Investigating the Stability of Six Phenolic TMZ Ester Analogues, Incubated in the Presence of Porcine Liver Esterase and Monitored by HPLC.

Previous published data from our group showed the encouraging in vitro activities of six phenolic temozolomide (TMZ) ester analogues (ES8-ES12 and ES14) with up to a five-fold increase in potency compared to TMZ against glioblastoma multiform cell lines and TMZ-resistant O6-methylguanine-DNA methyl transferase (MGMT)-positive primary cells. This study investigated the stabilities of the six phenolic TMZ ester analogues in the presence of porcine liver esterase (PLE) as a hydrolytic enzyme, using high-performance liquid chromatography (HPLC), monitored by a diode-array detector (DAD). Determining the rates of hydrolysis of the esters provided a useful insight into the feasibility of progressing them to the next phase of drug development. Fifty percent of TMZ esters consisting of para nitro, chloro, phenyl and tolyl groups (ES9, ES10, ES12 and ES14) were hydrolysed within the first 4.2 min of PLE exposure, while the TMZ esters consisting of para methoxy and nitrile groups (ES8 and ES11) demonstrated increased stability, with 50% hydrolysis achieved in 7.3 and 13.7 min, respectively. In conclusion, the survival of these phenolic TMZ esters on route to the target site of a brain tumor would be a challenge, mainly due to the undesirable rapid rate of hydrolysis. These findings therefore pose a question regarding the effectiveness of these esters in an in vivo setting.

Temperature and solvent facilitated extrusion based 3D printing for pharmaceuticals.

On demand manufacturing of patient-specific oral doses provides significant advantages to patients and healthcare staff. Several 3D printing (3DP) technologies have been proposed as a potential digital alternative to conventional manufacturing of oral tablets. For an additive manufacturing approach to be successful for on-demand preparation, a facile process with minimal preparation steps and training requirements is needed. A novel hybrid approach to the 3D printing process is demonstrated here based on combining both a solvent and heating to facilitate extrusion. The system employed a moderate elevated temperature range (65-100 °C), a brief drying period, and a simple set-up. In this approach, a compact material cylinder is used as a pharmaceutical ink to be extruded in a temperature-controlled metal syringe. The process proved compatible with hygroscopic polymers [Poly(vinyl alcohol (PVA) and polyvinylpyrrolidone (PVP)] and a number of pharmaceutical fillers (lactose, sorbitol and D-mannitol). The fabricated tablets demonstrated acceptable compendial weight and content uniformity as well as mechanical resistance. In vitro drug release of theophylline from 3D printed tablets was dependent on the nature of the polymer and its molecular weight. This reported approach offers significant advantages compared to other 3DP technologies: simplification of pre-product, the use of a moderate temperature range, a minimal drying period, and avoiding the use of mechanically complicated machinery. In the future, we envisage the use of this low-cost and facile approach to fabricate small batches of bespoke tablets.

A novel series of phenolic temozolomide (TMZ) esters with 4 to 5-fold increased potency, compared to TMZ, against glioma cells irrespective of MGMT expression.

The standard of care treatment for patients diagnosed with glioblastoma multiforme (GBM) is temozolomide (TMZ). Tumour resistance to TMZ results in significantly limited clinical effectiveness. There is therefore an inherent need for alternatives to TMZ capable of overcoming resistance associated with MGMT and MMR. In the present study, a series of ester and amide analogues of TMZ, modified at position 8 on the imidazole ring, were prepared and investigated for antiproliferative properties. It was found that phenolic ester analogues of TMZ displayed increased potency, of up to 5-fold, against specified glioblastoma cell lines. The encouraging results displayed by the phenolic TMZ esters prompted further investigations against patient-derived primary glioblastoma cultures. The primary cultures, BTNW914 and BTNW374, were MGMT positive and MGMT negative, respectively. Lead phenolic TMZ esters were found to decrease viability in primary cells at clinically relevant concentrations, irrespective of MGMT expression. Furthermore, TMZ was found to be ineffective against the same primary cells at clinically relevant concentrations. The novel phenyl ester analogues of TMZ, described in this study, could have potential chemotherapeutic properties for the treatment of GBM, overcoming the resistance associated with the expression of MGMT.

Identifying Reliable Diagnostic/Predictive Biomarkers for Rheumatoid Arthritis.

INTRODUCTION AND OBJECTIVE: Elevated C-reactive protein is usually a good indicator of rheumatoid arthritis (RA); however, there are limitations that compromise its specificity and therefore there is an urgent need to identify more reliable diagnostic biomarkers to detect early stages of RA. In addition, identifying the correct therapeutic biomarker for the treatment of RA using methotrexate (MTX) would greatly increase the benefits experienced by the patients. MATERIALS AND METHODS: Primary normal synoviocytes human fibroblast-like synoviocytes (HFLS) and its phenotype rheumatic HFLS-RA cells were chosen for this study. The HFLS-RA-untreated and MTX-treated cells were subjected to microarray analysis. RESULTS: Microarray data identified 74 differentially expressed genes. These genes were mapped against an RA inflammatory pathway, shortlisting 10 candidate genes. Gene expression profiling of the 10 genes were studied. Fold change (FC) was calculated to determine the differential expression of the samples. DISCUSSION: The transcription profiles of the 10 candidate genes were highly induced in HFLS-RA cells compared with HFLS cells. However, on treating the HFLS-RA cells with MTX, the transcription profiles of these genes were highly downregulated. The most significant expression FC difference between HFLS and HFLS-RA (treated and untreated) was observed with HSPA6, MMP1, MMP13, and TNFSF10 genes. CONCLUSIONS: The data from this study suggest the use of HSPA6, MMP1, MMP13, and TNFSF10 gene expression profiles as potential diagnostic biomarkers. In addition, these gene profiles can help in predicting the therapeutic efficacy of MTX.

Could the Anti-Chaperone VER155008 Replace Temozolomide for Glioma Treatment.

Cancer inducible molecular chaperone HSP90 is of great importance as an anticancer target. Proteomic analysis showed that inhibiting HSP90 by the geldanamycin derivative, 17-AAG elevated the expression of the co-chaperone Hsp70. In this study we used HSP90 selective inhibitor 17-AAG and HSP70/90 dual inhibitor, VER155008 (VER) in U87-MG glioma cells. miRNAs microarray technology was used to evaluate the efficacy of these inhibitory drugs compared with temozolomide (TMZ), used as a standard treatment for glioma. Microarrays data identified 154 differentially expressed miRNAs using stringent or unstringent parameters. 16 miRNAs were overlapped between treatments, 13 upregulated and one downregulated miRNA were overlapped between TMZ and VER. The miRNA target prediction software was used for these overlapped miRNAs and identified 6 of the 13 upregulated miRNAs target methyltransferase genes. The IC50, together with Akt and HSP70 and 90 protein level data favour VER and TMZ to 17-AAG, however due to the selectivity of VER to cancer cells as a potent antichaperon, it may be more favourable to the standard TMZ.

Can RNAi-mediated hsp90α knockdown in combination with 17-AAG be a therapy for glioma?

Heat shock protein 90 promotes tumor progression and survival and has emerged as a vital therapeutic target. Previously we reported that the combinatorial treatment of 17AAG/sihsp90α significantly downregulated Hsp90α mRNA and protein levels in Glioblastoma Multiforme (GBM). Here we investigated the ability of cell penetrating peptide (Tat48-60 CPP)-mediated siRNA-induced hsp90α knockdown as a single agent and in combination with 17-allylamino-17-demethoxygeldanamycin (17-AAG) to induce tumor growth inhibition in GBM and whether it possessed therapeutic implications. GBM and non-tumorigenic cells exposed to siRNA and/or 17-AAG were subsequently assessed by qRT-PCR, immunofluorescence, FACS analysis, quantitative Akt, LDH leakage and cell viability assays. PAGE was performed for serum stability assessment. A combination of siRNA/17-AAG treatment significantly induced Hsp90α gene and protein knockdown by 95% and 98%, respectively, concomitant to 84% Akt kinase activity attenuation, induced cell cycle arrest and tumor-specific cytotoxicity by 88%. Efficient complex formation between CPP and siRNA exhibited improved serum stability of the siRNA with minimal intrinsic toxicity in vitro. The preliminary in vivo results showed that combination therapy induced hsp90α knockdown and attenuated Akt kinase activity in intracranial glioblastoma mouse models. The results imply that RNAi-mediated hsp90α knockdown increases 17-AAG treatment efficacy in GBM. In addition, the cytotoxic response observed was the consequence of downregulation of hsp90α gene expression, reduced Akt kinase activity and S-G2/M cell cycle arrest. These results are novel and highlight the ability of Tat to efficiently deliver siRNA in GBM and suggest that the dual inhibition of Hsp90 has therapeutic potentials.

The complexity of identifying cancer stem cell biomarkers.

The efficacy of glioma therapy can be considerably improved if it eliminates cancer stem cells (CSCs); however, to achieve this, CSCs markers are required. This study investigated the influence of micro-environmental changes on CSCs in hypoxic, serum deprived U87-MG and the corresponding control cells. Proteomic analysis produced a wide dataset, depicting the changes that occur at the proteomic level in the differentiated and undifferentiated U87-MG cell line. With the IPA analysis, HPRD and literature reviews, 11 proteins were proposed as potential differentiated biomarkers for CSCs namely Hsp90ß1, vimentin, PGK1, GAPDH, EIF4e, TPI1, HspA8, HNRNPK, NAMPT, CCSNK2A1, and ANXA2.

Proteomic studies coupled with RNAi methodologies can shed further light on the downstream effects of telomerase in glioma.

A comprehensive proteomic study utilizing 2D-DIGE and MALDI-TOF was used to assess the effect of inhibiting two different regulatory mechanisms of telomerase in glioma. RNAi was used to target hTERT and hsp90α. Inhibition of telomerase activity resulted in downregulation of various cytoskeletal proteins with correlative evidence of the involvement of telomerase in regulating the expression of vimentin. Inhibition of telomerase via sihTERT resulted in the downregulation of vimentin expression in glioma cell lines in a grade-specific manner. This study identified novel downstream role of telomerase in regulating the expression of vimentin, thereby affecting tumor progression and metastasis.

A novel therapeutic strategy for the treatment of glioma, combining chemical and molecular targeting of hsp90a.

Hsp90a's vital role in tumour survival and progression, together with its highly inducible expression profile in gliomas and its absence in normal tissue and cell lines validates it as a therapeutic target for glioma. Hsp90a was downregulated using the post-transcriptional RNAi strategy (sihsp90a) and a post-translational inhibitor, the benzoquinone antibiotic 17-AAG. Glioblastoma U87-MG and normal human astrocyte SVGp12 were treated with sihsp90a, 17-AAG and concurrent sihsp90a/17-AAG (combined treatment). Both Hsp90a gene silencing and the protein inhibitor approaches resulted in a dramatic reduction in cell viability. Results showed that sihsp90a, 17-AAG and a combination of sihsp90a/17-AAG, reduced cell viability by 27%, 75% and 88% (p < 0.001), respectively, after 72 h. hsp90a mRNA copy numbers were downregulated by 65%, 90% and 99% after 72 h treatment with sihsp90a, 17-AAG and sihsp90a/17-AAG, respectively. The relationship between Hsp90a protein expression and its client Akt kinase activity levels were monitored following treatment with sihsp90a, 17-AAG and sihsp90a/17-AAG. Akt kinase activity was downregulated as a direct consequence of Hsp90a inhibition. Both Hsp90a and Akt kinase levels were significantly downregulated after 72 h. Although, 17-AAG when used as a single agent reduces the Hsp90a protein and the Akt kinase levels, the efficacy demonstrated by combinatorial treatment was found to be far more effective. Combination treatment reduced the Hsp90a protein and Akt kinase levels to 4.3% and 43%, respectively, after 72 h. hsp90a mRNA expression detected in SVGp12 was negligible compared to U87-MG, also, the combination treatment did not compromise the normal cell viability. Taking into account the role of Hsp90a in tumour progression and the involvement of Akt kinase in cell signalling and the anti-apoptotic pathways in tumours, this double targets treatment infers a novel therapeutic strategy.

Can hsp90alpha-targeted siRNA combined with TMZ be a future therapy for glioma?

Hsp90alpha's vital role in cell cycle progression and apoptosis together with its presence in gliomas and absence in normal tissue, make it a credible target for cancer therapy. Three sets of dsRNA oligos designed to align different regions of the hsp90alpha sequence were used to downregulate hsp90alpha. SiRNA 1, 2, and 3 resulted in significant levels of silencing of hsp90alpha after 48 hr treatment (p < .0001). Concurrent treatment of the glioma cell line U87-MG with siRNA 1 and temozolomide (TMZ) resulted in a 13-fold reduction in the dose of TMZ required to achieve a similar effect if TMZ was used alone.

Telomerase downregulation in cancer brain stem cell.

Cancer stem cells (CSCs) are a minute sub-population of self-renewing, immortal cells, which can be responsible for chemoresistance observed in the treatment of cancer. CSCs are similar to cancer cells requiring telomerase activity or alternative mechanisms for their proliferation and regeneration. This study explored the correlation between CD133 (stem cell marker) and telomerase expression using CD133+ cells isolated from the glioma GOS-3 cell line with magnetic affinity cell sorting (MACS). GOS-3 CD133+ showed a transcription downregulation of hTERT ( approximately 100-fold decrease) compared with CD133- cells. In order to further substantiate the novel finding, serum deprivation was adopted to enrich CD133 expression in GOS-3 cells. A pronounced upregulation of cd133 and downregulation of telomerase expression were produced as a consequence of decreasing serum supplement levels in GOS-3 cells. These findings showed for the first time that telomerase is downregulated in brain cancer stem cells compared to cancer cells.

To determine the cytotoxicity of chlorambucil and one of its nitro-derivatives, conjugated to prasterone and pregnenolone, towards eight human cancer cell-lines.

Four ester prodrugs derived from the bifunctional alkylating agent chlorambucil, and one of its nitro-derivatives, 3-nitrochlorambucil conjugated to prasterone and pregnenolone, were synthesized and tested for their cytotoxic activity against eight human cell lines, using the standard MTT assay. A comparison between the esters and the controls, namely chlorambucil and 3-nitrochlorambucil would suggest that all four esters possess to varying degrees, specificity towards the breast adenocarcinoma cell line (MDA-mb468) than the other seven cells' lines tested. The overall findings are encouraging since it infers that these lipophilic esters not only have the ability to traverse specific cell membranes but also exhibit cytotoxicity towards most of the cell lines tested.

Can the lack of HSP90alpha protein in brain normal tissue and cell lines, rationalise it as a possible therapeutic target for gliomas?

Despite studies suggesting a role for HSP90alpha in tumorigenesis, there are no reports as to its expression in normal human brain tissue. In this study, the expression of HSP90alpha was evaluated in both cell lines (3 gliomas and 2 controls) and brain tissue specimens of 10 patients (8 gliomas and 2 normal brain tissues). No HSP90alpha protein was detected in either normal cell lines or normal brain tissue. However, 8/8 glioma tissues and 3/3 glioma cell lines did express HSP90alpha. These findings provide a rationale for targeting HSP90alpha protein as a therapeutic candidate for glioma.

Epigenetic silencing of telomerase and a non-alkylating agent as a novel therapeutic approach for glioma.

5-Aza-2'-deoxycytidine (5azadC) inhibits DNA methyltransferase and subsequently induces the expression of genes silenced by methylation. While treatment with 5azadC downregulated hTERT and upregulated MGMT expression in two glioma cell lines, there was no change in the expression of these two genes in the normal cell line. However, cell viability was reduced as a result of 5azadC treatment in all three cell lines. 5azadC treatment reduced telomerase expression and activity and subsequently enhanced chemosensitivity towards cisplatin, taxol and tamoxifen but not with the alkylating agents temozolomide (TMZ), carmustine and chlorambucil. To further evaluate the effect of these findings, the level of hTERT and MGMT expression was measured in a recurrent anaplastic ependymoma, seven glioblastoma and two normal brain tissues. While four of eight gliomas and one of the normal tissues expressed MGMT, hTERT was expressed in all gliomas but not in the normal brain tissue. Results of this study suggest that taxol together with 5azadC may be a good therapeutic combination for glioma. In addition, the work on cell lines can be repeated on tissues utilizing hTERT as the therapeutic target for demethylation using 5azadC in glioma.

Telomerase subunits expression variation between biopsy samples and cell lines derived from malignant glioma.

Although scientific advances have recognised the prognostic power of telomerase activity in different cancers, as yet there has been no investigation regarding the expression variation of telomerase subunits in glioma tissues and cell lines. In this study, a recurrent anaplastic ependymoma and seven glioblastoma biopsy samples, four cell lines and four controls including two normal brain tissues were analysed for telomerase subunit expression profiles together with telomerase activity. Since telomerase activity is linked to tumourgenesis, the genes were analysed with respect to their expression variation. TEP1 was expressed in all glioma cell lines and 70% of glioblastoma tissues, in addition to the control brain tissues. Tankyrase was expressed in 85% of the glioblastoma tissues and was down-regulated in the recurrent anaplastic ependymoma tissue control cell lines. However, it was expressed in the control tissues. Dyskerin was expressed in all cell lines and tissues apart from U87-MG and NHA cells and the recurrent anaplastic ependymoma tissue. As expected, PARP1 and GAPDH showed constitutive expression throughout all cell lines and tissues since both are known to be housekeeping genes. hTERT was expressed in all glioma cell lines and tissues but was absent in the control cells and tissues. Telomerase activity was absent in IPDDC-A2 cells and 57% of the glioblastoma tissues. These results suggest that hTERT expression and not telomerase activity possibly represents a simple and reliable biological diagnostic tool.

The simultaneous separation and determination of five quinolone antibiotics using isocratic reversed-phase HPLC: application to stability studies on an ofloxacin tablet formulation.

A rapid and reliable HPLC method was developed for the simultaneously separation and quantitation of five quinolones antibiotics; nalidixic acid, norfloxacin, ofloxacin, ciprofloxacin and lomefloxacin. All five tablet formulations of individual quinolone antibiotics were routinely assayed without interference. The calibration curves were linear (r2> or =0.999) over the concentration range of 1.20-4.8 mg/100 ml. Selectivity, precision, sensitivity and accuracy were established and the method is stability indicating with respect to ofloxacin. The limit of detection and quantitation for ofloxacin was 18 and 36 microg/100 ml, respectively. The separation was performed on a Phenomenex ODS C18 column using an isocratic, ion-pairing mobile phase consisting of 35% (v/v) aqueous acetonitrile together with tetrabutylammonium acetate, sodium dodecyl sulphate and citric acid (pH* 3.4). All analyses were conducted at ambient temperature and was monitored using a Diode Array UV/VIS detector set at wavelengths 235, 254, 275 and 300 nm.