scholarly journals Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Britton Boras ◽  
Rhys M. Jones ◽  
Brandon J. Anson ◽  
Dan Arenson ◽  
Lisa Aschenbrenner ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Protease Inhibitor ◽  
Single Agent ◽  
Systemic Exposure ◽  
Synergistic Activity ◽  
Activity Data ◽  
3Cl Protease

AbstractCOVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential across a broad spectrum of coronaviruses with no close human analogs. PF-00835231, a 3CL protease inhibitor, has exhibited potent in vitro antiviral activity against SARS-CoV-2 as a single agent. Here we report, the design and characterization of a phosphate prodrug PF-07304814 to enable the delivery and projected sustained systemic exposure in human of PF-00835231 to inhibit coronavirus family 3CL protease activity with selectivity over human host protease targets. Furthermore, we show that PF-00835231 has additive/synergistic activity in combination with remdesivir. We present the ADME, safety, in vitro, and in vivo antiviral activity data that supports the clinical evaluation of PF-07304814 as a potential COVID-19 treatment.

scholarly journals Discovery of a Novel Inhibitor of Coronavirus 3CL Protease as a Clinical Candidate for the Potential Treatment of COVID-19

2020 ◽  
Author(s):  
Britton Boras ◽  
Rhys M. Jones ◽  
Brandon J. Anson ◽  
Dan Arenson ◽  
Lisa Aschenbrenner ◽  
...  
Keyword(s):  
Life Cycle ◽  
Antiviral Activity ◽  
Potent Inhibitor ◽  
Single Agent ◽  
Potential Treatment ◽  
Activity Data ◽  
Global Pandemic ◽  
3Cl Protease ◽  
Clinical Candidate

AbstractCOVID-19 caused by the SARS-CoV-2 virus has become a global pandemic. 3CL protease is a virally encoded protein that is essential to the viral life cycle across a broad spectrum of coronaviruses with no close human analogs. The designed phosphate prodrug PF-07304814 is metabolized to PF-00835321 which is a potent inhibitor in vitro of the coronavirus family 3CL pro, with selectivity over human host protease targets. Furthermore, PF-00835231 exhibits potent in vitro antiviral activity against SARS-CoV-2 as a single agent and it is additive/synergistic in combination with remdesivir. We present the ADME, safety, and in vitro antiviral activity data to warrant clinical evaluation.One Sentence SummaryThe phosphate prodrug PF-07304814 is disclosed as an investigational novel intravenous small molecule 3CL protease inhibitor for COVID-19.

Combined Fludarabine, Cyclophosphamide, and Alemtuzumab (FCC), an Active Regimen for Treated Patients with Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3133-3133 ◽  
Author(s):  
Marco Montillo ◽  
Sara Miqueleiz ◽  
Alessandra Tedeschi ◽  
Francesca Ricci ◽  
Eleonora Vismara ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Residual Disease ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Combination Regimen ◽  
Synergistic Activity ◽  
Color Flow ◽  
Grade Iii

Abstract Fludarabine (F) in combination with cyclophosphamide (C) showed a relevant advantage over single-agent F in pts with relapsed CLL. Although minimal residual disease (MRD) remains detectable in many pts achieving CR, the combination of F and C seems to reduce MRD more efficiently. Still, pts in CR eventually relapse and require treatment, demonstrating the need for improved treatments able to further reduce or eliminate MRD and induce “better quality” and thus more durable responses. Alemtuzumab (CAM), anti-CD52 monoclonal antibody, acts synergistically with F in vitro and appears to have synergistic activity in vivo. Additionally, CAM is highly effective at clearing disease from bone marrow, the usual site of residual disease following purine analogue-based treatment. Therefore, we designed a phase II study to determine feasibility and efficacy, overall response rate (ORR)-duration of response-ability at clearing MRD, of a 4-weekly combination regimen consisting of F, C, and CAM (FCC). The study population is represented by pts with B-CLL with relapsed or refractory disease after at least one line of treatment. Subcutaneous route of administration of CAM has been adopted in this trial. MRD was measured by 4-color flow cytometry in the bone marrow. The FCC regimen consisted of F 40 mg/m2/d os (d 1–3), C 250 mg/m2/d os (d 1–3) and CAM 10 mg sc (d 1–3). This combination was repeated on d 29 for up to 6 cycles. The dose of CAM was increased after the first cohort of 10 treated pts from 10 mg to 20 mg sc. Currently, 25 pts have been enrolled in this trial. Median age was 57 years (range 42–79), 15/25 (60%) were male, 23/25 (92%) were in Binet stage B or C, median number of prior treatment regimens was 2 (range 1–4). In six (24%) pts 17p deletion was detected. IgVH unmutated was observed in 17 (68%) pts. At the moment of writing 19 pts are eligible for evaluation of toxicity and response. The ORR was 79%, with 7 (37%) pts achieving CR, 7 (37%) pts a PR, 1 (5%) pt a PRn. Three pts had SD, while 1 showed progression of the disease. MRD negativity was achieved in the bone marrow of 4/15 (27%) pts. Grade III-IV neutropenia episodes were observed in 43% of the administered courses while grade III-IV thrombocytopenia episodes were detected only in 8% of cycles. Four major infections were recorded: two sustained by Mycobacterium tuberculosis (1 cutis, 1 lung), one by Nocardia (lung) and one by E. coli (sepsis). The patient with pneumonia due to M. tuberculosis died because of respiratory failure. CMV reactivation occurred in 6 pts: no CMV disease was recorded. After a median follow up of 10 m (range 1–22) 73% of responding pts did not progressed. In conclusion, results from the interim analysis of this new, 4-weekly dosing FCC regimen suggest that combination therapy with F, C and CAM is feasible, safe, and effective in treating pts with relapsed and refractory CLL, even in those patients with inherent poor prognostic factors and who had received.

The Novel Aurora Kinase Inhibitor ENMD-2076 Has Potent Single Agent Activity against Multiple Myeloma (MM) in Vitro and in Vivo, and Shows Synergistic Activity in Combination with Lenalidomide

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3660-3660 ◽  
Author(s):  
Xiaojing Wang ◽  
Anthony L. Sinn ◽  
Attaya Suvannasankha ◽  
Colin D. Crean ◽  
Li Chen ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Caspase 3 ◽  
Kinase Inhibitor ◽  
Single Agent ◽  
Synergistic Activity ◽  
Significant Activity ◽  
Aurora A Kinase ◽  
Free Base

Abstract ENMD-2076 is a novel, orally-active molecule that has been shown to have significant activity against Aurora A kinase as well as multiple receptor tyrosine kinases (RTK). We investigated the single agent activity of ENMD-2076 against MM cells in vitro and in vivo, and in combination with lenalidomide. ENMD-2076 free base showed significant cytotoxicity against MM cells with a mean LC50 of 3.84±0.86 μM at 48 hours in vitro. Cytotoxicity was associated with cleavage of caspase 3, 8, 9 and PARP, and loss of mitochondrial membrane potential as early as 6 hours. ENMD-2076 free base inhibited c-kit, FGFR-1, 3 and VEGFR1 and subsequently inhibition of downstream targets phosphorylated (p)-BAD, p-Foxo1a and p-GSK-3β was observed at 6 hours. NOD/SCID mice implanted with H929 human plasmacytoma xenografts and treated for 30 days with 50, 100, 200mg/kg/d ENMD-2076 showed a dose-dependent inhibition of tumor growth (Figure 1), with minimal toxicity as assessed by the stable weight of treated animals. Immunohistochemical staining of tumors from sacrificed animals showed significant reduction in Ki67 at all dose levels of treatment compared to control tumors. An increase in cleaved caspase-3 was observed on Western blot from the lysates of H929 tumors obtained from treated animals. ENMD-2076 free base also showed synergistic cytotoxic activity when combined with lenalidomide against H929, MM1.R and MM1.S cells as assessed by MTT assay and Annexin-V/PI staining. Using the Chou-Talalay method, the combination indices (CI) were < 1 for all three cell lines across a range of concentrations of ENMD-2076 free base (0.25–1.0 μM) plus lenalidomide (2.5–10 μM) indicating synergistic activity (CI=0.362 H929; CI=0.315 MM1.R; CI=0.415 MM1.S). Our results provide rationale for the investigation of ENMD-2076 alone and in combination with lenalidomide in patients with multiple myeloma. Figure Figure

The HIV-Protease Inhibitor Ritonavir(Norvir®) Is Active Against Human AML Blasts In Vitro and In Vivo and Has Synergistic Cytotoxic Activity with the Proteaseome Inhibitor Bortezomib (Velcade®).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2464-2464
Author(s):  
Christoph Driessen ◽  
Müller-Ide Hendrik ◽  
Gogel Jeanette ◽  
Kraus Marianne ◽  
Kanz Lothar ◽  
...  
Keyword(s):  
Protease Inhibitor ◽  
Cell Lines ◽  
Single Agent ◽  
Proteasome Inhibition ◽  
Hiv Protease ◽  
Malignant Cells ◽  
Short Period ◽  
Proteolytic Pathways

Abstract Upregulation of alternative proteolytic pathways characterizes malignant cells that overcome proteasome inhibition in vitro. The proteasome inhibitor Bortezomib (Velcade®), which selectively targets only one of the three active subunits of the proteasome, has shown limited activity in AML. Ritonavir (Norvir®) is an aspartate protease inhibitor used in intensive HIV-therapy, where therapeutic levels of 5–20μM are reached with an oral dose of 1200 mg/d. A cytotoxic effect of Ritonavir against malignant cells due to proteasome inhibition has been suggested (Gaedicke et al., Cancer Research 62, December 1, 2002). We have here tested the effect of Ritonavir on AML cells, both as single agent and in combination with Bortezomib. Ritonavir induced cytotoxic death in AML cell lines and primary AML blasts with an IC50 of 30–40 μM in vitro. The combination of Ritonavir and Bortezomib was synergistic in vitro, i.e. subtoxic concentrations of Ritonavir at 10 μM combined with subtoxic Bortezomib 5–10 nM induced robust cytotoxicity in AML cell lines and freshly isolated primary AML blasts. Using a novel chemical probe that for the first time allows to visualize the individual activity of proteasomal subunits in intact AML blasts, we show that Velcade selectively abrogates β5 proteasomal activity at 20 nM in AML cells, as expected. Ritonavir, by contrast, had no effect on active proteasomal subunits up to 50 μM. Thus, the synergistic effect of Ritonavir with Bortezomib on AML cells is not due to inhibition of the same proteasomal target by both drugs, but more likely mediated by blocking alternative proteolytic pathways. One individual patient aged 72 years with an early relaps of AML was treated with Ritonavir 400–600 mg/d p.o.. During treatment, the absolute leukocyte count dropped from 24000/μl to 8000/μl while the ANC raised from 185/μl to 1530/μl. Ritonavir was withdrawn due to diarrhoea and abdominal cramps, leading to a sharp increase in peripheral blood blasts and leukocytes. Retreatment with Ritonavir at a reduced dose of 200 mg/d combined with Velcade 1mg/sqm was tolerated and stabilized leukocyte counts for a short period of time. We conclude that Ritonavir has activity against chemotherapy-refractory AML in vitro and in vivo. The combination of Velcade and Ritonavir might allow to synergistically target the proteolytic machinery of AML blasts with tolerable toxicity.

Triple Combinations of the HDAC Inhibitor Panobinostat (LBH589) + Dexamethasone with Either Lenalidomide or Bortezomib Are Highly Effective in a Multiple Myeloma Mouse Model.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1514-1514
Author(s):  
Enrique M. Ocio ◽  
David Vilanova ◽  
Laura San-Segundo ◽  
Patricia Maiso ◽  
Mercedes Garayoa ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Median Survival ◽  
Hdac Inhibitor ◽  
Single Agent ◽  
Body Weight Loss ◽  
Control Group ◽  
Synergistic Activity ◽  
Median Volume

Abstract Introduction Panobinostat (LBH589) is a novel histone deacetylase (HDAC) inhibitor being evaluated in clinical trials in hematological and solid malignancies. In multiple myeloma (MM), investigators have demonstrated its in vitro antimyeloma effect in cell lines and patients cells. Cancer treatment is typically based on the concept of combining agents with different mechanisms of action to overcome drug resistance. This was the rationale of the present study in which the in vitro and in vivo benefit of combinations of pabinostat with conventional antimyeloma agents has been explored. Material and Methods The potential in vitro synergism of pabinostat with 6 antimyeloma agents (melphalan, doxorubicin, dexamethasone, thalidomide, lenalidomide, bortezomib) was analyzed in MM1S cell line. The two most favorable combinations were tested in 120 NOD/SCID mice implanted with a human subcutaneous plasmocytoma. Mice were randomized into 12 treatment groups. Drugs were given ip, 5 days/week × 7 weeks. Doses were: pabinostat: 10 mg/Kg × 3 weeks and 5 mg/Kg afterwards; dexamethasone (D): 1 mg/Kg; bortezomib (B): 0.1 mg/Kg; and lenalidomide (L): 15 mg/Kg. Tumor volumes clinical features and weight were monitored three times a week. Mice were sacrificed when their tumors reached 2 cm. Immunohistochemistry was performed in selected tumors. Results Three agents potentiated the effect of pabinostat in vitro: bortezomib, dexamethasone and, to a lesser extent, lenalidomide. Moreover, the triple combination of pabinostat+L+D and pabinostat+B+D resulted in high synergistic activity. These studies provided the rationale for testing these combinations in vivo: Single agent pabinostat at a dose of 10 mg/Kg completely abrogated the growth of plasmocytomas without significant toxicity. In fact, after three weeks of treatment, the median volume of tumors in the pabinostat group was 163±75 mm3 as compared to 1891±1182 mm3 in the control group (p=0.001). Immunohistochemistry of pabinostat treated tumors revealed a decrease in BrdU uptake, an increase in histone acetylation and phosphorylation of H2AX suggesting DNA damage. This antiproliferative action was associated with survival advantage: median survival 70±1.8 vs 30±2.1 days (p&lt;0.001) for the pabinostat and vehicle treated groups respectively. Subsequently the dose of pabinostat was decreased by 50% in order to gain further insights into the potential advantage of the combinations. Interestingly, the addition of D and suboptimal doses of either B or L significantly improved the antimyeloma effect of pabinostat. In this sense, median survival increased up to 86±2.6 days in pabinostat+D+B (p&lt;0.001) and 88±1.2 days for pabinostat+D+L (p&lt;0.001). The efficacy of these triple combinations was significantly higher than any of the respective double combinations (pabinostat+D; pabinostat+B; pabinostat+L; B+D; L+D). Some of these combinations (including or not pabinostat) initially induced a slight toxicity (5%–15% body weight loss) which spontaneously recovered after the third week of treatment. Conclusion Combinations of pabinostat + dexamethasone with either bortezomib or lenalidomide are safe and display promising antimyeloma efficacy. This study provides the rationale for the clinical development of triple combinations of these drugs to improve the outcome of MM patients.

scholarly journals Intracellular Antiviral Activity of Low-Dose Ritonavir in Boosted Protease Inhibitor Regimens

2014 ◽  
Vol 58 (7) ◽  
pp. 4042-4047 ◽  
Author(s):  
Amedeo De Nicolò ◽  
Marco Simiele ◽  
Andrea Calcagno ◽  
Adnan Mohamed Abdi ◽  
Stefano Bonora ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Protease Inhibitor ◽  
Protease Inhibitors ◽  
Antiviral Effect ◽  
Antiretroviral Drugs ◽  
Low Doses ◽  
Once Daily ◽  
Intracellular Concentrations

ABSTRACTProtease inhibitors are largely used for the treatment of HIV infection in combination with other antiretroviral drugs. Their improved pharmacokinetic profiles can be achieved through the concomitant administration of low doses of ritonavir (RTV), a protease inhibitor currently used as a booster, increasing the exposure of companion drugs. Since ritonavir-boosted regimens are associated with long-term adverse events, cobicistat, a CYP3A4 inhibitor without antiviral activity, has been developed. Recently, high intracellular concentrations of ritonavir in lymphocytes and monocytes were reported even when ritonavir was administered at low doses, so we aimed to compare its theoretical antiviral activity with those of the associated protease inhibitors. Intracellular concentrations of ritonavir and different protease inhibitors were determined through the same method. Inhibitory constants were obtained from the literature. The study enrolled 103 patients receiving different boosted protease inhibitors, darunavir-ritonavir 600 and 100 mg twice daily and 800 and 100 mg once daily (n= 22 and 4, respectively), atazanavir-ritonavir 300 and 100 mg once daily (n= 40), lopinavir-ritonavir 400 and 100 mg twice daily (n= 21), or tipranavir-ritonavir 500 and 200 mg twice daily (n= 16). According to the observed concentrations, we calculated the ratios between the intracellular concentrations of ritonavir and those of the companion protease inhibitor and between the theoretical viral protease reaction speeds with each drug, with and without ritonavir. The median ratios were 4.04 and 0.63 for darunavir-ritonavir twice daily, 2.49 and 0.74 for darunavir-ritonavir once daily, 0.42 and 0.74 for atazanavir-ritonavir, 0.57 and 0.95 for lopinavir-ritonavir, and 0.19 and 0.84 for tipranavir-ritonavir, respectively. Therefore, the antiviral effect of ritonavir was less than that of the concomitant protease inhibitors but, importantly, mostly with darunavir. Thus, furtherin vitroandin vivostudies of the RTV antiviral effect are warranted.

scholarly journals Characterization of in vitro and in vivo Antiviral Activity of Lactoferrin and Ribavirin upon Hantavirus.

2001 ◽  
Vol 63 (6) ◽  
pp. 637-645 ◽  
Author(s):  
Michael E. Murphy ◽  
Hiroaki Kariwa ◽  
Tetsuya Mizutani ◽  
Hiroki Tanabe ◽  
Kumiko Yoshimatsu ◽  
...  
Keyword(s):  
Antiviral Activity

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

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