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A theoretical discovery and development of an anti-ebola drug

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A theoretical discovery and development of an anti-ebola drug

  1. 1. 1 2010 Discovery & Development of a First in Class Anti-Ebola Drug- EBONAVIR® Summary: Ebola hemorrhagic fever (Ebola HF) caused by the Ebola virus is a severe, often-fatal viral hemorrhagic disease in humans and nonhuman primates with a fatality rate of 50-83%. The present document details the discovery and development of a novel small molecule organo-pharmaceutical that acts as a potent inhibitor of VP35 - a critical protein involved in EBOV pathogenesis through early preclinical studies as well as clinical studies. It provides an insight into the research, documentation and regulatory requirements pertaining to the commercial launch of the above mentioned small molecule drug. GAYATHRI VIJAYAKUMAR MS Biotechnology, NYU-POLY 5/1/2010
  2. 2. 2 CONTENTS 1) Introduction 5 a) Ebola Virus 5 b) Transmission 5 c) Symptoms 5 d) Diagnosis 6 e) Treatment 6 f) History 6 2) Current therapeutic advances for Ebola Hemorrhagic Fever 6 3) Promising targets for development of therapeutics 7 4) Target for drug development 8 a) Reason for selection of VP35 as a therapeutic target 8 b) Impairment of innate immunity 10 5) Medical hypothesis for developing an anti-Ebola therapeutic 12 6) Conclusion regarding medical hypothesis 14 7) Assays for HTS screening of potential anti-Ebola therapeutic 15 a) Luciferase Budding Assay 15 b) Immunocapture Assay 16 c) Cell based assay using recombinant GFP-ZEBOV 16 8) Luciferase Reporter based gene assay selected for HTS screening 18 a) Illustration of principle 21 9) Lead Compound 22 a) Selected Lead Compound candidate 24 10) Lead Optimization 25 a) Lead Optimization results 26 11) In vitro safety pharmacology profiling 28 a) In vitro SPP Profile results 33 12) Patent Application 35 13) Selection of appropriate animal model for efficacy evaluation of the selected NCE 36 a) Mouse model used for preclinical studies 38 b) Non-human primate model used for preclinical studies 40
  3. 3. 3 14) Biomarkers 42 a) Disease Biomarkers 43 b) Surrogate Biomarker 45 c) Toxicity Biomarker 45 d) Target Biomarker 46 e) Mechanism Biomarker 46 f) Efficacy Biomarker 47 g) Translational Biomarker 47 h) Stratification Biomarker 47 15) Efficacy studies for Minimum Effective Dose 48 a) Minimum Effective Dose 49 16) Principal studies in animal models with NCT1087 49 a) Animal models in safety studies 50 b) Route of Administration of NCT1087 51 c) Safety Pharmacology Studies 51 d) Acute Toxicity Study 53 e) Repeated Dose Toxicity Study 53 f) Genetoxicity Study 55 g) Carcinogenicity Study 57 h) Reproduction Toxicity Study 57 i) Immunotoxicity Study 58 j) Phototoxicty Study 59 17) Maximum Tolerated Dose 60 a) Toxicity studies for determining the MTD 60 18) Repeated dose toxicity studies for 2 months 62 19) Estimation of the first human dose 64 20) IND Application 65 21) Phase I Clinical Trials 67 22) Phase II Clinical Trials 71 23) Phase III Clinical Trials 77 24) Conclusion 83 25) Route of Delivery, Administration Regimen & Dose Concentration 83 26) NDA Application 84 27) Life Cycle Management of NCT1087 or Ebonavir® 85 28) The Drug Development process 86
  4. 4. 4 29) Preclinical & Requirements for IND, NDA &Clinical studies 88 30) CMC Requirements for IND, NDA & Clinical studies 89 31) Possible Emergence of Resistance to Ebonavir® 90 32) References 91
  5. 5. 5 EBOLA HEMORRHAGIC FEVER Ebola hemorrhagic fever (Ebola HF) is a severe, often-fatal viral hemorrhagic Figure 1. [3] disease in humans and nonhuman primates (monkeys, gorillas, and chimpanzees) that has appeared sporadically since its initial recognition in the Democratic Republic of Congo in 1976 [1]. It is caused by the Ebola Virus which interferes with the interior endothelial cell lining of blood vessels and coagulation which leads to hypovolemic shock [2]. Destruction of endothelial surfaces is associated with disseminated intravascular coagulation, and this may contribute to the hemorrhagic manifestations [5]. Fatality rate differs from 50-83% [2]. [3] EBOLA VIRUS (shown in image [6]) Figure 2. [6] The Ebola virus belongs to the Filoviridae family (filovirus) and is comprised of five distinct species: Zaire, Sudan, Côte d’Ivoire, Bundibugyo and Reston. [4] The Zaire virus (ZEBOV) is the most lethal with an average case fatality rate of 83% [2]. Ebola virus has a non-segmented, negative-stranded, RNA genome containing 7 structural and regulatory genes [5]. The exact origin, locations, and natural habitat (known as the "natural reservoir") of Ebola virus remain unknown. Researchers believe that the virus is zoonotic (animal-borne) [4]. Electron microscopic image of an Ebola infected cell in [9] (Figure 3). TRANSMISSION 1) Direct contact with the blood and/or secretions of an infected person/ animal [1]. 2) Contact with objects, such as needles, that have been contaminated with infected secretions [1]. 3) Nosocomial transmission refers to the spread of a disease within a health- care setting, due to incorrect infection control precautions and adequate barrier nursing procedures [1, 4] Figure 3: [9] SYMPTOMS The incubation period for Ebola HF ranges from 2 to 21 days. The onset of illness is abrupt and is characterized by fever, headache, joint and muscle aches, sore throat, and weakness, followed by diarrhea, vomiting, and stomach pain. A rash, red eyes, hiccups and internal and external bleeding may be seen [1].
  6. 6. 6 DIAGNOSIS Diagnosis is usually done with inactivated blood or saliva specimens to detect the viral antigen/antibody or genetic material [4]. They can also be isolated in cell cultures. Diagnostic laboratory tests such as ELISA, RT-PCR, IgM ELISA, Immunohistochemistry testing, Coagulation studies; Complete Blood Count, etc are usually performed [1,7]. TREATMENT There is no standard treatment for Ebola hemorrhagic fever. Treatment is primarily supportive and includes minimizing invasive procedures, balancing electrolytes, and, since patients are frequently dehydrated, replacing lost coagulation factors to help stop bleeding, maintaining oxygen and blood levels, and treating any complicating infections. Convalescent plasma, administration of an inhibitor of coagulation (rNAPc2), Morpholino antisense drugs have shown some promise in treatment for Ebola HF [2]. HISTORY History of Ebola virus outbreaks have been shown in the table to the side (Figure 4). Confirmed cases of Ebola HF have been reported in the Democratic Republic of the Congo, Gabon, Sudan, the Ivory Coast, Uganda, and the Republic of the Congo. [1] Because of its high morbidity, it is a potential agent for bioterrorism and since no approved vaccine or drugs are available it is classified as a biosafety level 4 agents as well as Category A bioterrorism agent by the CDC [2]. The last reported outbreak was in December 2008 in the Western Kasai province of the Democratic Republic of Congo [2]. Figure 4: From [8] CURRENT THERAPEUTIC ADVANCES FOR EBOLA HEMORRHAGIC FEVER To date, beyond supportive care, no effective treatments, therapies, or vaccines are approved to treat or prevent Ebola virus infections because natural immunity to this infection is difficult to find, plus there are no immune correlates in humans [10]. However, several drugs are under development as summarized:
  7. 7. 7 • USAMRID investigated a drug called recombinant nematode anticoagulant protein C2 (rNAPC2) which blocks the harmful effects of tissue factors and targets the disease process. It showed reasonable success in infected rhesus monkeys [11]. • AVI BioPharma’s proprietary NEUGENE drug targets a key Ebola gene providing complete protection in mice when administered either before or after an otherwise lethal infection with Ebola virus. NEUGENE antisense compounds are synthetic polymers designed to match up perfectly with a specific gene or viral sequence, blocking the function of the target gene or virus [12]. • Phosphorodiamidate morpholino oligomers (PMO) are a class of uncharged single-stranded DNA analogs modified such that each subunit includes a phosphorodiamidate linkage and morpholinering. Data suggest that antisense PMO and P-PMO have the potential to control EBOV infection and are promising therapeutic candidates [13]. A combination of EBOV-specific PMOs targeting sequences of viral mRNAs for the viral proteins (VPs) VP24, VP35 has been used as both pre- and post- exposure therapeutic regimens in non-human primates [14]. • NanoViricides, Inc. has come out with broad-spectrum nanoviricides(TM) drug candidates that have been found to be highly effective in cell culture studies by USAMRID. These antivirals use biomimetic technology (i.e. they mimic the host cell features) [15]. • 3-Deazaneplanocin A, an analog of adenosine, is a potent inhibitor of Ebola virus replication. A single dose early in infection prevents progression of the disease in EBOV infected mice. Protective effect of the drug results from massively increased production of interferon-α [17]. • Cyanovirin-N (CV-N) which is an HIV inactivating protein has been found to have both in vitro and in vivo antiviral activity against the Zaire strain of the Ebola Virus by inhibiting the development of viral cytopathic effects (CPEs). It has been found to delay fatality in

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