Getting the Dose Right in Drug Development for Rare Diseases: Barriers and Enablers.

In the relentless pursuit of optimizing drug development, the intricate process of determining the ideal dosage unfolds. This involves "dose-finding" studies, crucial for providing insights into subsequent registration trials. However, the challenges intensify when tackling rare diseases. The complexity arises from poorly understood pathophysiologies, scarcity of appropriate animal models, and limited natural history understanding. The inherent heterogeneity, coupled with challenges in defining clinical end points, poses substantial challenges, hindering the utility of available data. The small affected population, low disease awareness, and restricted healthcare access compound the difficulty in conducting dose-finding studies. This white paper delves into critical dose selection aspects, focusing on key therapeutic areas, such as oncology, neurology, hepatology, metabolic rare diseases. It also explores dose selection challenges posed by pediatric rare diseases as well as novel modalities, including enzyme replacement therapies, cell and gene therapies, and oligonucleotides. Several examples emphasize the pivotal role of clinical pharmacology in navigating the complexities associated with these diseases and emerging treatment modalities.

Functionalization of Ceramic Scaffolds with Exosomes from Bone Marrow Mesenchymal Stromal Cells for Bone Tissue Engineering.

The functionalization of bone substitutes with exosomes appears to be a promising technique to enhance bone tissue formation. This study investigates the potential of exosomes derived from bone marrow mesenchymal stromal cells (BMSCs) to improve bone healing and bone augmentation when incorporated into wide open-porous 3D-printed ceramic Gyroid scaffolds. We demonstrated the multipotent characteristics of BMSCs and characterized the extracted exosomes using nanoparticle tracking analysis and proteomic profiling. Through cell culture experimentation, we demonstrated that BMSC-derived exosomes possess the ability to attract cells and significantly facilitate their differentiation into the osteogenic lineage. Furthermore, we observed that scaffold architecture influences exosome release kinetics, with Gyroid scaffolds exhibiting slower release rates compared to Lattice scaffolds. Nevertheless, in vivo implantation did not show increased bone ingrowth in scaffolds loaded with exosomes, suggesting that the scaffold microarchitecture and material were already optimized for osteoconduction and bone augmentation. These findings highlight the lack of understanding about the optimal delivery of exosomes for osteoconduction and bone augmentation by advanced ceramic scaffolds.

Effect of N-Vinyl-2-Pyrrolidone (NVP), a Bromodomain-Binding Small Chemical, on Osteoblast and Osteoclast Differentiation and Its Potential Application for Bone Regeneration.

The human skeleton is a dynamic and remarkably organized organ system that provides mechanical support and performs a variety of additional functions. Bone tissue undergoes constant remodeling; an essential process to adapt architecture/resistance to growth and mechanical needs, but also to repair fractures and micro-damages. Despite bone's ability to heal spontaneously, certain situations require an additional stimulation of bone regeneration, such as non-union fractures or after tumor resection. Among the growth factors used to increase bone regeneration, bone morphogenetic protein-2 (BMP2) is certainly the best described and studied. If clinically used in high quantities, BMP2 is associated with various adverse events, including fibrosis, overshooting bone formation, induction of inflammation and swelling. In previous studies, we have shown that it was possible to reduce BMP2 doses significantly, by increasing the response and sensitivity to it with small molecules called "BMP2 enhancers". In the present study, we investigated the effect of N-Vinyl-2-pyrrolidone (NVP) on osteoblast and osteoclast differentiation in vitro and guided bone regeneration in vivo. We showed that NVP increases BMP2-induced osteoblast differentiation and decreases RANKL-induced osteoclast differentiation in a dose-dependent manner. Moreover, in a rabbit calvarial defect model, the histomorphometric analysis revealed that bony bridging and bony regenerated area achieved with NVP-loaded poly (lactic-co-glycolic acid (PLGA) membranes were significantly higher compared to unloaded membranes. Taken together, our results suggest that NVP sensitizes BMP2-dependent pathways, enhances BMP2 effect, and inhibits osteoclast differentiation. Thus, NVP could prove useful as "osteopromotive substance" in situations where a high rate of bone regeneration is required, and in the management of bone diseases associated with excessive bone resorption, like osteoporosis.

A Dynamic Quantitative Systems Pharmacology Model of Inflammatory Bowel Disease: Part 2 - Application to Current Therapies in Crohn's Disease.

Inflammatory bowel disease (IBD) is a heterogeneic disease with a variety of treatments targeting different mechanisms. A multistate, mechanistic, mathematical model of IBD was developed in part 1 of this two-part article series. In this paper, application of the model to predict response of key clinical biomarkers following different treatment options for Crohn's disease was explored. Five therapies, representing four different mechanisms of action, were simulated in the model and longitudinal profiles of key clinical markers, C-reactive protein and fecal calprotectin were compared with clinical observations. Model simulations provided an accurate match with both central tendency and variability observed in biomarker profiles. We also applied the model to predict biomarker and clinical response in an experimental, combination therapy of existing therapeutic options and provide possible mechanistic basis for the increased response. Overall, we present a validated, modular, mechanistic model construct, which can be applied to explore key biomarkers and clinical outcomes in IBD.

First-in-human, randomized dose-escalation study of the safety, tolerability, pharmacokinetics, pharmacodynamics and immunogenicity of PF-06480605 in healthy subjects.

AIMS: Human genetic, tissue expression, proteomics, transcriptomics and nonclinical studies implicate tumour necrosis factor α-like ligand 1A (TL1A) as a novel target in inflammatory bowel disease (IBD). PF-06480605, a fully human immunoglobulin G1 monoclonal antibody, targets TL1A. This first-in-human, Phase 1, dose-escalation study assessed safety, tolerability, pharmacokinetics, pharmacodynamics and immunogenicity of intravenous (IV) and subcutaneous (SC) PF-06480605 in healthy subjects (NCT01989143). METHODS: Ninety-two subjects were randomized to single ascending doses (SAD), PF-06480605 1 mg, 3 mg, 10 mg, 30 mg, 100 mg, 300 mg, 600 mg or 800 mg IV, or multiple ascending doses (MAD), PF-06480605 3 × 500 mg IV, or 3 × 30 mg, 3 × 100 mg, or 3 × 300 mg SC every 2 weeks for three doses, or placebo. Safety, tolerability, pharmacokinetics, immunogenicity profiles and total TL1A, anti-drug antibody (ADA) and neutralizing antibody (NAb) levels were assessed at pre-determined times. RESULTS: PF-06480605 SAD up to 800 mg IV and MAD up to 300 mg ×3 SC and 500 mg ×3 IV were well tolerated. Overall, there were 45 and 44 treatment-emergent adverse events in SAD and MAD cohorts, respectively, and no deaths or serious adverse events. PF-06480605 exposure generally increased dose-dependently. ADA and NAb levels did not impact safety, pharmacokinetics, or pharmacodynamics at higher doses. Target engagement was demonstrated through dose-dependent differences in serum total soluble TL1A concentrations for PF-06480605 vs placebo cohorts. CONCLUSIONS: PF-06480605 was generally well tolerated, and binding of soluble TL1A was maintained throughout the dose interval, supporting further study of PF-06480605 in patients with IBD and other inflammatory conditions.

The Use of Adipose Tissue-Derived Progenitors in Bone Tissue Engineering - a Review.

2500 years ago, Hippocrates realized that bone can heal without scaring. The natural healing potential of bone is, however, restricted to small defects. Extended bone defects caused by trauma or during tumor resections still pose a huge problem in orthopedics and cranio-maxillofacial surgery. Bone tissue engineering strategies using stem cells, growth factors, and scaffolds could overcome the problems with the treatment of extended bone defects. In this review, we give a short overview on bone tissue engineering with emphasis on the use of adipose tissue-derived stem cells and small molecules.

Applying the best of oncology drug development paradigms to the non-malignant space.

With 80-90% of drugs entering the clinic not meeting regulatory approval (a high cost of failure), there is a major need for innovation in the clinical development space. Features of the new era of practice-changing innovation in oncology have included novel clinical trial designs incorporating multiple new molecular entities and/or multiple patient types, patient selection strategies (which allow detection of early signs of efficacy), and use of surrogate endpoints to achieve speedy regulatory approval. Disease areas beyond oncology could benefit from the application of specific aspects of these approaches. Here, we describe several such potential adaptations of the approaches, with scenarios and prerequisites, which could help reduce the costs of, and accelerate, clinical drug development with confidence.

The bromodomain inhibitor N-methyl pyrrolidone reduced fat accumulation in an ovariectomized rat model.

BACKGROUND: Weight gain is one of the consequences of estrogen deficiency and constitutes a major health problem. The present study highlights the effects of N-methyl pyrrolidone (NMP) on adipogenesis in osteoporosis induced by estrogen deficiency in an ovariectomized rat model. RESULTS: Ovariectomy resulted in body weight gain, increased femoral marrow adipocytes, and hypertrophic adipocytes in white adipose tissue, distorted serum leptin, and TNF-α and PPARγ levels. Treatment with NMP normalized these parameters similar to the control group. In vitro, NMP inhibited the differentiation of 3T3-L1 pre-adipocytes and hMSCs, indicating its anti-adipogenic effect. Moreover, PPARγ was significantly reduced with NMP treatment in in vivo and in vitro experiments. NMP inhibited BRD2 and BRD4 binding in an AlphaScreen assay, with an IC50 of 3 and 4 mM, respectively. The effect of NMP was consistent with its role as a bromodomain inhibitor. CONCLUSIONS: Our data indicates that NMP inhibits the adipogenic effect of estrogen deficiency at the level of PPARγ expression by BRD4 inhibition.

Basal mTORC2 activity and expression of its components display diurnal variation in mouse perivascular adipose tissue.

In adipose tissue mTOR complex 2 (mTORC2) contributes to the regulation of glucose/lipid metabolism and inflammatory molecule expression. Both processes display diurnal variations during the course of the day. RICTOR and mSIN1 are unique and essential components of mTORC2, which is activated by growth factors including insulin. To assess whether mTORC2 components display diurnal variations, we analyzed steady state mRNA expression levels of Rictor, mSin1, and mTor in various adipose tissues during a 24 h period. Diurnally regulated expression of Rictor was detected in brown adipose tissues displaying highest mRNA expression levels at the beginning of the 12 h light period (zeitgeber time 2, ZT2). Gene expression patterns of mSin1 and mTor displayed a similar diurnal regulation as Rictor in PVAT while smaller changes were detected for these genes in aorta during the course of the day. Basal mTORC2 activity was measured by phosphorylation of protein kinase C (PKC) α at serine 657 was higher at ZT14 as compared with ZT2 in PVAT. In line, gene expression of inflammatory molecules nitric oxide synthase 2 and tumor necrosis factor α was lower at ZT 14 compared to ZT2. Our findings provide evidence for a diurnal regulation of expression of mTORC2 components and activity. Hence, mTORC2 is possibly an integral part of diurnally regulated signaling pathways in PVAT and possibly in other adipose tissues.

Hypoxia potentiates tumor necrosis factor-α induced expression of inducible nitric oxide synthase and cyclooxygenase-2 in white and brown adipocytes.

Obesity involves hypoxic adipose tissue and low-grade chronic inflammation. We investigated the impact of hypoxia on inflammatory response to TNF-α in white and brown adipocytes. In response to TNF-α, the expression of the inducible enzymes iNOS and COX-2 was prominently and selectively potentiated during hypoxia while only moderately under normoxia. Levels of their products, nitrite and prostaglandinE2 were elevated accordingly. NS398, a selective COX-2 inhibitor, reduced nitrite levels. The expression of PGC-1α, a transcriptional co-activator involved in mitochondrial biogenesis, and PPARγ, a transcription factor involved in adipocyte homeostasis, was reduced by TNF-α during hypoxia. These results suggest that hypoxia potentiates the inflammatory response by TNF-α in both white and brown adipocytes and downregulates the transcription factors involved in adipocyte function.

Reassessment of tigecycline bone concentrations in volunteers undergoing elective orthopedic procedures.

The goal of the this study was to re-evaluate tigecycline bone concentrations in subjects undergoing elective orthopedic surgery, using multiple doses and a more robust bone assay than was used in a previous study. Each subject received three intravenous doses of tigecycline (one 100-mg infusion followed by two 50-mg infusions, each administered over 30 minutes). A single bone sample was collected from each subject at one of the following times: 1, 2, 4, 6, 8, or 12 hours after the third dose. Four blood samples were collected from each subject: before the first dose, before and after the third dose, and within 15 minutes of the collection time of the bone sample. Noncompartmental pharmacokinetic analysis serum and bone area under the curve for the given dose interval (AUCτ ) values were 2,402 ng h/mL and 11,465 ng h/g, and maximum concentration (Cmax ) values were 974 ng/mL and 2,262 ng/g, respectively. The bone to serum ratio calculated using the AUCτ values was 4.77, confirming tigecycline penetration into bone.

Rictor in perivascular adipose tissue controls vascular function by regulating inflammatory molecule expression.

OBJECTIVE: Perivascular adipose tissue (PVAT) wraps blood vessels and modulates vasoreactivity by secretion of vasoactive molecules. Mammalian target of rapamycin complex 2 (mTORC2) has been shown to control inflammation and is expressed in adipose tissue. In this study, we investigated whether adipose-specific deletion of rictor and thereby inactivation of mTORC2 in PVAT may modulate vascular function by increasing inflammation in PVAT. APPROACH AND RESULTS: Rictor, an essential mTORC2 component, was deleted specifically in mouse adipose tissue (rictor(ad-/-)). Phosphorylation of mTORC2 downstream target Akt at Serine 473 was reduced in PVAT from rictor(ad-/-) mice but unaffected in aortic tissue. Ex vivo functional analysis of thoracic aortae revealed increased contractions and impaired dilation in rings with PVAT from rictor(ad-/-) mice. Adipose rictor knockout increased gene expression and protein release of interleukin-6, macrophage inflammatory protein-1α, and tumor necrosis factor-α in PVAT as shown by quantitative real-time polymerase chain reaction and Bioplex analysis for the cytokines in the conditioned media, respectively. Moreover, gene and protein expression of inducible nitric oxide synthase was upregulated without affecting macrophage infiltration in PVAT from rictor(ad-/-) mice. Inhibition of inducible nitric oxide synthase normalized vascular reactivity in aortic rings from rictor(ad-/-) mice with no effect in rictor(fl/fl) mice. Interestingly, in perivascular and epididymal adipose depots, high-fat diet feeding induced downregulation of rictor gene expression. CONCLUSIONS: Here, we identify mTORC2 as a critical regulator of PVAT-directed protection of normal vascular tone. Modulation of mTORC2 activity in adipose tissue may be a potential therapeutic approach for inflammation-related vascular damage.

Repeat-dose sirolimus pharmacokinetics and pharmacodynamics in patients with hepatic allografts.

PURPOSE: To determine sirolimus steady-state pharmacokinetics, and to assess the relationship between time-normalized trough sirolimus concentration (C(min,TN)) and evidence of efficacy (rejection and death) and adverse reactions (stomatitis and pneumonia) in liver allograft patients. METHODS: Dense sampling of sirolimus was performed over a single daily-dosing interval in 11 hepatic allograft recipients on day 28 and at 3 months after start of treatment. Serial trough concentration sampling was performed in 380 hepatic allograft recipients on days 1, 7, 14, 28, 42, 60, 90, 180, 270 and 360 after start of treatment. Occurrence of stomatitis, pneumonia, rejection, and death were collected for 360 days after start of treatment. Noncompartmental pharmacokinetic parameters were analyzed in the 11 densely sampled patients; C(min,TN) was determined in the 380 patients. RESULTS: Mean maximum concentration (C(max)), time to C(max) (t(max)), area under the curve for the given dose interval (AUC(tau)), and whole blood oral clearance at 3 months were 20.8 ± 7.6 ng/mL, 3 ± 1 h, 338 ± 144 ng·h/mL, and 10.0 ± 5.6 L/hr, respectively. In the 11 densely sampled patients, linear regression showed that C(min,TN) was highly predictive of AUC(tau) (r² = 0.77, P < 0.0001) at each analysis time point. Logistic regression showed a relationship between C(min,TN) in the 380 patients and pneumonia occurrence, but not between C(min,TN) and stomatitis, rejection, or death. CONCLUSIONS: In this study, the pharmacokinetic profile of sirolimus in hepatic allograft patients was consistent with that of renal transplantation recipients. With the exception of pneumonia, no correlation was observed between C(min,TN) and the occurrence of adverse events of interest.

Regulatory role of G protein-coupled estrogen receptor for vascular function and obesity.

We found that the selective stimulation of the intracellular, transmembrane G protein-coupled estrogen receptor (GPER), also known as GPR30, acutely lowers blood pressure after infusion in normotensive rats and dilates both rodent and human arterial blood vessels. Stimulation of GPER blocks vasoconstrictor-induced changes in intracellular calcium concentrations and vascular tone, as well as serum-stimulated cell proliferation of human vascular smooth muscle cells. Deletion of the GPER gene in mice abrogates vascular effects of GPER activation and is associated with visceral obesity. These findings suggest novel roles for GPER in protecting from cardiovascular disease and obesity.

Choroidal melanoma metastatizing to the biliary system: A diagnostic dilemma.

Metastatic melanoma to the gall bladder is extremely rare and is associated with a very poor prognosis. We report a case of choroidal melanoma metastatizing to the hepatobiliary system, with an unusual presentation. Our patient presenting with obstructive jaundice was misdiagnosed as having carcinoma of the gall-bladder, but the diagnosis of metastatic melanoma to the gallbladder was confirmed by ultrasonography guided fine needle aspiration cytology (USG-FNAC). On reviewing the past history, the patient had a history of enucleation for choroidal melanoma. Even though the liver 'is' a common site for metastasis of choroid melanoma, the patient presenting with a suspected gall bladder mass 'is' a rare presentation. Hence, gastrointestinal symptoms and a history of melanoma should be investigated for the presence of gastrointestinal or liver metastases, even if the original primary malignancy was diagnosed years before the patient's presentation.

Distinct roles of estrogen receptors alpha and beta mediating acute vasodilation of epicardial coronary arteries.

This study investigated the contribution of estrogen receptors (ERs) alpha and beta for epicardial coronary artery function, vascular NO bioactivity, and superoxide (O(2)(-)) formation. Porcine coronary rings were suspended in organ chambers and precontracted with prostaglandin F(2alpha) to determine direct effects of the selective ER agonists 4,4',4''-(4-propyl-[(1)H]pyrazole-1,3,5-triyl)tris-phenol (PPT) or 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) or the nonselective ER agonist 17beta-estradiol. Indirect effects on contractility to U46619 and relaxation to bradykinin were assessed and effects on NO, nitrite, and O(2)(-) formation were measured in cultured cells. Within 5 minutes, selective ERalpha activation by PPT, but not 17beta-estradiol or the ERbeta agonist DPN, caused rapid, NO-dependent, and endothelium-dependent relaxation (49+/-5%; P<0.001 versus ethanol). PPT also caused sustained endothelium- and NO-independent vasodilation similar to 17beta-estradiol after 60 minutes (72+/-3%; P<0.001 versus ethanol). DPN induced endothelium-dependent NO-independent relaxation via endothelium-dependent hyperpolarization (40+/-4%; P<0.01 versus ethanol). 17beta-Estradiol and PPT, but not DPN, attenuated the responses to U46619 and bradykinin. All of the ER agonists increased NO and nitrite formation in vascular endothelial but not smooth muscle cells and attenuated vascular smooth muscle cell O(2)(-) formation (P<0.001). ERalpha activation had the most potent effects on both nitrite formation and inhibiting O(2)(-) (P<0.05). These data demonstrate novel and differential mechanisms by which ERalpha and ERbeta activation control coronary artery vasoreactivity in males and females and regulate vascular NO and O(2)(-) formation. The findings indicate that coronary vascular effects of sex hormones differ with regard to affinity to ERalpha and ERbeta, which will contribute to beneficial and adverse effects of hormone replacement therapy.

Differential effects of 17beta-estradiol on function and expression of estrogen receptor alpha, estrogen receptor beta, and GPR30 in arteries and veins of patients with atherosclerosis.

Venous complications have been implicated in the adverse effects of hormone replacement therapy. This study investigated acute effects of the natural estrogen, 17beta-estradiol, on function, estrogen receptors/GPR30 expression, and kinase activation in vascular rings and cultured smooth muscle cells from arteries and veins of patients with coronary artery disease. Changes in vascular tone of internal mammary arteries and saphenous veins exposed to the steroid were recorded. 17Beta-estradiol caused concentration-dependent, endothelium-independent relaxation in arteries (P<0.05 versus solvent control) but not in veins (P not significant). 17Beta-estradiol enhanced contractions to endothelin-1 in veins but not in arteries. The novel membrane estrogen receptor GPR30 was detected in both vessels. Moreover, gene expression of estrogen receptor beta was 10-fold higher than that of estrogen receptor alpha or GPR30 (P<0.05). Expression of all 3 of the receptors was reduced after exposure to 17beta-estradiol in arteries but not in veins (P<0.05). Basal phosphorylation levels of extracellular signal-regulated kinase were higher in venous than in arterial smooth muscle cells and were increased by 17beta-estradiol in arterial cells only. In summary, this is the first study to report that, in human arteries but not in veins, 17beta-estradiol acutely affects vascular tone, estrogen receptor expression, including GPR30, and extracellular signal-regulated kinase phosphorylation. These data indicate that effects of natural estrogens in humans differ between arterial and venous vascular beds, which may contribute to the vascular risks associated with menopause or hormone therapy.

Endothelin-1 inhibits adipogenesis: role of phosphorylation of Akt and ERK1/2.

In adipogenesis, growth factors play a crucial role. Using serum depleted condition, we studied the causal role of endothelin-1 (ET-1) and epidermal growth factor (EGF), separately or together, in adipocyte differentiation of 3T3-L1 cells. ET-1 stimulation caused an anti-adipogenic response and this effect was potentiated upon treatment with EGF. Co-treatment with EGF and ET-1 blocked the expression of C/EBPalpha and PPARgamma, the adipogenic markers. The inhibition of adipogenesis was preceded by a biphasic (early and late) attenuation of Akt phosphorylation. We suggest that treatment with ET-1 and EGF together induce a more potent anti-adipogenic response, involving increased Erk1/2 phosphorylation and biphasic attenuation of Akt phosphorylation.