown, PA, U.S.A.) for 1 hr at 4°C and separated by Ficoll gradient centrifugation. Cells (106 cells/ml) were cultured in RPMI (Gibco Laboratories, Grand Island, NY, U.S.A.) supplemented with 10 AB+ serum (Biocell, Rancho Dominguez, CA, U.S.A.), 2 mM l-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Gibco).
In Vitro Cytokine Induction
Monocytes were stimulated with 1 µg/ml of LPS prepared from Salmonella minnesota R595 as previously described (2) (List Biological Labs, Campbell, CA, U.S.A.). Triplicate cultures were incubated with LPS for 18–20 hr at 37°C in 5% CO2. Supernatants were then harvested and assayed for cytokines levels. In some experiments, supernatants were kept frozen at −70°C until assay.
In Vitro Cytokine Inhibition
Thalidomide and analogs were synthesized (Celgene Corporation, Warren, NJ, U.S.A.) as described (10). The drugs were dissolved in DMSO (Sigma Chemical, St Louis, MO, U.S.A.); further dilutions were done in culture medium as previously described (3). The final DMSO concentration in all assays was 0.25%. Drugs were added to cell 1 hr before LPS stimulation. The toxicity of the compounds for the cells was assayed by trypan blue exclusion dye method, immediately after harvesting of the culture supernatant.
The concentration of the cytokines TNFα, IL-1β, IL-6, granulocyte macrophage-colony-stimulating factor (GM-CSF), and IL-10 in culture supernatants was determined by commercial enzyme-linked immunosorbent assay (ELISA) kits (Endogen, Boston, MA, U.S.A.) according to the manufacturer’s directions. The concentration of the cytokines TNFα and IL-10 in mouse serum was determined similarly by commercial ELISA kits (Endogen). Cytokine levels are expressed as ng/ml. Percentage inhibition was determined as 100 × [1 − (cytokine experimental/cytokine control)].
Northern Blot Hybridization
Cells were treated with thalidomide or analogs and stimulated with LPS for 6 hr, and total cellular RNA was extracted using RNAzol (Cina/Biotecx Lab. Inc., Houston, TX, U.S.A.). RNA was size fractionated by electrophoresis in 1% formaldehyde/agarose gel. RNA was transferred overnight onto nylon membranes (BioRad Labs., Richmond, CA, U.S.A.). Membranes were hybridized for TNFα (1.1-kb PstI fragment), IL-1β (0.6-kb BamHI + SmaI fragment), IL-6 (1.0-kb EcoRI fragment), and β-actin (1.4-kb EcoRI/XhoI fragment) (ATCC, Rockville, MD, U.S.A.). Filters were washed as previously described (3). Blots were exposed to X-ray film for 2 to 24 hr at −70°C. Densitometry of the Northern blots were done using a phosphorimager (Molecular Dynamics, Sunnyvale, CA, U.S.A.). The density units for the cytokines mRNA were normalized to the density for β-actin mRNA.
Human monocytes were stimulated with LPS (1 µg/ml) for 18–20 hr in culture in the presence of anti-IL-10 neutralizing antibody (10 µg/ml) or control IgG (10 µg/ml) (Endogen). Thalidomide or thalidomide analogs were added at varying concentrations 1 hr prior to LPS stimulation of the cells. Supernatants were harvested and assayed for TNFα levels by ELISA as described above.
Female BALB/c mice, 7–9 weeks old, weighing 18–20 g, were obtained from Charles River Laboratories (Wilmington, MA, U.S.A.) and housed under pathogen-free conditions.
Drug Stability in Human Plasma
Human plasma (Sigma) was spiked with thalidomide or analogs and kept at 37°C. Samples were taken every 30 min, and the levels of the drugs were determined by HPLC as described (11).
In Vivo Cytokine Induction
Cytokines production in the circulation of mice was induced by administration of Salmonella abortus equi-derived LPS (Sigma) at 5 mg/kg. LPS was injected intraperitoneally (i.p.) in a volume of 200 µl of PBS/mouse. Blood was collected by cardiac puncture under anesthesia 1.5 hr after LPS injection. Blood samples were allowed to clot at room temperature for 2 hr and centrifuged. Sera were collected, immediately frozen, and kept at −70°C. Cytokine levels in serum were determined by ELISA as described above.
In Vivo Cytokine Inhibition
Thalidomide and analogs were suspended in sterile saline and administered by i.p. injection (0.5 ml/mouse) 2 hours before LPS challenge. Control mice were injected with the same volume of vehicle.
LPS-Induced Death and Protection Studies
For the lethality experiments, an LD100 dose of LPS (7.5 mg/kg) was injected i.p. in a volume of 200 µl PBS. For the protection studies, drugs were given i.p. at a single dose of 100 mg/kg in 0.5 ml of saline, 2 hr before LPS injection. Survival of treated mice was assessed two to three times a day. A minimum of 10 animals per group was used in each experiment.
Student’s t test was used to determine statistical significance and p values of <0.05 were considered significant.
Selection of Thalidomide Analogs with Enhanced Capacity to Inhibit TNFα Production in Vitro
Dose-response curves for CC-1069, CC-1104, and CC-1115 are the standard sigmoidal curves seen for classical pharmacological antagonists compared with the dose-response curve for thalidomide, which shows a partial antagonist type dose-dependent inhibition (Fig. 2A). Although thalidomide inhibition of TNFα production never reaches 100% (even at concentrations up to 780 µM; not shown), all three analogs completely inhibited TNFα production (Fig. 2A). Treatment of the cells at this range of concentrations with the parent drug or with any of the analogs did not affect cell viability as assayed by trypan blue exclusion.
In our experiments, TNFα released by LPS-stimulated cells was usually monitored after about 20 hr of incubation with the drugs. However, inhibition of TNFα production by thalidomide (2) and its analogs could be documented as early as 2 hr after LPS stimulation throughout the 20 hr incubation period (Fig. 2B).
Effect of Thalidomide and the Analogs on LPS-Induced Cytokine Production in Vitro
Effect of thalidomide and analogs on cytokine production by LPS-stimulated human monocytes
LPS (1 µg/ml) +:
0.25% DMSO (control)
6.1 ± 2.0 ng/ml
0.4 ± 0.4 ng/ml
23.8 ± 7.0 ng/ml
540 ± 11 ng/ml
1.9 ± 1.1 ng/ml
Thalidomide (194 µM)
49 ± 4.0%a,b
86 ± 5.50%
86 ± 20.3%
100 ± 13.5%
149 ± 26.0%b
CC-1069 (12.5 µM)
40 ± 5.9%b
56 ± 1.50%b
77 ± 4.60%b
97 ± 5.60%
130 ± 16.4%
CC-1104 (2.7 µM)
50 ± 8.6%b
62 ± 13.9%
91 ± 13.9%
88 ± 7.00%
140 ± 21.8%
CC-1115 (0.5 µM)
44 ± 8.9%b
25 ± 0.90%b
99 ± 10.9%
102 ± 8.60%
160 ± 14.9%b
We next studied the kinetics of IL-10 and TNFα production in human monocytes in vitro. Production of the two cytokines in response to LPS stimulation of human monocytes followed different kinetics (Fig. 3, insert) confirming previously reported results (12). TNFα was measurable in the culture supernatant as early as 2 hr after LPS stimulation and continued to accumulate in the supernatant for up to 12 hr and then the concentration waned. In contrast, IL-10 first appeared in the culture supernatant at 6 hr after LPS stimulation and peaked at 18 hr (Fig. 3, insert). These observations taken together suggest that thalidomide and its analogs directly inhibit TNFα production, rather than acting via the induction of IL-10.
Effect of Thalidomide and Its Analogs on Cytokine mRNA Expression in Vitro
Effect of Thalidomide and Thalidomide Analogs on TNFα and IL-10 Production in Vivo
Effect of thalidomide and analogs on serum cytokine levels in LPS treated mice
Cytokine Level (ng/ml)
LPS (5 mg/kg) +:
4.5 ± 1.5
19.9 ± 5.1
2.2 ± 0.7a
25.6 ± 5.6
2.1 ± 0.8a
25.3 ± 5.6
1.7 ± 0.5a
32.4 ± 4.8a
3.1 ± 1.0
25.7 ± 2.1
1.5 ± 0.2a
28.7 ± 4.1a
0.6 ± 0.3a
44.8 ± 5.6a
4.9 ± 0.8
13.0 ± 0.3
3.2 ± 0.3
16.7 ± 3.8
1.9 ± 0.4a
17.9 ± 2.3
2.7 ± 0.9
26.6 ± 2.8
2.7 ± 0.4
29.9 ± 2.5a
1.6 ± 0.4a
32.4 ± 2.4a
Stability of thalidomide and analogs in human plasma
Half-Lives (t1/2) of Compounds (hr)
1.52 ± 0.11
7.80 ± 0.66
2.57 ± 0.53
3.77 ± 0.32
To determine the relative stability of thalidomide and the three analogs in vivo, the drugs were evaluated for half-life in human plasma. The three thalidomide analogs showed improved stability in human plasma over the parent drug (Table 3). While thalidomide had a half-life (t1/2) of less than 2 hr under these assay conditions, the analogs had longer t1/2. Analog CC-1069 was the most stable with a t1/2 of 8 hr in human plasma (Table 3). This improved stability of CC-1069 in human plasma could be due to the fact that CC-1069 is an amide while the other analogs are esters and may therefore be more readily degraded by plasma esterases.
Effect of Thalidomide and CC-1069 on the Survival of Mice Treated with a Lethal Dose of LPS
Since CC-1069 was both most active at modulating cytokine levels in vivo and more stable in plasma, we tested its efficacy in protecting mice from death due to LPS-induced shock.
It has long been known that reducing the levels of TNFα and other cytokines during an inflammatory episode can improve clinical outcome by ameliorating the toxic effects of the inflammatory cascade (13). Strategies for the design of optimal TNFα inhibitors aim at the synthesis of drugs which are effective at low doses, nontoxic, and specific enough to modulate the inflammatory response without shutting down the protective immune response. Recently one drug, thalidomide, has been shown to selectively, albeit partially, inhibit TNFα production in vitro. Following treatment with thalidomide, we have observed a decrease in TNFα and other cytokines, and a clinical improvement in leprosy (ENL) patients (4) and tuberculosis patients (5), in the absence of significant toxicities. We have also observed partial inhibition of TNFα release as well as only partial reduction in inflammatory manifestations in the central nervous system following administration of thalidomide in rabbits with bacterial meningitis (14). To obtain drugs which are more efficient than thalidomide and retain the specificity of the parent drug and will therefore not be immune suppressive, we have modified the parent drug and examined some of the resulting compounds for the desired profile of activities.
In this report, we describe three new drugs with significantly improved (up to 400-fold) capacity to inhibit TNFα production in vitro. In addition to the effect on TNFα production, the new compounds along with thalidomide produced an enhancement in the production of IL-10 in LPS-stimulated cells. IL-10 is known to suppress the production of TNFα by human and mouse cells (15,16). TNFα inhibition by these compounds, however, was not mediated by IL-10 since comparable inhibition was found in the presence of neutralizing IL-10 antibodies. The enhancement of IL-10 production by LPS-stimulated cells in association with TNFα inhibition has been reported previously in LPS-stimulated macrophages in vitro (16,17). TNFα inhibition and IL-10 enhancement by thalidomide and analogs was also observed in vivo. The increased production of IL-10 may be partially responsible for the protection from LPS lethality achieved by these drugs since IL-10 neutralization is associated with increased lethality in endotoxemia (18,19). The ability of CC-1069 to protect mice from death due to LPS-induced shock suggests that this class of compounds might be useful in similar clinical situations in humans. Since the potent teratogenic effect of thalidomide is well documented, the analogs are now being tested for their teratogenic potential. Preliminary results are exciting because several analogs appear to be less teratogenic than the parent compound, thalidomide.
Although the focus of the present study was on identifying molecules that inhibit TNFα production more efficiently than thalidomide, it is obvious from these studies, as well as from other published studies (20), that thalidomide and possibly some of its analogs are pleiotropic in their effects. Thalidomide has been shown to act as a teratogen and as a sedative, and also is capable of inhibiting angiogenesis (1,21). It is not clear that these varied physiologic effects are mediated by a common mechanism. The separation of the different effects of thalidomide in the different physiologic situations is a central focus of the ongoing rational drug design program. This should enable us to design drugs that retain the positive effects and eliminate or minimize toxicities and side effects.
This study was supported in part by U.S. Public Health Service Grant AI-33124 and the Celgene Corporation. Andre Luis Moreira is a Villares Fellow. We would like to thank Marguerite Nulty for secretarial help, Judy Adams for helping in preparing the figures, and Dr. Victoria Freedman for critical and helpful discussions during the preparation of this manuscript.
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