SO078APABETALONE, A BROMODOMAIN AND EXTRA-TERMINAL (BET) PROTEIN INHIBITOR, REDUCES ALKALINE PHOSPHATASE IN CVD PATIENTS, IN MICE, AND IN CELL CULTURE SYSTEMS

Abstract Background and Aims Elevated serum alkaline phosphatase (ALP) independently predicts major adverse cardiac events (MACE) by contributing to vascular calcification and endothelial dysfunction arising in chronic kidney disease (CKD) and cardiovascular disease (CVD). Apabetalone is an orally active inhibitor of bromodomain and extraterminal (BET) proteins – epigenetic readers that modulate gene expression involved in vascular inflammation and calcification. Here we examined apabetalone’s effects on ALP post-hoc in recent clinical trials, then performed mechanistic studies into apabetalone’s impact on tissue non-specific ALP (TNALP) expression in mice and cell culture. Method Serum ALP was determined in CVD patients in phase 2 trials (3 month ASSERT and 6 month SUSTAIN & ASSURE) and in the phase 3 BETonMACE CVD outcomes trial, including subpopulations with CKD (eGFR<60 mL/min/1.73m2). Apabetalone’s effect on expression of TNALP (gene symbol ALPL) was examined in mice, cultured primary human hepatocytes (PHH), HepaRG, HepG2, vascular smooth muscle cells (VSMCs), and vascular endothelial cells by real-time PCR. TNALP protein levels were assessed by immunoblots and flow cytometry. ALP enzyme activity was measured in enzymatic assays. Results In phase 2 trials, baseline serum ALP independently predicted MACE (hazard ratio [HR] 1.6, 95% CI 1.2-2.2, p=0.001). In the 3 month ASSERT trial, apabetalone dose dependently reduced serum ALP (p<0.001 vs placebo). Prominent reductions in ALP were apparent in patients on apabetalone (n=331) vs placebo (n=166) in combined analysis of the ASSURE & SUSTAIN trials (median % change -11 vs -3.2; p<0.001). In the subset with CKD, patients on apabetalone (n=69) had greater reduction in serum ALP than placebo (n=22; p=0.008). Strikingly, ALP reductions in phase 2 correlated with reduction in MACE (HR 0.58, 95% CI 0.44-0.77, p<0.001). Consistent with phase 2, BETonMACE saw serum ALP reduced by 6.8 U/L with apabetalone (n=1082) vs placebo (n=1070; p<0.001) at 24 weeks. At the conclusion of BETonMACE, fewer MACE occurred in the CKD subgroup with apabetalone (n=124) vs placebo (n=164; HR 0.50 95% CI 0.26-0.96 p=0.032). Neither apabetalone nor statins that control low-density lipoprotein cholesterol inhibited recombinant TNALP enzyme activity, implying that decreased serum ALP activity in patients reflected reduction in TNALP production rather than inhibition of the enzyme. Liver-derived TNALP accounts for ≈50% of circulating ALP. In the liver of mice on high fat diet, apabetalone or JQ1 (BET inhibitors with different chemical scaffolds) reduced Alpl mRNA (p<0.001) with corresponding trends in TNALP activity. In PHH, HepaRG, & HepG2 cells, apabetalone dose dependently suppressed ALPL expression by 60-80%. In HepG2 cells, apabetalone reduced TNALP protein (>55%, p<0.001), enzyme activity (> 40%; p<0.001), and % of TNALP positive cells (15-30%; p<0.001). MZ1, which promotes degradation of BET proteins, downregulated ALPL / TNALP similar to apabetalone. In VSMCs, apabetalone or JQ1 suppressed ALPL gene expression, TNALP protein levels, and enzyme activity, leading to decreases in extracellular calcium deposition. In addition, apabetalone downregulated ALPL expression in human aortic, umbilical vein, and brain microvascular endothelial cells by 50-70%. Conclusion Apabetalone lowers serum ALP in clinical trials, which is consistent with reduced hepatic production of TNALP - the most abundant ALP isoform. Further, apabetalone downregulates ALPL gene expression in vascular cell types while reducing calcification. Together, BET-dependent epigenetic modulation of ALP by apabetalone can affect several pathogenetic processes, and thereby improve cardiovascular outcomes. This study provides insights to the CVD event reductions observed in the CKD subpopulation in the BETonMACE Phase 3 trial.

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Clinical Drug Development for the Treatment of Pulmonary Arterial Hypertension: Collaboration With the Pharmaceutical Industry and Regulatory Agencies

Advances in Pulmonary Hypertension ◽

10.21693/1933-088x-9.4.214 ◽

2010 ◽

Vol 9 (4) ◽

pp. 214-219

Author(s):

Robyn J. Barst

Keyword(s):

Clinical Trials ◽

Pulmonary Arterial Hypertension ◽

Drug Development ◽

Phase 1 ◽

Preclinical Studies ◽

Phase 2 ◽

Phase 3 ◽

Preclinical Testing ◽

New Drug ◽

Pulmonary Arterial

Drug development is the entire process of introducing a new drug to the market. It involves drug discovery, screening, preclinical testing, an Investigational New Drug (IND) application in the US or a Clinical Trial Application (CTA) in the EU, phase 1–3 clinical trials, a New Drug Application (NDA), Food and Drug Administration (FDA) review and approval, and postapproval studies required for continuing safety evaluation. Preclinical testing assesses safety and biologic activity, phase 1 determines safety and dosage, phase 2 evaluates efficacy and side effects, and phase 3 confirms efficacy and monitors adverse effects in a larger number of patients. Postapproval studies provide additional postmarketing data. On average, it takes 15 years from preclinical studies to regulatory approval by the FDA: about 3.5–6.5 years for preclinical, 1–1.5 years for phase 1, 2 years for phase 2, 3–3.5 years for phase 3, and 1.5–2.5 years for filing the NDA and completing the FDA review process. Of approximately 5000 compounds evaluated in preclinical studies, about 5 compounds enter clinical trials, and 1 compound is approved (Tufts Center for the Study of Drug Development, 2011). Most drug development programs include approximately 35–40 phase 1 studies, 15 phase 2 studies, and 3–5 pivotal trials with more than 5000 patients enrolled. Thus, to produce safe and effective drugs in a regulated environment is a highly complex process. Against this backdrop, what is the best way to develop drugs for pulmonary arterial hypertension (PAH), an orphan disease often rapidly fatal within several years of diagnosis and in which spontaneous regression does not occur?

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The role of matrix metalloproteinase activity in the maturation of human capillary endothelial cells in vitro

Journal of Cell Science ◽

10.1242/jcs.112.10.1599 ◽

1999 ◽

Vol 112 (10) ◽

pp. 1599-1609 ◽

Cited By ~ 2

Author(s):

B.M. Kraling ◽

D.G. Wiederschain ◽

T. Boehm ◽

M. Rehn ◽

J.B. Mulliken ◽

...

Keyword(s):

Cell Culture ◽

Cell Proliferation ◽

Endothelial Cells ◽

Endothelial Cell ◽

Endothelial Cell Proliferation ◽

Protein Levels ◽

Matrix Deposition ◽

Capillary Endothelial Cells ◽

Vessel Maturation

Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor epsilon-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.

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1480 ONSET OF ACTION AND TIME TO EFFICACY OF AVANAFIL, A NOVEL, RAPID-ONSET PDE5 INHIBITOR IN MEN WITH MILD TO SEVERE ERECTILE DYSFUNCTION DATA FROM PHASE 2 AND PHASE 3 CLINICAL TRIALS

The Journal of Urology ◽

10.1016/j.juro.2012.02.2002 ◽

2012 ◽

Vol 187 (4S) ◽

Cited By ~ 1

Author(s):

Wayne J.G. Hellstrom ◽

Matthew T. Freier ◽

Ege Can Serefoglu ◽

Ronald W. Lewis ◽

Craig A. Peterson ◽

...

Keyword(s):

Clinical Trials ◽

Erectile Dysfunction ◽

Pde5 Inhibitor ◽

Rapid Onset ◽

Phase 2 ◽

Phase 3 ◽

Onset Of Action ◽

Severe Erectile Dysfunction

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3D bioprinting of mesenchymal stem cells and endothelial cells in an alginate-gelatin-based bioink

Journal of 3D Printing in Medicine ◽

10.2217/3dp-2020-0026 ◽

2021 ◽

Author(s):

Sindhuja D Eswaramoorthy ◽

Nandini Dhiman ◽

Akshay Joshi ◽

Subha N Rath

Keyword(s):

Gene Expression ◽

Stem Cells ◽

Alkaline Phosphatase ◽

Endothelial Cells ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Alkaline Phosphatase Activity ◽

Histochemical Staining ◽

Vital Role ◽

3D Bioprinting

Aim: Bioink is one of the essential factors in 3D bioprinting that determines the fate of cells, in our case, umbilical cord-derived mesenchymal stem cells (UMSC). The aim was to determine if the presence of the osteoinductive factors in the bioink enhances osteodifferentiation as compared with adding them postprinting and if the UMSC and endothelial cells (EC) coculture result in better osteodifferentiation. Materials & methods: Alginate-gelatin along with UMSC–EC were bioprinted using an extrusion 3D bioprinter. Results & conclusion: The UMSC–EC interaction, as well as intrinsic addition of the differentiation components in the bioink, were observed to play a vital role in increasing the osteogenic differentiation as shown by the histochemical staining, alkaline phosphatase activity and gene expression of osteogenic markers.

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OR18-01 Effect of Teprotumumab on Proptosis Reduction Across Various Demographic Sub-Groups

Journal of the Endocrine Society ◽

10.1210/jendso/bvaa046.1103 ◽

2020 ◽

Vol 4 (Supplement_1) ◽

Author(s):

George J Kahaly ◽

Terry J Smith ◽

Robert Holt ◽

Saba Sile ◽

Raymond S Douglas

Keyword(s):

Clinical Trials ◽

Smoking Status ◽

Reduction Rate ◽

Continuous Variable ◽

Clinical Activity ◽

Phase 2 ◽

Phase 3 ◽

Multicenter Studies ◽

Using Data ◽

Intent To Treat

Abstract Introduction: Teprotumumab, an insulin-like growth factor 1 receptor inhibitory monoclonal antibody, was recently shown to significantly reduce proptosis in patients with active, moderate-to-severe thyroid eye disease (TED) in phase 2 and phase 3 clinical trials.1,2 Prior analyses have demonstrated a combined trial proptosis response (≥2 mm reduction) rate of 77.4% in the teprotumumab group and 14.9% in the placebo group after 24 weeks of therapy (p < 0.001).3 The current analysis was performed to investigate whether or not patient demographic characteristics influence the teprotumumab proptosis response. Methods: Data from two 24-week randomized, double-masked, placebo-controlled, parallel-group, multicenter studies (Phase 2 [NCT01868997], Phase 3 [NCT03298867[) were combined. All patients had active TED associated with Graves’ disease. The study eye designated at baseline manifested more severe TED and a clinical activity score of > 4. Subjects were divided into subgroups based on gender, smoking status, and age at baseline (younger: <65, older: ≥65). The percentage of proptosis (≥2 mm) responders and proptosis change from baseline were examined in each of these subgroups. Because most of both teprotumumab (85%) and placebo (87%) subjects were white, there were insufficient numbers of subjects to examine the effect of race on the teprotumumab proptosis response. All analyses were performed on the intent-to-treat (ITT) population using data from the study eye. Results: A total of 171 patients comprised the population from the two studies. Eighty-four and 87 patients were randomized to the teprotumumab and placebo groups, respectively, and the treatment groups had balanced baseline characteristics. At week 24, significantly more teprotumumab than placebo patients were proptosis responders in all examined subgroups (male: 73.1% vs. 5.0%, female: 79.3% vs. 17.9%, smokers: 70.0% vs. 23.1%, non-smokers 79.7% vs. 11.5%, younger: 76.1% vs. 16.2%, older: 84.6% vs. 7.7%; all p < 0.001). In continuous variable analyses, the mean proptosis reduction from baseline was also significantly greater at week 24 in teprotumumab-treated patients than placebo patients (male: -3.34 vs. -0.07 mm, female: -3.10 vs. -0.42 mm, smokers: -2.99 vs. -0.72 mm, non-smokers: -3.20 vs. -0.31 mm, younger: -3.10 vs. -0.39 mm, older: -3.55 vs. -0.22 mm; all p < 0.001). Conclusion: Teprotumumab was effective across subgroups of age, gender, and smoking status in the pooled 24-week clinical trials. Reference: (1) Smith TJ, et al. N Engl J Med 2017;376:1748-1761. (2) Douglas RS, et al. AACE 2019 late-breaking abstract. (3) Kahaly GJ, et al. Thyroid 2019;29(Suppl1):A-1 [abstract].

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Atezolizumab Versus Docetaxel in Pretreated Patients With NSCLC: Final Results From the Randomized Phase 2 POPLAR and Phase 3 OAK Clinical Trials

Journal of Thoracic Oncology ◽

10.1016/j.jtho.2020.09.022 ◽

2021 ◽

Vol 16 (1) ◽

pp. 140-150

Author(s):

Julien Mazieres ◽

Achim Rittmeyer ◽

Shirish Gadgeel ◽

Toyoaki Hida ◽

David R. Gandara ◽

...

Keyword(s):

Clinical Trials ◽

Phase 2 ◽

Phase 3 ◽

Pretreated Patients ◽

Randomized Phase 2

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Effect of age on pharmacokinetics, efficacy, and safety of galcanezumab treatment in adult patients with migraine: results from six phase 2 and phase 3 randomized clinical trials

The Journal of Headache and Pain ◽

10.1186/s10194-020-01148-9 ◽

2020 ◽

Vol 21 (1) ◽

Cited By ~ 1

Author(s):

Virginia L. Stauffer ◽

Ira Turner ◽

Phebe Kemmer ◽

William Kielbasa ◽

Kathleen Day ◽

...

Keyword(s):

Clinical Trials ◽

Randomized Clinical Trials ◽

Adult Patients ◽

Phase 2 ◽

Efficacy And Safety ◽

Phase 3 ◽

Effect Of Age

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Are There Circumstances in Which Phase 2 Study Results Should Be Practice-Changing?

Hematology ◽

10.1182/asheducation-2007.1.489 ◽

2007 ◽

Vol 2007 (1) ◽

pp. 489-492 ◽

Cited By ~ 9

Author(s):

Marcie R. Tomblyn ◽

J. Douglas Rizzo

Keyword(s):

Clinical Trials ◽

Treatment Modalities ◽

Phase 2 ◽

Phase 3 ◽

Randomized Controlled Clinical Trials ◽

Phase 2 Trial ◽

Treatment Indications ◽

Study Results ◽

Patient Resources ◽

Large Phase

Abstract New pharmaceuticals, innovative combinations of approved agents, and novel treatment modalities have resulted in a marked increase in the need for clinical trials. Evidence for treatment efficacy is best derived from large phase 3 randomized, controlled clinical trials. However, phase 3 investigations are lengthy and expensive, and consume patient resources. Furthermore, some diseases and treatment indications are rare, and adequate numbers of patients for a definitive phase 3 trial do not exist. Consequently, it is imperative for clinicians to understand phase 2 trial design, since their interpretation is required to apply the findings in clinical practice appropriately. The complexity of phase 2 studies is explored, including unique designs, possible use of randomization, and other key elements necessary for interpretation of phase 2 trials. Specific examples and application of these concepts are discussed in this review.

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Assessing Sunscreen Protection Using UV Photography: Descriptive Study (Preprint)

10.2196/preprints.24653 ◽

2020 ◽

Author(s):

Caitlin Horsham ◽

Helen Ford ◽

Jeremy Herbert ◽

Alexander Wall ◽

Sebastian Walpole ◽

...

Keyword(s):

Clinical Trials ◽

New Zealand ◽

Phase 1 ◽

Sun Protection ◽

Trial Registration ◽

Phase 2 ◽

Phase 3 ◽

The Public ◽

Uv Photography ◽

Clinical Trials Registry

BACKGROUND Photography using a UV transmitting filter allows UV light to pass and can be used to illuminate UV blocking lotions such as sunscreens. OBJECTIVE The aim of this study is to compare currently available UV photography cameras and assess whether these devices can be used as visualization tools for adequate coverage of sun protection lotions. METHODS This study was conducted in 3 parts: in phase 1, 3 different UV cameras were tested; in phase 2, we explored whether UV photography could work on a range of sun protection products; and in phase 3, a UV webcam was developed and was field-tested in a beach setting. In phase 1, volunteers were recruited, and researchers applied 3 sun protection products (ranging from sun protection factor [SPF] 15 to 50+) to the participants’ faces and arms. UV photography was performed using 3 UV cameras, and the subsequent images were compared. In phase 2, volunteers were recruited and asked to apply their own SPF products to their faces in their usual manner. UV photographs were collected in the morning and afternoon to assess whether the coverage remained over time. Qualitative interviews were conducted to assess the participants’ level of satisfaction with the UV image. In phase 3, a small portable UV webcam was designed using a plug-and-play approach to enable the viewing of UV images on a larger screen. The developed webcam was deployed at a public beach setting for use by the public for 7 days. RESULTS The 3 UV camera systems tested during phase 1 identified the application of a range of sun protection lotions of SPF 15 to 50+. The sensitivity of the UV camera devices was shown to be adequate, with SPF-containing products applied at concentrations of 2 and 1 mg/cm<sup>2</sup> clearly visible and SPF-containing products applied at a concentration of 0.4 mg/cm<sup>2</sup> having lower levels of coverage. Participants in phase 2 reported high satisfaction with the UV photography images, with 83% (29/35) of participants likely to use UV photography in the future. During phase 2, it was noted that many participants used tinted SPF-containing cosmetics, and several tinted products were further tested. However, it was observed that UV photography could not identify the areas missed for all tinted products. During phase 3, the electrical components of the UV webcam remained operational, and the camera was used 233 times by the public during field-testing. CONCLUSIONS In this study, we found that UV photography could identify the areas missed by sun protection lotions with chemical filters, and participants were engaged with personalized feedback. CLINICALTRIAL Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12619000975190; http://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=377089 ; Australian New Zealand Clinical Trials Registry (ANZCTR) ACTRN12619000145101; https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=376672.

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