Evaluation of Solubility, Dissolution Rate, and Oral Bioavailability of ß-Cyclodextrin and Hydroxypropyl ß-Cyclodextrin as Inclusion Complexes of the Tyrosine Kinase Inhibitor, Alectinib.

This study aims to improve the solubility and dissolution rate of alectinib (ALB), a tyrosine kinase inhibitor commonly used for treating non-small-cell carcinoma (NSCLC). Given ALB's low solubility and bioavailability, complexation with ß-cyclodextrin (ßCD) and hydroxy propyl ß-cyclodextrin (HPßCD) was evaluated. Some of the different preparation methods used with varying ALB-to-CD ratios led to the formation of complexes that were characterized using Fourier-Transform Infrared (FTIR) techniques and Differential Scanning Calorimetry (DSC) to prove complex formation. The encapsulation efficiency was also determined. The simulations were carried out for ALB's interactions with ßCD and HPßCD. This study identified the most soluble complex (ALB-HPßCD; 1:2 ratio) and evaluated its dissolution. The bioavailability of the ALB-HPßCD complex was evaluated in Wistar rats relative to free ALB. Pharmacokinetic profiles revealed increased Cmax (240 ± 26.95 ng/mL to 474 ± 50.07 ng/mL) and AUC0-48 (5946.75 ± 265 ng.h/mL to 10520 ± 310 ng.h/mL) with no change in the elimination rate constant. In conclusion, the complexation of ALB-HPßCD manages to increase in vitro solubility, the dissolution rate, and oral bioavailability, providing a favorable approach to improving ALB administration.

Osteopontin can act as an effector for a germline mutation of BRCA1 in malignant transformation of breast cancer-related cells.

Breast cancer-associated 1 (BRCA1) plays an important role in breast cancer initiation and progression through its functions in the cell cycle and DNA repair processes; however, its role in metastatic development in human breast cancer is still poorly understood. We have previously shown that osteopontin (OPN) expression was suppressed by wild-type BRCA1 (Wt.BRCA1) and that a natural mutant allele of BRCA1 (Mut.BRCA1) diminished the effect of Wt.BRCA1 on OPN in vitro. In this study, we show that while Wt.BRCA1 suppresses OPN-induced metastasis in a rat syngeneic system, Mut.BRCA1 enhances the development of metastasis through OPN, suggesting that OPN and BRCA1 work closely to regulate metastatic development in the rat. To test whether these findings are relevant to human breast cancer, we have investigated the relationship between BRCA1, OPN, and metastatic properties in human breast cancer-related cells. Using western blot analysis, we show that Wt.BRCA1 suppresses, while Mut.BRCA1 enhances, OPN protein expression; and in parallel that Wt.BRCA1 suppresses, while Mut.BRCA1 enhances, OPN-mediated in vitro properties associated with the metastatic state in both MCF-7 and MDA MB435s cells. Overall, these results suggest that Mut.BRCA1 can elicit some of the changes involved in metastatic progression in human breast cancer via the overexpression of OPN.

Identification of genes differentially expressed between benign and osteopontin transformed rat mammary epithelial cells.

BACKGROUND: Osteopontin is a secreted, integrin-binding and phosphorylated acidic glycoprotein which has an important role in tumor progression. FINDINGS: In this study, we have utilized suppressive subtractive hybridization (SSH) to evaluate OPN regulated gene expression, using the Rama 37 benign non-invasive rat mammary cell line and a subclone, Rama 37-OPN. Rama 37-OPN was produced by stably transfecting Rama 37 with an OPN expression vector and it demonstrates increased malignant properties in vitro. Sequence and expression array analysis of the respective cDNA libraries of over 1600 subtracted cDNA fragments revealed 982 ESTs, 45 novel sequences and 659 known genes. The known up-regulated genes in the Rama 37-OPN library code for proteins with a variety of functions including those involved in metabolism, cell adhesion and migration, signal transduction and in apoptosis. Four of the most differentially expressed genes between the benign and in vitro malignant rat mammary cell lines are tumor protein translationally controlled I (TPTI), aryl hydrocarbon receptor nuclear translocator (ARNT), ataxia telangiectasia mutated (ATM) and RAN GTPase (RAN). The largest difference (ca 10,000 fold) between the less aggressively (MCF-7, ZR-75) and more aggressively malignant (MDA MB 231, MDA MB 435S) human breast cancer cell lines is that due to RAN, the next is that due to osteopontin itself. CONCLUSION: The results suggest that enhanced properties associated with the malignant state in vitro induced by osteopontin may be due to, in part, overexpression of RAN GTPase and these biological results are the subject of a subsequent publication 1.

Osteopontin: a new role for a familiar actor.

Our understanding of the events that occur in cancer progression has been enhanced by the use of cell lines in vitro. Changes in gene expression, induction of signalling, and cell motility can all be investigated in this setting. However, other aspects of progression can be revealed only in vivo, especially the interactions of tumour cells with host cells and organ systems. In one such in vivo model, described by McAllister and colleagues, it proved possible to establish a novel function of an already well-characterised protein, osteopontin, adding to its attractiveness as a target in cancer therapy.

Role of osteopontin in cellular signaling and metastatic phenotype.

Osteopontin (opn) is a glyco-phosphoprotein that is expressed and secreted by numerous human cancers. Opn has pivotal role in cell adhesion, chemotaxis, prevention of apoptosis, invasion, migration and anchorage-independent growth of tumor cells. Extensive research has demonstrated the pivotal role of opn in regulation of cellular signaling, which controls neoplastic and malignant transformation. The elevated expression of opn has been observed in a variety of cancers. Recently, substantial evidence has linked opn with tumor metastasis and poor prognosis. However, the understanding of the molecular mechanisms that define the role of opn in cell invasion and metastasis is incomplete. The following review will discuss the molecular structure of opn, its function role in tumor cell metastasis and its downstream target genes that activate invasive mechanisms. Understanding of the role of opn in neoplastic transformation and its cellular target genes may enable development of novel anti-cancer therapy approaches.

Osteopontin as a target for cancer therapy.

Osteopontin (OPN) is a glycophosphoprotein cytokine that has multiple functions. OPN is expressed and secreted by various cells, and has a role in cell adhesion, chemotaxis, prevention of apoptosis, invasion, migration and anchorage-independent growth of tumor cells. Extensive research has demonstrated the pivotal participation of OPN in the regulation of cell signaling which controls neoplastic and malignant transformation. The elevated expression of OPN has been observed in a variety of cancers. OPN has been linked with tumor metastasis and signifies a poor prognosis for the patient. This review details the mechanisms by which OPN facilitates these pathological events. It will also show that gaining an understanding of the mechanism of OPN's action at a cellular level has led to the development of a number of therapeutic strategies against the cytokine. These include inhibiting its expression, antagonizing cell surface receptor activation and blocking downstream cell signaling pathways. In addition to the potential of these therapies, serum levels of OPN could be used as a diagnostic and prognostic marker. The authors propose that with further research and development, osteopontin directed treatment could greatly enhance outcomes for cancer patients.

The role of LEF/TCF factors in neoplastic transformation.

The T cell factor 4 (Tcf-4) interacts with beta-catenin in the Wnt signalling pathway and coactivates downstream target genes in diverse systems including the breast. This activity is important during normal development but its deregulation plays a pivotal role in cancer progression. In a rat model for breast cancer it has been shown that metastasis-inducing DNA (Met-DNA) sequesters the endogenous inhibitory Tcf-4 and thereby promotes transcription of the secreted extracellular matrix glycophosphoprotein, osteopontin, the direct effector of metastasis in this model system. Permanent transfection of the benign rat mammary cell line with a fragment from the Met-DNA containing the Tcf recognition sequence CAAAG induces the cells to metastasize in syngeneic rats in vivo. Tcf-4 expression in human breast carcinomas is inversely associated with osteopontin protein levels. High Tcf-4 expression impedes both OPN promoter activity and protein expression in rat mammary carcinoma cells. Understanding the role of Tcf-4 in cancer development and its transcription regulation should lay the foundation for novel therapeutic approaches in the future.

Interaction between transcription factor, basal transcription factor 3, and the NH2-terminal domain of human estrogen receptor alpha.

The estrogen receptor (ER), like other members of the nuclear receptor superfamily, possesses two separate transcriptional activation functions, AF-1 and AF-2. Although a variety of coactivators and corepressors of AF-2 have been identified, less is known of the mechanism of action of AF-1. We have used the yeast two-hybrid system to isolate a cDNA coding for a protein that binds specifically to the AF-1 region of human ERalpha. This cDNA codes for the transcription factor basal transcription factor 3 (BTF3). The specificity of the interaction between BTF3 and ERalpha has been confirmed in vivo and in vitro. Transient transfection experiments reveal that overexpression of BTF3 modulates the transcriptional response of reporter genes to ERalpha. BTF3 interacts with ERalpha that has been activated either by 17beta-estradiol (ligand-dependent activation) or by epidermal growth factor (ligand-independent activation). The effects of BTF3 on the reporter genes requires the presence of ERalpha containing an active AF-1 function. BTF3 may be a component of the mechanism by which the AF-1 function of ERalpha stimulates gene transcription.

The regulation and role of osteopontin in malignant transformation and cancer.

Osteopontin (OPN) is a predominantly secreted extracellular matrix glycophosphoprotein which binds to alpha v-containing integrins and has an important role in malignant cell attachment and invasion. High OPN expression in the primary tumor is associated with early metastasis and poor outcome in human breast and other cancers. Forced OPN overexpression in benign cells may induce neoplastic-like cell behaviour including increased attachment and invasion in vitro as well as the ability to metastasize in vivo. Conversely, OPN inhibition by antisense cDNA impedes cell growth and tumor forming capacity. OPN is not mutationally activated in cancer but its expression is regulated by Wnt/Tcf signaling, steroid receptors, growth factors, ras, Ets and AP-1 transcription factors. Presumably these factors are implicated in induction of OPN overexpression in cancer. Greater understanding of the role of OPN in neoplastic change and its transcriptional regulation may enable development of novel cancer treatment strategies.

BRCA1 suppresses osteopontin-mediated breast cancer.

BRCA1 is a well described breast cancer susceptibility gene thought to be involved primarily in DNA repair. However, mutation within the BRCA1 transcriptional domain is also implicated in neoplastic transformation of mammary epithelium, but responsible mechanisms are unclear. Here we show in a rat mammary model system that wild type (WT) BRCA1 specifically represses the expression of osteopontin (OPN), a multifunctional estrogen-responsive gene implicated in oncogenic transformation, particularly that of the breast. WT.BRCA1 selectively binds OPN-activating transcription factors estrogen receptor alpha, AP-1, and PEA3, inhibits OPN promoter transactivation, and suppresses OPN mRNA and protein both from an endogenous gene and a relevant model inducible gene. WT.BRCA1 also inhibits OPN-mediated neoplastic transformation characterized by morphology change, anchorage-independent growth, adhesion to fibronectin, and invasion through Matrigel. A mutant BRCA1 allele (Mut.BRCA1) associated with familial breast cancer lacks OPN suppressor effects, binds to WT.BRCA1, and impedes WT.BRCA1 suppression of OPN. Stable transfection of rat breast tumor cell lines with Mut.BRCA1 dramatically up-regulates OPN protein and induces anchorage independent growth. In human primary breast cancer, BRCA1 mutation is significantly associated with OPN overexpression. Taken together, these data suggest that BRCA1 mutation may confer increased tissue-specific cancer risk, in part by disruption of BRCA1 suppression of OPN gene transcription.