Drug Dissolution in Oral Drug Absorption: Workshop Report.

The in-person workshop "Drug Dissolution in Oral Drug Absorption" was held on May 23-24, 2023, in Baltimore, MD, USA. The workshop was organized into lectures and breakout sessions. Three common topics that were re-visited by various lecturers were amorphous solid dispersions (ASDs), dissolution/permeation interplay, and in vitro methods to predict in vivo biopharmaceutics performance and risk. Topics that repeatedly surfaced across breakout sessions were the following: (1) meaning and assessment of "dissolved drug," particularly of poorly water soluble drug in colloidal environments (e.g., fed conditions, ASDs); (2) potential limitations of a test that employs sink conditions for a poorly water soluble drug; (3) non-compendial methods (e.g., two-stage or multi-stage method, dissolution/permeation methods); (4) non-compendial conditions (e.g., apex vessels, non-sink conditions); and (5) potential benefit of having both a quality control method for batch release and a biopredictive/biorelevant method for biowaiver or bridging scenarios. An identified obstacle to non-compendial methods is the uncertainty of global regulatory acceptance of such methods.

Regulation of human 3 alpha-hydroxysteroid dehydrogenase (AKR1C4) expression by the liver X receptor alpha.

Type I human hepatic 3alpha-hydroxysteroid dehydrogenase (AKR1C4) plays a significant role in bile acid biosynthesis, steroid hormone metabolism, and xenobiotic metabolism. Utilization of a hidden Markov model for predictive modeling of nuclear hormone receptor response elements coupled with chromatin immunoprecipitation/microarray technology revealed a putative binding site in the AKR1C4 promoter for the nuclear hormone receptor known as liver X receptor alpha, (LXRalpha [NR1H3]), which is the physiological receptor for oxidized cholesterol metabolites. The putative LXRalpha response element (LXRE), identified by chromatin immunoprecipitation, was approximately 1.5 kilobase pairs upstream of the transcription start site. LXRalpha was shown to bind specifically to this LXRE and mediate transcriptional activation of the AKR1C4 gene, leading to increased AKR1C4 protein expression. These data suggest that LXRalpha may modulate the bile acid biosynthetic pathway at a unique site downstream of CYP7A1 and may also modulate the metabolism of steroid hormones and certain xenobiotics.

Investigational PPAR-gamma agonists for the treatment of Type 2 diabetes.

The tremendous increase in the global prevalence of Type 2 diabetes (T2D) and its conglomeration of metabolic disorders has dramatically intensified the search for innovative therapies to fight this emerging epidemic. Over the last decade, the family of nuclear receptors, especially the peroxisome proliferator-activated receptors (PPARs), has emerged as one of the most important drug targets aimed at combating the metabolic syndrome. Consequently, compounds that activate the PPARs have served as potential therapeutics for the treatment of T2D and the metabolic anomalies associated with this disorder. This review focuses on the currently marketed compounds and also describes the discovery and development of the next generation of PPAR ligands that are under investigation for the potential treatment of T2D and the metabolic syndrome.

Identification and characterization of noncalcemic, tissue-selective, nonsecosteroidal vitamin D receptor modulators.

Vitamin D receptor (VDR) ligands are therapeutic agents for the treatment of psoriasis, osteoporosis, and secondary hyperparathyroidism. VDR ligands also show immense potential as therapeutic agents for autoimmune diseases and cancers of skin, prostate, colon, and breast as well as leukemia. However, the major side effect of VDR ligands that limits their expanded use and clinical development is hypercalcemia that develops as a result of the action of these compounds mainly on intestine. In order to discover VDR ligands with less hypercalcemia liability, we sought to identify tissue-selective VDR modulators (VDRMs) that act as agonists in some cell types and lack activity in others. Here, we describe LY2108491 and LY2109866 as nonsecosteroidal VDRMs that function as potent agonists in keratinocytes, osteoblasts, and peripheral blood mononuclear cells but show poor activity in intestinal cells. Finally, these nonsecosteroidal VDRMs were less calcemic in vivo, and LY2108491 exhibited more than 270-fold improved therapeutic index over the naturally occurring VDR ligand 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] in an in vivo preclinical surrogate model of psoriasis.

Colocalization of muscleblind with RNA foci is separable from mis-regulation of alternative splicing in myotonic dystrophy.

Myotonic dystrophy type I (DM1), which is caused by a non-coding CTG-repeat expansion in the dystrophia myotonica-protein kinase (DMPK) gene, is an RNA-mediated disease. Expanded CUG repeats in transcripts of mutant DMPK form nuclear foci that recruit muscleblind-like (MBNL) proteins, a family of alternative splicing factors. Although transcripts of mutant DMPK and MBNL proteins accumulate in nuclear RNA foci, it is not clear whether foci formation is required for splicing mis-regulation. Here, we use a co-transfection strategy to show that both CUG and CAG repeats form RNA foci that colocalize with green fluorescent protein (GFP)-MBNL1 and endogenous MBNL1. However, only CUG repeats alter splicing of the two tested pre-mRNAs, cardiac troponin T (cTNT) and insulin receptor (IR). Using FRAP, we demonstrate that GFP-MBNL1 in CUG and CAG foci have similar half-times of recovery and fractions of immobile molecules, suggesting that GFP-MBNL1 is bound by both CUG and CAG repeats. We also find an immobile fraction of GFP-MBNL1 in DM1 fibroblasts and a similar rapid exchange in endogenous CUG RNA foci. Therefore, formation of RNA foci and disruption of MBNL1-regulated splicing are separable events.

Coactivation of the human vitamin D receptor by the peroxisome proliferator-activated receptor gamma coactivator-1 alpha.

The vitamin D receptor (VDR) belongs to the superfamily of steroid/thyroid hormone receptors that is activated by 1alpha,25-dihydroxyvitamin D(3). Traditional targets for 1alpha,25-dihydroxyvitamin D(3) action include tissues involved in the maintenance of calcium homeostasis and bone development and remodeling. Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha), a transcriptional coactivator that plays a role in mitochondrial biogenesis and energy metabolism, is predominantly expressed in kidney, heart, liver, and skeletal muscle. Because VDR and PGC-1alpha display an overlapping pattern of expression, we investigated the possibility that PGC-1alpha could serve as a coactivator for VDR. Transient cotransfection assays demonstrate that PGC-1alpha augments ligand-dependent VDR transcription when either full-length VDR or Gal4 DNA binding domain-VDR-ligand binding domain chimeras were analyzed. Furthermore, mammalian two-hybrid assays, coimmunoprecipitation analyses, and biochemical coactivator recruitment assays demonstrate a ligand-dependent interaction between the two proteins both in cells and in vitro. The coactivation potential of PGC-1alpha requires an intact AF-2 domain of VDR and the LXXLL motif in PGC-1alpha. Taken together, these results indicate that PGC-1alpha serves as a coactivator for VDR.

Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor.

The pyruvate dehydrogenase complex (PDC) functions as an important junction in intermediary metabolism by influencing the utilization of fat versus carbohydrate as a source of fuel. Activation of PDC is achieved by phosphatases, whereas, inactivation is catalyzed by pyruvate dehydrogenase kinases (PDKs). The expression of PDK4 is highly regulated by the glucocorticoid and peroxisome proliferator-activated receptors. We demonstrate that the farnesoid X receptor (FXR; NR1H4), which regulates a variety of genes involved in lipoprotein metabolism, also regulates the expression of PDK4. Treatment of rat hepatoma cells as well as human primary hepatocytes with FXR agonists stimulates the expression of PDK4 to levels comparable to those obtained with glucocorticoids. In addition, treatment of mice with an FXR agonist significantly increased hepatic PDK4 expression, while concomitantly decreasing plasma triglyceride levels. Thus, activation of FXR may suppress glycolysis and enhance oxidation of fatty acids via inactivation of the PDC by increasing PDK4 expression.

Regulation of carbohydrate metabolism by the farnesoid X receptor.

The farnesoid X receptor (FXR; NR1H4) is a nuclear hormone receptor that functions as the bile acid receptor. In addition to the critical role FXR plays in bile acid metabolism and transport, it regulates a variety of genes important in lipoprotein metabolism. We demonstrate that FXR also plays a role in carbohydrate metabolism via regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression. Treatment of either H4IIE or MH1C1 rat hepatoma cell lines as well as primary rat or human hepatocytes with FXR agonists led to stimulation of PEPCK mRNA expression to levels comparable to those obtained with glucocorticoid receptor agonists. We examined the physiological significance of FXR agonist-induced enhancement of PEPCK expression in primary rat hepatocytes. In addition to inducing PEPCK expression in primary hepatocytes, FXR agonists stimulated glucose output to levels comparable to those observed with a glucocorticoid receptor agonist. Consistent with these observations, treatment of C57BL6 mice with GW4064 significantly increased hepatic PEPCK expression. Activation of FXR initiated a cascade involving induction of peroxisome proliferator-activated receptor alpha and TRB3 expression that is consistent with stimulation of PEPCK gene expression via interference with a pathway that may involve Akt-dependent phosphorylation of Forkhead/winged helix transcription factor (FOXO1). The FXR-peroxisome proliferator-activated receptor alpha-TRB3 pathway was conserved in rat hepatoma cell lines, mice, as well as primary human hepatocytes. Thus, in addition to its role in the regulation of lipid metabolism, FXR regulates carbohydrate metabolism.

Ligand-dependent coactivation of the human bile acid receptor FXR by the peroxisome proliferator-activated receptor gamma coactivator-1alpha.

Peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1alpha) has been shown to play an important role in energy metabolism by coordinating transcriptional programs involved in mitochondrial biogenesis, adaptive thermogenesis, gluconeogenesis, and fatty acid oxidation. PGC-1alpha also plays a crucial role in cholesterol metabolism by serving as a coactivator of the liver X receptor-alpha and inducing the expression of cholesterol 7-alpha-hydroxylase. Here, we demonstrate that PGC-1alpha also functions as an effective coactivator of farnesoid X receptor (FXR), the bile acid receptor. Transient cotransfection assays demonstrate that PGC-1alpha enhances ligand-mediated FXR transcription when either full-length FXR or Gal4 DNA binding domain-FXR-ligand binding domain chimeras were analyzed. Mammalian two-hybrid analyses, glutathione S-transferase affinity chromatography and biochemical coactivator recruitment assays demonstrate ligand-dependent interaction between the two proteins both in vivo and in vitro. PGC-1alpha-mediated coactivation of FXR was highly ligand-dependent and absolutely required an intact activation function-2 (AF-2) domain of FXR and the LXXLL motif in PGC-1alpha. The integrity of the charge clamp was required, further illustrating the role of the ligand binding domain of FXR in PGC-1alpha recognition. Together, these results indicate that PGC-1alpha functions as a potent coactivator for FXR and further implicates its role in the regulation of genes that are involved in bile acid and lipid metabolism.

Pharmacology of nuclear receptor-coregulator recognition.

The nuclear receptor (NR) superfamily comprises approximately 50 members that are responsible for regulating a number of physiologic processes in humans, including metabolism, homeostasis, and reproduction. Included in the superfamily are the receptors for steroids, lipophilic vitamins, bile acids, retinoids, and various fatty acids. NRs exert their action as transcription factors that directly bind to the promoters of target genes and regulate their rate of transcription. To modulate transcription, however, NRs must recruit a number of accessory coregulators known as corepressors and coactivators. These coregulators harbor a variety of activities, such as the ability to modify chromatin structure, interact with basal transcriptional machinery, and modify RNA splicing. Recent studies have revealed that the pharmacological characteristics of various NR ligands are regulated by their ability to modulate the coregulator interaction profile of an NR.

Alternative splicing within the ligand binding domain of the human constitutive androstane receptor.

The human constitutive androstane receptor (hCAR; NR1I3) is a member of the nuclear receptor superfamily. The activity of hCAR is regulated by a variety of xenobiotics including clotrimazole and acetaminophen metabolites. hCAR, in turn, regulates a number of genes responsible for xenobiotic metabolism and transport including several cytochrome P450s (CYP 2B5, 2C9, and 3A4) and the multidrug resistance-associated protein 2 (MRP2, ABCC2). Thus, hCAR is believed to be a mediator of drug-drug interactions. We identified two novel hCAR splice variants: hCAR2 encodes a receptor in which alternative splice acceptor sites are utilized resulting in a 4 amino acid insert between exons 6 and 7, and a 5 amino acid insert between 7 and 8, and hCAR3 encodes a receptor with exon 7 completely deleted resulting in a 39 amino acid deletion. Both hCAR2 and hCAR3 mRNAs are expressed in a pattern similar to the initially described MB67 (hCAR1) with some key distinctions. Although the levels of expression vary depending on the tissue examined, hCAR2 and hCAR3 contribute 6-8% of total hCAR mRNA in liver. Analysis of the activity of these variants indicates that both hCAR2 and hCAR3 lose the ability to heterodimerize with RXR and lack transactivation activity in cotransfection experiments where either full-length receptor or GAL4 DNA-binding domain/CAR ligand binding domain chimeras were utilized. Although the role of hCAR2 and hCAR3 is currently unclear, these additional splice variants may provide for increased diversity in terms of responsiveness to xenobiotics.

Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing.

Myotonic dystrophy type 1 (DM1) is a dominant multisystemic disorder caused by a CTG expansion in the 3' untranslated region of the DMPK gene. A predominant characteristic of DM1 is myotonia resulting from skeletal muscle membrane hyperexcitability. Here we demonstrate loss of the muscle-specific chloride channel (ClC-1) mRNA and protein in DM1 skeletal muscle tissue due to aberrant splicing of the ClC-1 pre-mRNA. The splicing regulator, CUG binding protein (CUG-BP), which is elevated in DM1 striated muscle, binds to the ClC-1 pre-mRNA, and overexpression of CUG-BP in normal cells reproduces the aberrant pattern of ClC-1 splicing observed in DM1 skeletal muscle. We propose that disruption of alternative splicing regulation causes a predominant pathological feature of DM1.