Apremilast is a selective PDE4 inhibitor with regulatory effects on innate immunity.

Apremilast, an oral small molecule inhibitor of phosphodiesterase 4 (PDE4), is in development for chronic inflammatory disorders, and has shown efficacy in psoriasis, psoriatic arthropathies, and Behçet's syndrome. In March 2014, the US Food and Drug Administration approved apremilast for the treatment of adult patients with active psoriatic arthritis. The properties of apremilast were evaluated to determine its specificity, effects on intracellular signaling, gene and protein expression, and in vivo pharmacology using models of innate and adaptive immunity. Apremilast inhibited PDE4 isoforms from all four sub-families (A1A, B1, B2, C1, and D2), with IC50 values in the range of 10 to 100 nM. Apremilast did not significantly inhibit other PDEs, kinases, enzymes, or receptors. While both apremilast and thalidomide share a phthalimide ring structure, apremilast lacks the glutarimide ring and thus fails to bind to cereblon, the target of thalidomide action. In monocytes and T cells, apremilast elevated intracellular cAMP and induced phosphorylation of the protein kinase A substrates CREB and activating transcription factor-1 while inhibiting NF-κB transcriptional activity, resulting in both up- and down-regulation of several genes induced via TLR4. Apremilast reduced interferon-α production by plasmacytoid dendritic cells and inhibited T-cell cytokine production, but had little effect on B-cell immunoglobulin secretion. In a transgenic T-cell and B-cell transfer murine model, apremilast (5mg/kg/day p.o.) did not affect clonal expansion of either T or B cells and had little or no effect on their expression of activation markers. The effect of apremilast on innate immunity was tested in the ferret lung neutrophilia model, which allows monitoring of the known PDE4 inhibitor gastrointestinal side effects (nausea and vomiting). Apremilast significantly inhibited lung neutrophilia at 1mg/kg, but did not induce significant emetic reflexes at doses <30 mg/kg. Overall, the pharmacological effects of apremilast are consistent with those of a targeted PDE4 inhibitor, with selective effects on innate immune responses and a wide therapeutic index compared to its gastrointestinal side effects.

Apremilast, a cAMP phosphodiesterase-4 inhibitor, demonstrates anti-inflammatory activity in vitro and in a model of psoriasis.

BACKGROUND AND PURPOSE: Apremilast is an orally administered phosphodiesterase-4 inhibitor, currently in phase 2 clinical studies of psoriasis and other chronic inflammatory diseases. The inhibitory effects of apremilast on pro-inflammatory responses of human primary peripheral blood mononuclear cells (PBMC), polymorphonuclear cells, natural killer (NK) cells and epidermal keratinocytes were explored in vitro, and in a preclinical model of psoriasis. EXPERIMENTAL APPROACH: Apremilast was tested in vitro against endotoxin- and superantigen-stimulated PBMC, bacterial peptide and zymosan-stimulated polymorphonuclear cells, immunonoglobulin and cytokine-stimulated NK cells, and ultraviolet B light-activated keratinocytes. Apremilast was orally administered to beige-severe combined immunodeficient mice, xenotransplanted with normal human skin and triggered with human psoriatic NK cells. Epidermal skin thickness, proliferation index and inflammation markers were analysed. KEY RESULTS: Apremilast inhibited PBMC production of the chemokines CXCL9 and CXCL10, cytokines interferon-gamma and tumour necrosis factor (TNF)-alpha, and interleukins (IL)-2, IL-12 and IL-23. Production of TNF-alpha by NK cells and keratinocytes was also inhibited. In vivo, apremilast significantly reduced epidermal thickness and proliferation, decreased the general histopathological appearance of psoriasiform features and reduced expression of TNF-alpha, human leukocyte antigen-DR and intercellular adhesion molecule-1 in the lesioned skin. CONCLUSIONS AND IMPLICATIONS: Apremilast displayed a broad pattern of anti-inflammatory activity in a variety of cell types and decreased the incidence and severity of a psoriasiform response in vivo. Inhibition of TNF-alpha, IL-12 and IL-23 production, as well as NK and keratinocyte responses by this phosphodiesterase-4 inhibitor suggests a novel approach to the treatment of psoriasis.

Phase I study to determine the safety, tolerability and immunostimulatory activity of thalidomide analogue CC-5013 in patients with metastatic malignant melanoma and other advanced cancers.

We assessed the safety, tolerability and efficacy of the immunomodulatory drug, CC-5013 (REVIMID trade mark ), in the treatment of patients with metastatic malignant melanoma and other advanced cancers. A total of 20 heavily pretreated patients received a dose-escalating regimen of oral CC-5013. Maximal tolerated dose, toxicity and clinical responses were evaluated and analysis of peripheral T-cell surface markers and serum for cytokines and proangiogenic factors were performed. CC-5013 was well tolerated. In all, 87% of adverse effects were classified as grade 1 or grade 2 according to Common Toxicity Criteria and there were no serious adverse events attributable to CC-5013 treatment. Six patients failed to complete the study, three because of disease progression, two withdrew consent and one was entered inappropriately and withdrawn from the study. The remaining 14 patients completed treatment without dose reduction, with one patient achieving partial remission. Evidence of T-cell activation was indicated by significantly increased serum levels of sIL-2 receptor, granulocyte-macrophage colony-stimulating factor, interleukin-12 (IL-12), tumour necrosis factor-alpha and IL-8 in nine patients from whom serum was available. However, levels of proangiogenic factors vascular endothelial growth factor and basic foetal growth factor were not consistently affected. This study demonstrates the safety, tolerability and suggests the clinical activity of CC-5013 in the treatment of refractory malignant melanoma. Furthermore, this is the first report demonstrating T-cell stimulatory activity of this class of compound in patients with advanced cancer.

First Report of the Causal Agent of Huanglongbing ("Candidatus Liberibacter asiaticus") in Brazil.

Huanglongbing (ex-greening) disease is one of the most serious diseases of citrus. It is caused by the phloem-limited, gram-negative bacterium "Candidatus Liberibacter spp.". This bacterium is not well characterized mainly because it is still uncultured. There are two known strains, Asian ("Candidatus Liberibacter asiaticus") and African ("Candidatus Liberibacter africanus") that cause severe damage to citrus plants including twig dieback, decline, and death. Symptoms first appear as leaf mottling and chlorosis occurring in one shoot or sector of trees. Later, leaf symptoms resemble nutritional deficiencies (Zn, Ca, and N) that vary depending on the strains, with more severe symptoms caused by "Ca. L. asiaticus". The Asian strains are transmitted by the Asian citrus psyllid (AsCP), Diaphorina citri, which is present in Brazil. The bacterium has been detected in citrus plants in many geographic locations including China, Japan, Thailand, India, the Philippines, the Arabian Peninsula, and Africa. In 2004, plants showing Huanglongbing symptoms were observed in the Araraquara County, a central region of the State of Sao Paulo, the largest citrus-producing area in Brazil. To verify the presence of "Ca. L. spp." in these plants, leaf samples of sweet orange cvs. Hamlin and Valencia were used for DNA extraction and polymerase chain reaction amplification using the specific OI1 and Oi2c primers (1). Amplification of the 16S rDNA was positive for 2 (cvs. Hamlin and Valencia) of 10 analyzed plants. The amplified fragments were cloned and sequenced. The amplicons obtained from both plants showed the same sequence, which differed from "Ca. L. africanus", utilized as the positive control in the amplification experiment (27 divergent bases in 1,160). The sequences were used for BLAST searches, and the results showed identities ranging from 94.71 to 100% with "Ca. L. spp." sequences available at the National Center for Biotechnology Information database (on-line publication). The highest scores were obtained with "Ca. L. asiaticus sequences. These analyses confirmed the presence of such agent in the State of Sao Paulo. To our knowledge, this is the first report of "Ca. L. asiaticus" in Brazil as well as elsewhere in the Americas. The significance of this report relates to the potential damage that this pathogen could cause to the citrus industry in the largest citrus-producing country in the world. It remains unclear how and when the pathogen entered Brazil. Reference: (1) S. Jagoueix et al. Mol. Cell Probes 10:43, 1996.

Novel thalidomide analogues display anti-angiogenic activity independently of immunomodulatory effects.

The anti-tumour effects of thalidomide have been associated with its anti-angiogenic properties. Second generation thalidomide analogues are distinct compounds with enhanced therapeutic potential. Although these compounds are beginning to enter trials for the treatment of cancer there is very little information regarding the anti-angiogenic activity of these clinically relevant compounds. Furthermore, it is not known how the various immunomodulatory activities of these compounds relate to anti-angiogenic activity. In this study we assessed the anti-angiogenic activity of compounds from both IMiD and SelCID classes of analogues using a novel in vitro multicellular human assay system and the established rat aorta assay. Our results show that both the IMiDs and SelCIDs tested are significantly more potent than thalidomide. The anti-angiogenic potency of the analogues was not related to inhibition of endothelial cell proliferation, nor their TNF-alpha/PDE type 4 inhibitory properties. However, anti-migratory effects in vitro and inhibition of tumour growth in vivo was observed with the analogue IMiD-1 (clinically known as REVIMID). Our results show that anti-angiogenic activity spans both currently defined classes of thalidomide analogue and is not related to their previously described immunomodulatory properties. Identification of the differential effects of these compounds will enable targeting of such compounds into the appropriate clinical setting.

Potent inhibition of cytokine production from intestinal lamina propria T cells by phosphodiesterase-4 inhibitory thalidomide analogues.

In Crohn's disease, intestinal lamina propria (LP) T cells overproduce TNF-alpha and IFN-gamma, and clinical and animal studies indicate that this is pathogenic. Thalidomide influences cytokine production by leukocytes, inhibiting macrophage TNF-alpha, and is beneficial in treating Crohn's disease. Chemical analogues have been synthesized that may lack teratogenic and other side effects of thalidomide. We tested three analogues [selective cytokine inhibitory drugs (SelCIDs) A, B, and C, all potent PDE4 inhibitors] for effect on TNF-alpha, IFN-gamma, and IL-10 production by and on proliferation of intestinal LP mononuclear cells after T-cell stimulation and results were compared with those for peripheral blood leukocytes (PBL). While thalidomide itself had little effect, the SelCIDs were potent inhibitors, with relative inhibitory potencies: A> or =B>>C. The LP T cells were less sensitive to inhibition by the SelCIDs than were PBL. Since highly pre-activated PBL were even less sensitive, activation state alone can account for the responsiveness of intestinal LP T cells. Thalidomide analogues could play a role in treating Crohn's disease and other inflammatory disorders.

Thalidomide and immunomodulatory derivatives augment natural killer cell cytotoxicity in multiple myeloma.

The antiangiogenic activity of thalidomide (Thal), coupled with an increase in bone marrow angiogenesis in multiple myeloma (MM), provided the rationale for the use of Thal in MM. Previously, the direct anti-MM activity of Thal and its analogues (immunomodulatory drugs, IMiDs) on MM cells was demonstrated, suggesting multiple mechanisms of action. In this study, the potential immunomodulatory effects of Thal/IMiDs in MM were examined. It was demonstrated that Thal/IMiDs do not induce T-cell proliferation alone but act as costimulators to trigger proliferation of anti-CD3-stimulated T cells from patients with MM, accompanied by an increase in interferon-gamma and IL-2 secretion. However, an increase in autologous T-cell killing of patient MM cells could not be demonstrated. A role for natural killer (NK)- and LAK-cell-mediated killing is suggested because IL-2-primed peripheral blood mononuclear cells (PBMCs) treated with Thal/IMiDs demonstrated significantly increased lysis of MM cell lines. Cold target inhibition assays suggested NK- rather than LAK-cell-mediated killing. Furthermore, this killing was not major histocompatibility complex-class restricted, and the depletion of CD56(+) cells blocked the drug-induced MM cell lysis. It was significant that increased killing of patient MM cells by autologous PBMCs treated with Thal/IMiDs was also observed. Although the in vivo relevance of NK-cell-mediated MM cell killing is unknown, phenotypic analysis performed in MM patients receiving Thal therapy demonstrated an increase in CD3(-)CD56(+) cells in patients responding to therapy. Thus in vitro and in vivo data support the hypothesis that Thal may mediate its anti-MM effect, at least in part, by modulating NK cell number and function.

Amino-substituted thalidomide analogs: potent inhibitors of TNF-alpha production.

Thalidomide, (1), is a known inhibitor of TNF-alpha release in LPS stimulated human PBMC. Herein we describe the TNF-alpha inhibitory activity of amino substituted analogs of thalidomide (1) and its isoindolin-1-one analog, EM-12 (2). The 4-amino substituted analogs were found to be potent inhibitors of TNF-alpha release in LPS stimulated human PBMC.

Differential cytokine modulation and T cell activation by two distinct classes of thalidomide analogues that are potent inhibitors of TNF-alpha.

TNF-alpha mediates both protective and detrimental manifestations of the host immune response. Our previous work has shown thalidomide to be a relatively selective inhibitor of TNF-alpha production in vivo and in vitro. Additionally, we have recently reported that thalidomide exerts a costimulatory effect on T cell responses. To develop thalidomide analogues with increased anti-TNF-alpha activity and reduced or absent toxicities, novel TNF-alpha inhibitors were designed and synthesized. When a selected group of these compounds was examined for their immunomodulatory activities, different patterns of cytokine modulation were revealed. The tested compounds segregated into two distinct classes: one class of compounds, shown to be potent phosphodiesterase 4 inhibitors, inhibited TNF-alpha production, increased IL-10 production by LPS-induced PBMC, and had little effect on T cell activation; the other class of compounds, similar to thalidomide, were not phosphodiesterase 4 inhibitors and markedly stimulated T cell proliferation and IL-2 and IFN-gamma production. These compounds inhibited TNF-alpha, IL-1beta, and IL-6 and greatly increased IL-10 production by LPS-induced PBMC. Similar to thalidomide, the effect of these agents on IL-12 production was dichotomous; IL-12 was inhibited when PBMC were stimulated with LPS but increased when cells were stimulated by cross-linking the TCR. The latter effect was associated with increased T cell CD40 ligand expression. The distinct immunomodulatory activities of these classes of thalidomide analogues may potentially allow them to be used in the clinic for the treatment of different immunopathological disorders.

Thalidomide analogs and PDE4 inhibition.

N-Phthaloyl 3-amino-3-arylpropionic acid analogs of thalidomide that are potent inhibitors of tumor necrosis factor-alpha are reported. These compounds were found to be potent inhibitors of phosphodiesterase 4.

Thalidomide and thalidomide analogs reduce HIV type 1 replication in human macrophages in vitro.

Thalidomide is currently being evaluated for efficacy in alleviating some manifestations of HIV-1 infection. To determine whether thalidomide has any direct effects on HIV-1 infection, we investigated the effect of thalidomide and also of three structural analogs of thalidomide on HIV-1 replication in vitro in human monocyte-derived macrophages. The thalidomide analogs were previously shown to inhibit TNF-alpha production in vitro at much lower concentrations than thalidomide. In HIV-1-infected macrophages treated with thalidomide or thalidomide analogs, viral replication was reduced by 60 to 80% as determined by measuring viral RT activity in the culture supernatants. In all experiments the analogs inhibited HIV-1 replication more efficiently than did thalidomide. The drugs also reduced HIV-1 gag mRNA expression. Furthermore, the drugs caused a decrease in NF-kappaB-binding activity in nuclear extracts of HIV-1-infected macrophages. The role of NF-kappaB in the drug-induced inhibition of HIV-1 replication was confirmed using an NF-kappaB-defective mutant virus to infect macrophages.

Selection of novel analogs of thalidomide with enhanced tumor necrosis factor alpha inhibitory activity.

BACKGROUND: Tumor necrosis factor alpha (TNF alpha) is thought to mediate both protective and detrimental manifestations of the inflammatory response. Recently, thalidomide (alpha-N-phthalimidoglutarimide) was shown to partially inhibit monocyte TNF alpha production (by 50-70%) both in vivo and in vitro. More efficient inhibition of TNF alpha may, however, be necessary to rescue the host from more acute and extensive toxicities of TNF alpha-mediated inflammation. MATERIALS AND METHODS: Three structural analogues of thalidomide were selected for study based on increased activity against TNF alpha 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. RESULTS: The new compounds (two esters and one amide) showed increased inhibition of TNF alpha 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-1 beta protein and mRNA production. When tested in vivo, the amide analog protected 80% of LPS-treated mice against death from endotoxin induced shock. CONCLUSIONS: Analogs of thalidomide designed to better inhibit TNF alpha 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 TNF alpha production such as tuberculous meningitis or toxic shock.

Carboxylic acid replacement structure-activity relationships in suosan type sweeteners. A sweet taste antagonist. 1.

N-(4-Cyanophenyl)-N'-(2-carboxyethyl)urea (2), an analogue of suosan [1,N-(4-nitrophenyl)-N'-(2-carboxyethyl)urea], is a known high-potency sweetener derived from beta-alanine. Sulfonic and phosphonic acid analogues of 2 were prepared to develop structure-activity relationships through modification of the carboxylic acid region of this family of sweeteners. Neither of the carboxylic acid replacements resulted in sweet analogues. However, we found that N-(4-cyanophenyl)-N'-[(sodiosulfo)methyl]urea (7) is an antagonist of the sweet taste response. The bitter taste response to caffeine, quinine, and naringin was also antagonized. Antagonist 7 was found to inhibit the sweet taste perception of a variety of sweeteners. Antagonist 7 had no effect on the sour or salty taste response.

N,N'-disubstituted guanidine high-potency sweeteners.

The role and function of the aryl group in the highly potent trisubstituted guanidine sweeteners 7a-d was investigated. Four disubstituted guanidines, lacking the aryl group, were prepared. These guanidines contain a hydrophobic substituent on one nitrogen and a carboxymethyl group substituted on one of the other nitrogens. They were found to be tasteless or to have a significantly lower sweetness potency than the corresponding trisubstituted compounds. Possible rationales for the effects of structure on the sweet taste activity are discussed.

[The value of diagnostic computerized tomography in tumor nephrectomy patients]. / Stellenwert der computertomographischen Diagnostik bei tumornephrektomierten Patienten.

In 84 patients after nephrectomy due to renal cell carcinoma a computerized tomography was performed to evaluate local tumor recurrence. The findings are classified stage-dependent. Other metastatic lesions were found by conventional radiologic methods. Based on stage-dependent different occurrence of metastases or local recurrences recommendations are made to the use of computerized tomography in the follow-up of patients suffering from renal cell carcinoma after nephrectomy.

[Dynamic computerized tomography in staging of urinary bladder cancer]. / Dynamische Computertomographie beim Staging des Harnblasenkarzinoms.

In 25 patients suffering from bladder cancer a dynamic computerized tomography was performed under constant conditions in a selected tumor layer level. It was the aim of this study to improve the efficacy of preoperative staging of bladder cancer. In the tumors of stages pT1 and pT2 (12 patients) different courses in the time-density-diagrams were shown in comparison with the stages pT3a + b (13 patients). Furthermore, a different intake and delivery of contrast medium was observed and differences in the vascularization of the individual tumor stages may be suspected. Thereby a separation between operable (pT1/2) and inoperable tumor (pT3a/b) seem to be possible.

[The value of computerized tomography and renovasography in kidney injuries]. / Stellenwert von Computertomographie und Renovasographie bei Nierenverletzungen.

Computerized tomography (CT) and renal angiography are compared in the staging of traumatic renal lesions. After evaluation of 44 angiographic and 74 CT investigations in 111 patients it was concluded, that CT is superior to angiography in the diagnosis of blunt renal injuries. However, if vascular injuries are suspected an angiographic investigation should be done.

Actin synthesis rate and mRNA level increase during early recovery of atrophied muscle.

The purpose of this study was to correlate actin synthesis rate and alpha-actin mRNA level in the gastrocnemius-plantaris muscles of limbs during their recovery from 7 days of immobilization in 200- to 280-g female rats. The fractional synthesis rate of actin in control muscle was 1%/day. Actin synthesis rate was 33% of control level at the 7th day of hindlimb immobilization, returned to control value at the 2nd recovery day, and was three times higher than control on the 4th day of recovery. The alpha-actin mRNA was 53% of control at the 7th day of immobilization, and its increase during the 1st 2 recovery days paralleled the increase in actin synthesis rate; this suggests that pretranslational mechanisms caused the initial increase in actin synthesis. Further increases in actin synthesis from the 2nd to the 4th day appear to be under translational control, since actin synthesis was 300% of control on the 4th recovery day and alpha-actin mRNA was only 128% of control.

[Value of angiography and computerized tomography for preoperative evaluation of bladder cancer]. / Aussagewert der Angiographie und der Computertomographie für die präoperative Beurteilung des Harnblasenkarzinoms.

In a comparative study in 30 patients with tumour of the urinary bladder the value of angiographic and computer-tomographic investigation in the proof of tumours and for staging was controlled. In 30% of the cases the angiographic proof was not successful. A concordance to the histologically recognized degree of infiltration could be established by means of angiography in 36.6% and by computed tomography in 86.3%. When it is possible to apply computed tomography for staging we regard the performance of the angiography of the pelvis as no more indicated.