Pharmacology education in the medical curriculum: Challenges and opportunities for improvement.

The knowledge and application of pharmacology is essential for safe prescribing and administration of drugs. In this narrative review, the challenges to pharmacology education in the medical curricula were broadly identified to include issues around content and pedagogies. The increasing number of approved drugs and drug targets, expanding field of pharmacology and the often-changing treatment guidelines and board-defined competencies can make pharmacology education in the medical curriculum daunting. There has been a consensus around the deployment of innovative medical curricula with emphasis on vertical and horizontal integration. This strategy, effective as it has been, presents new challenges to pharmacology education. As a discipline often perceived by students to be hard-to-learn, the future of pharmacology education must include heavy reliance on active learning strategies. The continuing utilization of problem-based, team-based and case-based learning can be complemented with personalized learning which aims to identify the learning gaps in individual students. Technology-inspired student engagement can foster pharmacology learning and retention. Early exposure to pharmacology from premedical preparation through an enduring across-the-level integration can be an effective way to enhance pharmacology learning in the medical curricula.

Repression of CFTR activity in human MMNK-1 cholangiocytes induces sulfotransferase 1E1 expression in co-cultured HepG2 hepatocytes.

Mouse models of cystic fibrosis (CF) indicate that sulfotransferase (SULT) 1E1 is significantly induced in livers of many mice lacking cystic fibrosis transmembrane receptor (CFTR) activity. Increased SULT1E1 activity results in the alteration of estrogen-regulated protein expression in the livers of these mice. In this study, human MMNK-1 cholangiocytes with repressed CFTR function were used to induce SULT1E1 expression in human HepG2 hepatocytes to investigate whether SULT1E1 can be increased in human CF liver. CFTR expression was inhibited in MMNK-1 cholangiocytes using CFTR-siRNA, then the MMNK-1 and HepG2 cells were co-cultured in a membrane-separated Transwell system. Expression of SULT1E1 and selected estrogen-regulated proteins were then assayed in the HepG2 cells. Results demonstrate that inhibition of CFTR expression in MMNK-1 cells results in the induction of SULT1E1 message and activity in HepG2 cells in the Transwell system. The expression of estrogen-regulated proteins including insulin-like growth factor (IGF)-1, glutathione-S-transferase (GST) P1 and carbonic anhydrase (CA) II expression are repressed in the HepG2 cells cultured with the CFTR-siRNA-MMNK-1 cells apparently in response to the increased sulfation of beta-estradiol. Thus, we have shown that co-culture of HepG2 hepatocytes with MMNK-1 cholangiocytes with siRNA repressed CFTR expression results in the selective induction of SULT1E1 in the HepG2 cells. Loss of CFTR function in cholangiocytes may have a paracrine regulatory effect on hepatocytes via the induction of SULT1E1 and the increased sulfation of beta-estradiol. Experiments are presently underway in our laboratory to elucidate the identity of these paracrine regulatory factors.

Increased SULT1E1 activity in HepG2 hepatocytes decreases growth hormone stimulation of STAT5b phosphorylation.

Mouse models of cystic fibrosis (CF) display increased sulfotransferase 1E1 (SULT1E1) activity in hepatocytes of cystic fibrosis transmembrane receptor (CFTR)-deficient animals. SULT1E1 is responsible for the sulfation and inactivation of beta-estradiol (E2) at physiological concentrations. IGF-1 message levels in CFTR(-/-) mouse livers were positively correlated with body weight and negatively correlated with SULT1E1 activity. Growth hormone (GH) is important in the regulation of hepatic IGF-1 expression indicating that E2 levels are involved with GH signaling in hepatocytes. To investigate the effects of E2 and SULT1E1 activity on GH signal transduction in human hepatocytes, SULT1E1 was stably expressed in HepG2 cells. Effects of increased E2 sulfation on the GH signaling pathway and E2-regulated gene expression were examined. Pretreatment of HepG2 cells with 10nM E2 prior to GH stimulation increased STAT5b phosphorylation and IGF-1 expression. In SULT1E1-transfected HepG2 cells, GH-stimulated STAT5b phosphorylation was significantly decreased. E2 treatment had no effect on STAT5b phosphorylation in the absence of GH stimulation. E2 also had no effect on Jak-2 phosphorylation. E2 has an apparent rapid action on increasing GH-stimulated STAT5b phosphorylation that was not attenuated by the estrogen receptor antagonist, ICI 182,780. Physiological levels of E2 in HepG2 cells increase GH stimulation of IGF-1 production apparently through increased phosphorylated STAT5b levels and transcriptional activation of the IGF-1 gene. The enhanced SULT1E1 activity may have a role in inhibiting GH-stimulated STAT5b phosphorylation and IGF-1 synthesis via the sulfation and inactivation of E2.

Association of SULT2A1 allelic variants with plasma adrenal androgens and prostate cancer in African American men.

Dehydroepiandrosterone (DHEA) sulfate which is present at micromolar levels in the plasma, can be desulfated to supply free DHEA for metabolism to androgens or estrogens in peripheral tissues. Human cytosolic sulfotransferase (SULT) 2A1 catalyzes DHEA sulfation in the adrenal cortex. Three SULT2A1 nonsynonymous coding single nucleotide polymorphisms (SNPs), identified only in African Americans (AA), are associated with decreased levels of activity and expression as compared to wild-type cDNA when expressed in COS cells. To test whether the SNPs are associated with decreased plasma androgens, 124 normal AA men were genotyped and plasma DHEA, DHEA-sulfate and testosterone levels determined. The two SNPs identified in these participants occurred at allelic frequencies of 0.044 (G187C) and 0.101 (G781A). The G187C SNP was highly linked to the G781A SNP. Although no differences in hormone levels were associated with the individual SNPs, a significant increase in the DHEA:DHEA-sulfate ratio was observed in participants with a heterozygous G187C/G781A genotype. Increased free DHEA levels may result in increased testosterone synthesis and stimulation in the prostate, therefore a group of AA prostate cancer (PC) patients and controls were genotyped. No significant association of the presence of the different SULT2A1 alleles with the occurrence of PC was detected.

Regulation of gene expression in inflammatory bowel disease and correlation with IBD drugs: screening by DNA microarrays.

Potential biomarkers for Crohn's disease (CD) and ulcerative colitis (UC) were identified from two sets of full thickness pathologic samples utilizing DermArray and PharmArray DNA microarrays relative to uninvolved (Un) colon or normal colon. Seven of the over-expressed genes were verified using quantitative RT-PCR (i.e., TMPT, FABP1, IFI27, LCN2, COL11A2, HXB, and metallothionein). By correlating gene expression profiles between inflammatory bowel disease (IBD) tissue samples and IBD drug-treated cell cultures it might be possible to identify new candidate molecular target genes for IBD therapy and drug discovery. Potential biomarkers for CaCo2 cell cultures, which are routinely used as a GI tract surrogate model for in vitro pharmacokinetic studies, treated with azathioprine, 5-aminosalicylic acid, metronidazole, and prednisone were also identified from another experiment. Metallothionein mRNA expression was found to be down-regulated in azathioprine-treated CaCo2 cells, and was coincidentally up-regulated in the CD sample, thus resulting in an anti-correlation. These results suggest that this new screening methodology is feasible, that metallothioneins might be biomarkers for azathioprine therapy in vivo in CD, and that azathioprine might mechanistically down-regulate metallothionein gene expression. Correlations were also observed between IBD samples and either metronidazole- or 5-aminosalicylic acid-treated CaCo2 cells. Similar comparisons of disease tissue samples in vivo vs drug-treated cell cultures in vitro might reveal new mechanistic insights concerning established or experimental drug therapies. This affordable in vitro methodology is promising for expanded studies of IBD and other diseases.

Denosumab for the prevention of skeletal-related events in patients with bone metastasis from solid tumor.

Most patients with advanced malignancy develop bone metastases during the course of their disease. For the remainder of the patient's life, these bone metastases lead to skeletal-related events such as pathologic fractures and spinal cord compression, as well as bone pain or lesions requiring palliative radiation therapy or surgery to prevent or treat fractures. Skeletal-related events result in increased morbidity, mortality and health care costs. For the past decade, intravenous bisphosphonates (zoledronic acid, pamidronate) have been recognized as the primary pharmacologic options in the prevention or treatment of skeletal-related events in patients with bone metastasis. Recently, the United States Food and Drug Administration approved denosumab, a fully human monoclonal antibody, for the prevention of skeletal-related events in patients with bone metastases from solid tumors. Three prominent clinical trials were conducted to establish the efficacy of denosumab. In two of three trials, denosumab was found to delay the time to first skeletal-related event significantly more than zoledronic acid in patients with breast or castration-resistant prostate cancer with bone metastasis. The third trial found denosumab to be noninferior to zoledronic acid in patients with metastases from solid tumors, excluding breast and prostate solid tumors. Overall survival and progression-free survival were similar between zoledronic acid and denosumab. Thus, evidence is insufficient to prove a greater efficacy of one agent over the other. According to the American Society of Clinical Oncology and the National Comprehensive Cancer Network, patients with bone metastasis should have zoledronic acid, pamidronate, or denosumab (with calcium and vitamin D supplementation) added to their chemotherapy regimen if they have an expected survival of 3 months or longer and have adequate renal function.

Effects of denosumab on bone mineral density and bone turnover in postmenopausal women.

Osteoporosis is a degenerative bone disease affecting approximately 10 million American adults. Several options are available to prevent development of the disease or slow and even stop its progression. Nonpharmacologic measures include adequate intake of calcium and vitamin D, exercise, fall prevention, and avoidance of tobacco and excessive alcohol intake. Current drug therapy includes bisphosphonates, calcitonin, estrogen or hormone therapy, selective estrogen receptor modulators, and teriparatide. Denosumab, a receptor activator of nuclear factor-K B ligand (RANKL) inhibitor, was recently approved by the United States Food and Drug Administration for treatment of postmenopausal osteoporosis. Patients treated with denosumab experienced significant gains in bone mineral density, rapid reductions in markers of bone turnover, and a reduced risk for new vertebral fracture. Compared with placebo, patients receiving denosumab 60 mg subcutaneously once every 6 months experienced gains in bone mineral density of 6.5-11% when treated for 24-48 months. One trial demonstrated the superiority of denosumab compared with alendronate, but the differences were small. The most common adverse reactions to denosumab include back pain, pain in extremities, musculoskeletal pain, and cystitis. Serious, but rare, adverse reactions include the development of serious infections, dermatologic changes, and hypocalcemia. The recommended dosing of denosumab is 60 mg every 6 months as a subcutaneous injection in the upper arm, upper thigh, or abdomen. Although beneficial effects on bone mineral density and fracture rate have been established in clinical trials, the risks associated with denosumab must be evaluated before therapy initiation. Of concern is the risk of infection, and denosumab should likely be avoided in patients taking immunosuppressive therapy or at high risk for infection. Therefore, bisphosphonates will likely remain as first-line therapy. Denosumab should be considered in patients unable to tolerate or who have adherence issues or contraindications to other therapies.

Regulation of hepatic sulfotransferase (SULT) 1E1 expression and effects on estrogenic activity in cystic fibrosis (CF).

Cystic fibrosis (CF) is a major genetic disease in Caucasians affecting 1 in 2500 newborns. Hepatobiliary pathology is a major cause of morbidity and mortality in CF second only to pulmonary disease. SULT1E1 activity is significantly elevated, generally 20-30-fold, in hepatocytes of mouse models of CF. SULT1E1 is responsible for the inactivation of beta-estradiol (E2) at physiological concentrations via conjugation with sulfonate. The increase in SULT1E1 activity results in the alteration of E2-regulated protein expression in CF mouse liver. To investigate the mechanism by which the absence of CFTR in human cholangiocytes induces SULT1E1 expression in hepatocytes, a membrane-separated human MMNK-1 cholangiocyte and human HepG2 hepatocyte co-culture system was developed. The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in bile duct cholangiocytes but not hepatocytes, whereas SULT1E1 is expressed in hepatocytes but not cholangiocytes. CFTR expression in MMNK-1 cells was inhibited with siRNA by >90% as determined by immunoblot and immunohistochemical analysis. Control and CFTR-siRNA-MMNK-1 cells were co-cultured with HepG2 cells in a Transwell membrane-separated system. After 8h of co-culture, HepG2 cells were removed from exposure to MMNK-1 cells and placed in fresh medium. After 24-48h, expression of SULT1E1 and selected E2-regulated proteins was analyzed in the HepG2 cells. Results demonstrated that SULT1E1 message and activity were selectively induced in HepG2 cells co-cultured with CFTR-deficient MMNK-1 cells. The expression of E2-regulated proteins (IGF-1, GST-P1 and carbonic anhydrase II) was also altered in response to decreased E2 levels. Thus, the loss of CFTR activity in cholangiocytes stimulates the expression of SULT1E1 in hepatocytes by a paracrine mechanism. SULT1E1 expression in HepG2 cells is inducible by sterol mediated liver-X-receptor (LXR) activation although not by progestins that induce SULT1E1 in the endometrium. SULT1E1 induction in the human cholangiocyte/hepatocyte co-culture system is consistent with and supports the results observed in CF mice. The changes in hepatocyte gene expression affect liver biochemistry and may facilitate the development of CF liver disease.

Biomarkers of human cutaneous squamous cell carcinoma from tissues and cell lines identified by DNA microarrays and qRT-PCR.

Squamous cell carcinoma (SCC) is the second most common form of skin cancer in Caucasians. Here we report on the identification of biomarkers of human cutaneous SCC cell lines in vitro and tissue samples in vivo using DermArray and PharmArray DNA microarrays, consisting of ca. 7400 unique human cDNAs. Differentially expressed genes were identified in two facial skin SCC cell lines (SCC 12 and SCC 13) compared to normal keratinocytes, and three cutaneous SCC tissue samples compared to normal skin. Quantitative validations of up- and down-regulated biomarkers were performed by qRT-PCR on 23 biomarker genes for the cell lines and 20 biomarker genes for the tumor tissues. In addition, three oral SCC cell lines were also included in the qRT-PCR validations for comparison, and the biomarker profiles were highly similar between the cutaneous and the oral SCC cell lines for all 23 biomarkers examined. The expression profiles for a variety of non-cutaneous SCC types, such as head-and-neck, oral, and lung, have been previously published. This report is the first to describe biomarkers for cutaneous SCC in two contexts, in vitro and in vivo. Although there was minimal overlap between the two different contexts using DNA microarrays, five genes were found common to both the cell lines and tissues, namely fibronectin 1, annexin A5, glyceraldehyde 3-phosphate dehydrogenase, zinc-finger protein 254, and huntingtin-associated protein interacting protein. Some of our previously published biomarkers of normal keratinocytes were down-regulated in SCC, suggestive of the dedifferentiated status of the transformed cells. While recent reports have identified some of the same genes as SCC biomarkers, for instance in head-and-neck cancer, thereby validating our approach, we have identified some novel biomarkers for cutaneous disease. These biomarker lists may be useful in molecular diagnostics of non-melanoma skin cancer, and a subset of the biomarkers might serve as suitable targets for drug discovery efforts of therapies for SCC.

DNA microarrays and likelihood ratio bioinformatic methods: discovery of human melanocyte biomarkers.

In this article, some of the advantages and limitations of DNA microarray technologies for gene expression profiling are summarized. As a model experiment, DermArray DNA microarrays were utilized to identify potential biomarkers of cultured normal human melanocytes in two different experimental comparisons. In the first case, melanocyte RNA was compared with vastly dissimilar non-melanocytic RNA samples of normal skin keratinocytes and fibroblasts. In the second case, melanocyte RNA was compared with a primary cutaneous melanoma line (MS7) and a metastatic melanoma cell line (SKMel-28). The alternative approaches provide dramatically different lists of 'normal melanocyte' biomarkers. The most robust biomarkers were identified using principal component analysis bioinformatic methods related to likelihood ratios. Only three of 25 robust biomarkers in the melanocyte-proximal study (i.e. melanocytes vs. melanoma cells) were coincidentally identified in the melanocyte-distal study (i.e. melanocytes vs. non-melanocytic cells). Selected up-regulated biomarkers of melanocytes (i.e. TRP-1, melan-A/MART-1, silver/Pmel17, and nidogen-2) were validated by qRT-PCR. Some of the melanocytic biomarkers identified here may be useful in molecular diagnostics, as potential molecular targets for drug discovery, and for understanding the biochemistry of melanocytic cells.

Biomarkers of human skin cells identified using DermArray DNA arrays and new bioinformatics methods.

Biomarker genes of human skin-derived cells were identified by new simple bioinformatic methods and DNA microarray analysis utilizing in vitro cultures of normal neonatal human epidermal keratinocytes, melanocytes, and dermal fibroblasts. A survey of 4405 human cDNAs was performed using DermArray DNA microarrays. Biomarkers were rank ordered by "likelihood ratio" algorithms and stringent selection criteria that have general applicability for analyzing a minimum of three RNA samples. Signature biomarker genes (up-regulated in one cell type) and anti-signature biomarker genes (down-regulated in one cell type) were determined for the three major skin cell types. Many of the signature genes are known biomarkers for these cell types. In addition, 17 signature genes were identified as ESTs, and 22 anti-signature biomarkers were discovered. Quantitative RT-PCR was used to verify nine signature biomarker genes. A total of 158 biomarkers of normal human skin cells were identified, many of which may be valuable in diagnostic applications and as molecular targets for drug discovery and therapeutic intervention.

A method to improve selection of molecular targets by circumventing the ADME pharmacokinetic system utilizing PharmArray DNA microarrays.

DNA microarrays may be used to identify potential molecular targets for drug discovery. Yet, DNA microarray experiments provide massive amounts of data. To limit the choice of potential molecular targets, it may be desirable to eliminate genes coincidentally up-regulated in tissues implicated in absorption, distribution, metabolism, and excretion (ADME) pharmacokinetics. DNA microarray experiments were performed to demonstrate a gene-exclusion approach using as an example RNA samples of neural origin, i.e., a human neuroblastoma cell line (SK-N-SH) and brain tissue, as the intended hypothetical site(s) of drug action. Biomarkers were identified using PharmArray DNA microarrays. The lists of neuroblastoma and neural biomarkers were constrained by limiting selection to the subset of genes that were not highly expressed in three transformed cell lines from liver, colon, and kidney (HepG2, Caco-2, and 786-O, respectively) that are routinely used as representatives of the ADME system during in vitro pharmacology and toxicology experiments. Principal component analysis methods with likelihood ratio-related bioinformatic tools were utilized to identify robust potential biomarker genes for the three ADME-related cell lines, neuroblastoma, and normal brain. Biomarkers of each sample were identified and selected genes were validated by qRT-PCR. Hundreds of biomarkers of the three ADME-related cell types, representing hepatocytes, kidney epithelium, and gastrointestinal tract, may now be used as a valuable database to restrict selection of biomarkers as potential molecular targets from the intended samples (e.g., neuroblastoma in this work). In addition to biomarker discovery per se, this demonstration suggests that our model method may be viable to help restrict gene lists during selection of potential molecular targets for subsequent drug discovery.