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Discovery & Development of a First in Class Anti-Ebola

Drug- EBONAVIR ® Summary:

Ebola hemorrhagic fever (Ebola HF) caused by the Eb ola virus is a severe, often-fatal viral hemorrhagic disease in humans and nonhuman primates with a fatality rate of 50-83%. T he 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 w ell as clinical studies. It provides an insight into the r esearch, documentation and regulatory requirements pertainin g to the commercial launch of the above mentioned small mole cule drug.

2010

GAYATHRI VIJAYAKUMAR

MS Biotechnology, NYU-POLY

5/1/2010

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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 Hemorrhag ic Fever 6

3) Promising targets for development of therapeutic s 7

4) Target for drug development 8

a) Reason for selection of VP35 as a therapeutic ta rget 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 2 8

a) In vitro SPP Profile results 33

12) Patent Application 35

13) Selection of appropriate animal model for effic acy evaluation of the selected NCE 36

a) Mouse model used for preclinical studies 3 8

b) Non-human primate model used for preclinical stu dies 40

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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 6 0

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 & Dos e Concentration 83

26) NDA Application 84

27) Life Cycle Management of NCT1087 or Ebonavir® 85

28) The Drug Development process 86

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29) Preclinical & Requirements for IND, NDA &Clinic al studies 88

30) CMC Requirements for IND, NDA & Clinical studie s 89

31) Possible Emergence of Resistance to Ebonavir® 90

32) References 91

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EBOLA HEMORRHAGIC FEVER

Ebola hemorrhagic fever (Ebola HF) is a severe, often-fatal viral hemorrhagic

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].

EBOLA VIRUS (shown in image [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]

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].

Figure 1 . [3]

[3]

Figure 2 . [6]

Figure 3 : [9]

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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].

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:

Figure 4 : From [8]

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• 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 EBOV infected mice. It binds with considerable affinity to the

Ebola envelope glycoprotein GP1,2 . [19].

• EBOV infected immunocompetent mice treated with the adenosine analogue carbocyclic 3-deazaadenosine

protects it against fatality by blocking the cellular enzyme S-adenosyl-L -homocysteine hydrolase (SAH) [20].

• A vaccine based on recombinant vesicular stomatitis virus expressing the ZEBOV glycoprotein has been found

to confer immunity against aerosol challenge in cynomolgus macaques which is likely to be the port of entry in a

bioterrorist attack [21].

• Intranasal vaccine based on replication-competent human parainfluenza virus type 3 (HPIV3) expressing EBOV

glycoprotein GP (HPIV3/EboGP) and showed that it is immunogenic and protective against a high dose

parenteral EBOV challenge [22].

PROMISING TARGETS FOR DEVELOPMENT OF THERAPEUTICS

An eight amino acid sequence (TELRTFSI) present in the carboxy terminal end (aa 577–584) of membrane-

anchored GP, the major structural protein of Ebola virus, was identified as an H-2k-specific murine cytotoxic T cell

epitope. Immunity can be stimulated by injecting irradiated Ebola virus encapsulated in liposomes containing lipid A

[16].

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• RNA is blocked by inhibiting the cellular enzyme SAH, indirectly

resulting in reduced methylation of the 5′ cap of viral mRNA [17].

• NSecretary glycoproteins can be targeted by neutralizing

monoclonal antibodies [18].

• VP24, VP30 and VP40 can induce protective immune

responses [23].

• SCripps Research Institute found that VP35 is critical in

allowing uncontrolled replication by fooling the human immune

system [24].

In my opinion there definitely exists a medical nee d as

1) there are no approved therapies yet in the market, 2) it has a fatality of 50-80%, 3) it is a potential bioterrorism

agent, but it is definitely not an impossible task which is clearly observable from all the latest developments in the

field and all the possible targets available for developing a potential therapy.

TARGET FOR DRUG DEVELOPEMENT

The target that I have selected for development of a therapy against EBOV infection is the virion protein VP35 .

Reason for selection of VP35 as a therapeutic targe t

The 18.9-kb RNA genome of EBOV is non-infectious and encodes

seven structural proteins and one non-structural protein in the following order

within the genome: 3′ non-coding region (leader), nucleoprotein (NP), virion

protein 35 (VP35), VP40, glycoprotein (sGP and GP), VP30, VP24, RNA-

dependant RNA-polymerase (L) protein and 5′ non-coding region (trailer)

[27]. Mature EBOV particles form long filamentous rods with a uniform

diameter of 80 nm and a mean length of 1250nm. Virus particles possess a

central core, known as the ribonucleoprotein (RNP) complex that consists of

NP, VP35, VP30, L and the viral RNA. This RNP complex is surrounded by a

lipid envelope, with which the remaining proteins GP1,2, VP40 and VP24 are

associated; these three proteins function as surface glycoprotein, major

matrix protein and minor matrix protein, respectively as shown in the figure

on the side. (Figure 6; [27])

Three pathogenic mechanisms that underlie EBOV infection are vascular instability, coagulopathy and

immunosupression (Figure 7; [27]. Besides the induction of cytopathic cytotoxicity in epithelial and endothelial

cells (which perhaps explains the hemorrhagic manifestations characteristic of filovirus infection), destruction

of the immune system is another significant feature of EBOV infection. The EBOV infects the mononuclear

phagocyte and fibroblastic reticular systems associated with lymph nodes which maximizes immune

Figure 5 . Ebola Virus Potential targets [10].

Figure 6 . [27]

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responses by activation of appropriate cytokine production and antigen trafficking. This results in inadequate

development of immunity, thus playing an important role in pathogenesis [25].

Recent data shows that the EBOV virus VP35 protein, which plays an essential role in viral RNA synthesis,

acts as a type 1 IFN antagonist in much the same way as the NS1 protein, another IFN antagonist, enhances the

replicative ability of influenza virus, indicating that the presence of such a protein could be required for full

expression of virulence [25]. Nucleoproteins VP-35 and -30 and RNA polymerase are required for RNA transcription

and replication [25]. Another study found that the formation of nucleocapsid structure doesn’t occur in the absence

of VP35, VP24 or NP [26].

Figure 7: Overview of the mechanisms that are involved in EBOV pathogenesis. taken from [27].

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Impairment of innate immunity

EBOV infection attacks the innate and adaptive immunity by

• Inflammatory responses that are accompanied by substantial cytokine production [27].

• DCs, which have a crucial role in both innate and adaptive immunity, fail to fulfill their function after

infection by not producing proinflammatory cytokines or express co-stimulatory molecules such as CD80

or CD86, are impaired in their ability to support T-cell proliferation and undergo anomalous maturation

[27].

• Depletion of NK cells and lymphocytes [27].

• EBOV selectively suppresses responses to IFN-α and IFN-γ and the production of IFN-α in response to

double-stranded RNA [27].

• Blockade of IFN signaling has an important role in the pathogenesis of EBOV infection in vivo [27].

VP35 blocks phosphorylation of the IFN-regulatory factor 3 (IRF3), which acts as a transcription factor for

IFN production, whereas VP24 seems to block IFN signaling indicating that the block of IFN signaling might be

due to inhibition of p38 phosphorylation, which is central in the mitogen-activated-protein-kinase (MAPK) p38

IFN-signaling pathway [27].

During virus infection, RNAi against the virus is activated

by the production of virus-specific double-stranded RNAs (RNA

interference (RNAi) mechanism). Similar to RNAi, the IFN

pathway is triggered by cytoplasmic viral dsRNAs and acts as a

sensitive and potent antiviral response that is involved in innate

and subsequent adaptive immunity. To overcome this antiviral

RNAi response, viruses encode RNA silencing suppressors

(RSSs) and these include the influenza A virus NS1 (NS1),

vaccinia virus E3L (E3L), hepatitis C virus Core, primate foamy

virus type 1 (PFV-1) Tas, and the HIV-1 Tat proteins, as well as

the adenovirus virus-associated RNAs I and II (VAI and VAII).

Strikingly, all RSS proteins from mammalian viruses possess IFN

or protein kinase R (PKR) antagonistic properties, suggesting

that RNAi and other innate antiviral responses are interrelated. A

study showed that Ebola virus (EBOV) VP35 protein is a potent

RNAi suppressor that is functionally equivalent to the HIV-1 Tat RSS function [28].

Figure 8. RNA Interference Mechanism [28]

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Although the importance of antiviral RNAi responses in plants, nematodes, and insects is firmly

established, it is still under debate whether RNAi has a similar function in mammalian cells. But all the same

the above study demonstrates the importance of VP35 in helping EBOV in avoiding detection by the immune

system [28].

Another study showed that the Ebola virus VP35 protein is a virus-encoded inhibitor of the type I IFN

response. VP35 subsequently was shown to block double-stranded RNA- and virus-mediated induction of an

IFN-stimulated response and to block double-stranded RNA- and virus-mediated induction of the IFN-β

promoter. Type I IFN is synthesized in response to viral infection; double-stranded RNA (dsRNA) or viral

infection activates latent transcription factors, including IRF-3 and NF-κB, resulting in the transcriptional up-

regulation of type I IFN, IFN-α, and IFN-β, genes. Secreted type I IFNs signal through a common receptor,

activating the JAK/STAT signaling pathway. This signaling stimulates transcription of IFN-sensitive genes,

including a number that encode antiviral proteins, and leads to the induction of an antiviral state. Among the

antiviral proteins induced in response to type I IFN are dsRNA-dependent protein kinase R (PKR), 2′,5′-

oligoadenylate synthetase (OAS), and the Mx proteins [29].

This study validates that Ebola virus VP35 is therefore likely to inhibit induction of type I IFN in Ebola

virus-infected cells and may be an important determinant of Ebola virus virulence in vivo [29].

Figure 9. This image shows the crystal structure of the Ebola virus protein VP35 bound to a molecule of double-stranded helical RNA (green). Two VP35 molecules come together to mask the end of the double-stranded RNA helix from detection by the immune system. The interface between the two VP35s (white) may provide new targets for drugs to counteract the virus. [30]

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Medical Hypothesis for developing an anti-EBOV Ther apeutic

The Ebola VP35 protein is multifunctional, acting as a component of the viral RNA polymerase complex,

a viral assembly factor, and an inhibitor of host interferon (IFN) production [31]. All the studies above have

established the fact the VP35 is absolutely essential for Ebola virus pathogenesis as well virulence.

Therefore I conclude that it makes an excellent target for development of a therapeutic. From whatever

scientific publications I have read so far, I think that there are two ways you can tackle this problem.

1. Using antisense Phosphorodiamidate morpholino ol igomers

They are a class of uncharged single-stranded DNA analogs modified such that each subunit

includes a phosphorodiamidate linkage and morpholine ring [13]. Data suggests that antisense

PMO and P-PMO (PMO conjugated to arginine-rich cell-penetrating peptide) have the potential to

control EBOV infection by binding to the translation start site region of EBOV VP35 positive-sense

RNA and generating sequence-specific and time- and dose-dependent inhibition of EBOV

amplification in cell culture [13]. AVI BioPharma’s proprietary NEUGENE drug uses a combination

of EBOV-specific PMOs targeting sequences of viral mRNAs for the viral proteins (VPs) VP24,

VP35, and RNA polymerase L and found it to have reasonably good success in EBOV-infected

rodents and primates. It is currently under clinical trials [14].

2. Targeting C-terminal of VP35 IID

Mutation of select basic residues within the C-terminal half of VP35 inhibits its dsRNA-binding

activity, impairs VP35-mediated IFN antagonism, and attenuates EBOV growth in vitro and in vivo

[31]. A functional VP35 is required for efficient viral replication and pathogenesis; knockdown of

VP35 leads to reduced viral amplification and reduced lethality in infected mice [31]. VP35

contains an N-terminal coiled-coil domain required for its oligomerization (required for viral

replication) and a C-terminal dsRNA-binding region (blocks signaling that triggers IFN process)

[31]. Data suggest that the dsRNA binding activity mediated by the C terminus of VP35 is critical

for viral suppression of innate immunity and for virulence. There are 2 basic patches that are

highly conserved among members of the Filoviridae family (identical among EBOV isolates).

Biochemical and NMR-based structural analyses show that one patch contains residues that are

required for dsRNA binding and IFN inhibition, whereas the functional significance of the other

basic patch remains unknown [31]. Therefore a compound that would target the conserved region

in the C-terminal of VP35 IID which are critical for dsRNA binding may result in impaired IFN

suppression and yield attenuated viruses in vivo because of reduced dsRNA binding ([31]; see

Figures 10-12 below).

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Figure 10 . Crystal structure of VP35 C-terminal IFN-inhibitory domain (IID) reveals a fold that binds dsRNA. (A) Domain organization of VP35. (B) Ribbon representation of VP35 IID. Secondary structural elements that form the α-helical subdomain (orange) and the β-sheet subdomain (yellow). (C) Topology and delimiting sequence markers of VP35 IID [31].

Figure 12 . Structures of the Ebola VP35 IID dsRNA binding domain [31].

Figure 11 . Polymerase cofactor VP35, Key word 3FKE [32]. ]

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CONCLUSION CONCLUSION CONCLUSION CONCLUSION

A molecule or compound that inhibits VP35 of the Ebola virus will inhibit binding of the virus-specific dsRNA

produced by the host cell as part of RNA interference (RNAi) mechanism thus activating both double-stranded

RNA- and virus-mediated induction of an IFN-stimulated response as well as double-stranded RNA- and virus-

mediated induction of the IFN-β promoter [28][29]. This activated latent transcription factors, including IRF-3 and

NF-κB, resulting in the transcriptional up-regulation of type I IFN, IFN-α, and IFN-β, genes. Secreted type I IFNs

signal through a common receptor, activating the JAK/STAT signaling pathway. This signaling stimulates

transcription of IFN-sensitive genes, including a number that encode antiviral proteins, and leads to the induction of

an antiviral state [29]. This leads to up regulation of the innate immune system.

Virus-specific dsRNAs on the other hand are processed into small interfering RNAs (siRNAs; a 21-

nucleotide dsRNA duplex) by the RNAse III–like endonuclease-denoted Dicer. Subsequently, one strand of the

siRNA duplex, the guide-strand, is incorporated into the RNA-induced silencing complex (RISC) to target viral

mRNAs bearing complementary sequences for destruction. [28]

Thus I conclude that a compound that inhibits VP35

leads to activation and up regulation of the innate and

adaptive immune system. In addition inhibition of V P35

exposes viral mRNAs to host cell antiviral response . This

leads to reduction in the viral load, and thus will stop

progression of the infection .

Why is my target better?

I think that this target is better than the others

• As scientific evidence doesn’t prove that inhibition

of other viral targets will elicit the same degree of

recovery as VP35 inhibition.

• VP35 is very integral to EBOV replication and

pathogenesis.

• C-terminal of VP35 IID is conserved among members

of the Filoviridae family (identical among EBOV isolates).

Figure 13 : Model for VP35-mediated IFN inhibition and immune suppression [31].

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ASSAY FOR HTS SCREENING OF POTENTIAL ANTI-EBOLA THE RAPEUTICS

There are a few assays that are in use for Ebola research. It is listed as follows along with their principle and a brief

description of their methodology.

LUCIFERASE BUDDING ASSAY

It is a functional budding assay that has been developed based on the ability of VP40 matrix protein of EBOV in

bringing about the budding of virus-like particles (VLP) from mammalian cells. This well-defined assay has been

modified for potential use in a high-throughput format in which the detection and quantification of firefly luciferase

protein in VLPs represents a direct measure of VP40 budding efficiency. Luciferase was found to be incorporated

into budding VP40 VLPs. In contrast, when luciferase is co-expressed with a budding deficient mutant of VP40,

luciferase levels in the VLP fraction decrease significantly. Although VP40 does not require the presence of

additional viral proteins to achieve VLP egress, the contribution of glycoprotein (GP), nucleoprotein (NP), VP35 and

VP24 to budding has been studied.

BRIEF DESCRIPTION OF THE METHODOLOGY

Human 293 T cells are grown in DMEM supplemented with

10% FCS and 1% penicillin-streptomycin at 5% CO2at 37OC.

pCAGGS-luciferase vectors are constructed by insertion of

luciferase into the SacI and NsiI sites of the pCAGGS vector.

Subsequently, VP40 is joined to the C-terminal end of

luciferase by insertion into the NsiI and XhoI sites of pCAGGS-

luciferase. One hundred millimeter plates of 293T cells are

transfected with 10µg of each plasmid DNA using

Lipofectamine in OptiMem. Cells are metabolically labeled 24h

post-transfection with Met-Cys. Six hours later, media was

harvested, clarified, and layered over a 20% sucrose cushion

in STE buffer, then centrifuged. The pellet was resuspended in

STE buffer and lysed with radioimmunoprecipitation assay

(RIPA) buffer. Cells were washed twice with PBS and then

lysed in RIPA buffer. Both cells and VLP lysates were

immunoprecipitated with the appropriate antibodies and

analyzed by SDS polyacrylamide gel electrophoresis (PAGE)

[33].

Subsequently Protease Protection assay and VLP Luciferase Assay was performed [33],

Figure 14 . Luciferase activity in VLPs correlates with expression and budding efficiency of VP40. Human 293T cells were transfected with pCAGGS + luciferase, VP40 + luciferase, VP40 + GP + luciferase, or VP40d-PT/PY + luciferase. VLPs were isolated and then combined with luciferase reagent. Relative light units (RLU) were measured using a luminometer. The RLU value of luciferase + pCAGGS was normalized to one [33].

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IMMUNOCAPTURE ASSAY

It is basically a rapid ELISA assay that detects both Ebola virus and EBOV-like particles (VLPs) directly from cell-

culture supernatants with the VP40 matrix protein serving as antigen. This assay can be used in surrogate models

in non-biocontainment environment, facilitating both basic research on the mechanism of EBOV assembly and

budding as well as drug-discovery research. Such an assay could also replace or be complementary to the

conventional viral plaque assays for virus detection, which require more than a week to accomplish [34].

BRIEF DESCRIPTION OF METHODOLOGY

cDNA for EBOV Zaire NP, VP30, VP35, and VP24 was cloned

into pWRG7077 vector. Polyclonal anti-VP40 antibody was

generated by immunizing rabbits with a peptide corresponding to

the N-terminal 15 amino acids of VP40 protein. Sera from

convalescent EBOV-infected guinea pigs were used as the

polyclonal anti-EBOV antibody. Bacterial expression of EBOV

VP40 was done by using a pET16b-VP40 clone to introduce a

His6-fusion at the N-terminus in E.coli. Subsequently purification

and extraction of EBOV VP40 was done from the cell lysate.

Fractions were analyzed by SDS-PAGE and appropriate

fractions were pooled [34].

HB 96-well plates were coated with AE11 monoclonal mouse

anti-VP40 antibodies. The plates were washed with PBST.

PBST containing 5% milk was added to block non-specific

binding sites. After binding, plates were washed with PBST.

Cellular extracts or culture supernatants were loaded with 1%

milk PBST. The samples were removed and the wells washed

three times for 5 min. Rabbit polyclonal anti-VP40 antibodies

were added in PBST/5% milk and incubated for 1 h. The plate

was washed again and anti-rabbit HRP-conjugated antibodies

were added in PBST for 30 min. The plate was washed with

PBST and PBS. HRP substrate was added to each well and

absorbance at 650 nm was read [34].

CELL-BASED ASSAY USING RECOMBINANT GFP-ZEBOV

In this assay the GFP acts as a receptor for virus replication. As such this assay can be used to screen compounds

that inhibit replication of the virus [35]

Figure 15 . Establishing an EBOV VP40 ELISA. (A) Coomassie staining of purified VP40 protein (lane 1). Immunoblotting of VP40 protein gel using anti-VP40 antibodies showing the monomeric and oligomeric forms of VP40 (lane 2). (B) Standard curve for VP40 ELISA generated using purified VP40 protein. (C) Standard curve for VP40 ELISA using inactivated EBOV lysed in 1.5% Triton X100. Capture antibodies were monoclonal mouse anti-VP40 and detection antibodies rabbit anti-VP40 polyclonal antisera [2].

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BRIEF DESCRIPTION OF METHODOLOGY

Confluent Vero E6 cells (96-well plate format) were pretreated

with compound (drug) diluted in cell-culture medium for 18h.

Cells were then infected with GFP-ZEBOV. Compound was

reapplied to cells after infection by adding media containing the

same pretreatment concentration. Cultures were incubated for

48h before fixation. After nuclear staining (Hoechst dye), viral

infection was determined using a high content imaging system,

Discovery 1, which compiles GFP fluorescence data from

approximately 25,000 cells per well. This procedure identifies

the total number of adherent cells, and fraction of infected

cells, thus providing an immediate assessment of efficacy and

toxicity. Validation of the results was done by means of Virus

Yield Reduction Assay. [35]

Another paper which utilizes the same principle engineered an

EboZ-eGFP by inserting eGFP open reading frame preceded

by a 90-nucleotide fragment containing an authentic

transcription stop sequence, to direct the termination of the NP

mRNA, and a transcription start sequence to direct the

initiation of the eGFP message into the cloned BsiW1

restriction site present in the NP 3′ UTR. The “wild-type like”

virus was termed EboZ-BsiW1. As a proof of principle for using

the EboZ-eGFP virus to rapidly screen antiviral compounds,

consensus interferon alpha (conIFN-α), a compound with

known EboV antiviral properties, was used at multiple

concentrations to treat Vero E6 cells infected with either EboZ-

eGFP virus or a control Zaire ebolavirus. The infected cells

were monitored for evidence of virus replication by either

fluorescence microscopy (for EboZ-eGFP virus) or a previously

published CPE-based assay [37].

Figure. 16 : FGI-106 blocks Ebola virus infection. (A) Vero E6 cells were infected with Zaire-Ebola virus (ZEBOV) for 72 h in the presence of 0–4 µM FGI-106. Antiviral efficacy was evaluated using plaque assays of infectious Ebola particles to assess the number of plaque forming units per unit volume (pfu/mL). (B) The number of viable Vero E6 cells was assessed following 72 h incubation in the presence of the indicated concentrations of FGI-106. (Drug) [35]

Figure 17 : (A) Time course of Vero E6 cells infected with EboZ-eGFP, EboZ-BsiW1, or EboZ-wt. Vero E6 cells were infected at a MOI of two and virus (B) Fluorescent microscopy of human PBMCs either mock-infected or infected with EboZ-eGFP and analyzed at the indicated times post-infection. (C) Electron micrograph of a human macrophage infected with EboZ-eGFP virus [37].

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PLAQUE ASSAY FOR EBOLA VIR

PLAQUE ASSAY

Confluent Vero cells are infected with an EBOV strain. After

absorption of the viral innoculum for 1h, 30 ml of EBM containing

5% FBS, antibiotics and HEPES buffer are incubated in flasks

for 10 days. Harvested medium is centrifuged and the

supernatant fluid is made into dilutions using L-15 medium and

inoculated into monolayers of cell lines being tested. After

various incubation periods, cell monolayers are stained using an

agarose overlay containing neutral red. Plates are then

examined for plaques 24 and 48 hours after adding the stain

overlay. The viral origin of the plaques is verified by inoculating

one of the plaques mixed with L-15 medium into suckling mice.

After death of the mice, the livers are removed, processed for

virus isolation, and examined for viral antigens with the IFA

procedure. Liver tissue collected from mice which had died post-

inoculation will show EV-specific fluorescence by IFA and from

this the PFUs can be counted [36].

THE ASSAY THAT IS SELECTED FOR HTS SCREENING-A LUCI FERASE

REPORTER BASED GENE ASSAY

PRINCIPLE:

The Ebola VP35 protein is multifunctional, acting as a

component of the viral RNA polymerase complex, a viral

assembly factor, and an inhibitor of host interferon (IFN)

production [31]. The EBOV virus VP35 protein, which plays an

essential role in viral RNA synthesis, acts as a type 1 IFN

antagonist by blocking phosphorylation of the IFN-regulatory

factor 3 (IRF3), which acts as a transcription factor for IFN

production and turns on a number of anti-viral genes [25, 27].

Figure 18: From [36].

Figure19 : From [31]

19

RF-3 is activated early during virus infection by the presence of dsRNA molecules within the cell, and it acts in

conjunction with other transcription factors to turn on the expression of immediate early antiviral genes such as IFN-

α/β, IL-15, ISG15, and ISG56. It was found that a C-terminal basic amino acid motif in VP35 is required for inhibition

of ISG56 reporter gene expression as well as IFN-β production [39].

Previous studies showed that infection of 293 T cells with Sendai virus (SeV), a potent inducer of the type I

interferon system, activates the IFN-responsive promotor ISG56 in an IRF-3-dependent manner, and this activation

is significantly inhibited by the presence of VP35 from Ebola virus. In this transfection-based reporter gene assay,

293T cells were transfected with

1. ISG56-firefly luciferase plasmid (pISG56/pGL3b)

2. a VP35 expression plasmid (pCEZ-VP35), and

3. a constitutively expressed Renilla luciferase plasmid (phRG-TK) [39].

After induction of the interferon pathway by SeV infection, firefly luciferase activity was measured and normalized

relative to Renilla expression. SeV infection induced approximately a 10-fold increase in firefly luciferase activity in

control wells, and this expression was significantly inhibited by the presence of VP35 but not the ebolavirus NP

protein [39]. So if VP35 is inhibited by a particular compound in EBOV-infected cells then fluorescence emitted will

be the same as that of SeV infected cells.

Figure 20 : The effect of C-terminal deletion mutations on IFN-antagonism mediated by VP35. (A) SeV induction of ISG56-

luciferase expression in the presence of an empty vector control plasmid, ebolavirus VP35, or ebolavirus NP. (B) Schematic

illustration of the 5 C-terminal deletion constructs. Full-length VP35 is illustrated on top. Stop codons were inserted at the

indicated residues. (C) Evaluation of each deletion construct in the ISG56-luciferase reporter gene assay. Error bars indicate

the average of each construct tested in duplicate; results shown here are representative of three independent experiments [39].

This study found that the 40 amino acids of the C Terminal is essential for the inhibitory activity of VP35 as

demonstrated by an experiment in which five C-terminal deletion constructs were generated by inserting a stop

20

codon at the indicated position using the overlap-extension PCR method. When each of these constructs was

tested in the reporter gene assay, all five truncated proteins lost the ability to block ISG56 activation. The largest of

the proteins, R300T, has a stop codon inserted at arginine 300, resulting in a deletion of the C-terminal 40 amino

acids [39].

METHODOLOGY: [39]

� 293T cells were transfected in suspension by the calcium phosphate method with the following:

� 2 µg of pCEZ-VP35

� 150 ng of pISG56/pGL3b

� 150 ng of phRG-TK

� 4.4 µg of salmon testes carrier DNA

� Combined plasmid DNA is diluted to 110µl in sterile water.

� 125 µl of 2 × HEPES-buffered saline was added, followed by 15.5 µl of 2 M CaCl2 added dropwise with gentle

shaking.

� The DNA mixture was allowed to sit at room temperature for 30 min before using it to resuspend a cell pellet

containing approximately 0.5–1.0 × 106 cells.

� The mixture of cells and DNA complexes was incubated at room temperature for 15 min, at which time 2.3 ml of

DMEM/10% FBS/Pen-Strep was added, and the cells were transferred to 6-well plates for incubation at 37 °C.

� Twenty-four hours following transfection, the cells were infected with 1024 HAU of Sendai virus per well.

� For each well, 50 µl of virus was diluted in PBS/BA (Dulbecco's PBS w/Ca2+/Mg2++, 0.2% Bovine Albumin,

Penicillin/Streptomycin) to a total volume of 600 µl.

� The media from each well was removed, and the cells were washed once with PBS before adding 600 µl of

virus mixture.

� The plates were incubated at room temperature for 1 h, at which time the inoculum was removed and replaced

with 2.3 ml of DMEM containing 0.2% BA and Penicillin-streptomycin.

� After 18–20 h at 37 °C, the cells were harvested i n 500 µl of passive lysis buffer, and 20 µl of each sample was

tested in the Dual-luciferase assay according to the manufacturer's instruction.

� The firefly luciferase values were normalized to the Renilla values for each sample.

� The relative fold-induction of reporter gene expression was assessed by comparing SeV-infected wells versus

uninfected control wells.

This methodology can be adapted for HTS screening of a library for compounds that effectively inhibits the activity

of VP35, as this assay specifically measures the inhibition of vp35 with regards to the particular pathway that it

inhibits.

21

Illustration of the principle for the luciferase-ba sed assay (Figure 21)

The firefly luciferase values are normalized to the Renilla values for each sample. The relative fold-induction of reporter gene expres sion is assessed by comparing SeV-infected wells versus uninfected control wells.

Infected with Se ndai virus. Image Taken from [40]

SeV induction of ISG56-luciferase expression [39]

22

LEAD COMPOUND

Several studies have found that the Influenza virus protein NS1 has striking similarity in terms of function and

structure of the effecter domains. Like VP35, NS1 induces antiviral RNAi response in the infected host cells. VP35

has a dsRNA-binding motif with high similarity to the dsRNA-binding domain of the NS1 protein [41]. Additionally,

the NS1 protein is able to prevent viral activation of IRF-3 and NF-κB, central components in promoting type I IFN

synthesis [38]. The NS1 protein of influenza A virus blocks both the production of type I IFN and the activation of

the IFN-induced antiviral proteins PKR and OAS [41].

A bioinformatics study identified an 8-residue motif in VP35 that has 75% sequence identity to the influenza NS1A

protein, including basic residues essential for binding of dsRNA by NS1 [38]. Mutation of these residues in both

NS1 and VP35 inhibited their IFN antagonist function [38]. Therefore like NS1, VP35 inhibits phosphorylation,

activation, and nuclear localization of IRF-3 and inhibits viral- and dsRNA-induced expression of the IFNβ gene.

VP35 also inhibits activation of the cellular antiviral kinase RNA-dependent protein kinase (PKR) and activation of

the RNAi pathway [38].

Bioinformatic comparison revealed that a 9-amino acid region

(a.a. 304–312) in the C-Terminal region shows remarkable

similarity to part of the N-terminal RNA-binding domain of the

influenza A virus NS1 protein (a.a. 36–46). No other known

viral interferon antagonists showed any similarity with VP35

[39].

Alanine substitution studies found that out of the three basic

amino acids changed to alanine, the R312A mutation resulted

in the greatest disruption to the inhibitory capacity of VP35.

Thus, of the three amino acids studied here, R312 appeared

to be the most important amino acid for inhibiting ISG56

promotor activation (IFN pathway) [39].

Double mutations in K309A and R312A completely abolished the inhibitory activity [39]. Within the 73-amino acid N-

terminal RNA-binding domain, substitution of the highly conserved residues R38 and K41 with alanines prevented

RNA binding, destroyed some of the ability to antagonize IFN [39].

From all of these findings we can safely conclude that NS1 and VP35 are very similar and therefore a drug that can

inhibit NS1 could also theoretically have some degree of inhibitory activity against VP35.

There is currently no approved NS1 protein inhibitor in the market, but I was able to find one that had been applied

for patent ship. So, technically this inhibitor should also be able to have some activity against VP35.

Figure 22 : A short C-terminal motif within VP35 displays high identity with part of the N-terminal RNA-binding domain of influenza virus NS1 protein. Part of the N-terminal RNA binding domain of NS1 from influenza A/WSN/33 (M12597) compared to the C-terminal domain from Zaire ebolavirus Mayinga VP35 (AF272001). Asterisks below NS1 sequence indicate residues that have been shown to be responsible for RNA-binding. Asterisks above VP35 sequence are the basic amino acids changed to alanine residues in this study [39].

23

In this invention the inventors have identified small “organopharmaceuticals” that inhibit the activity of NS1 protein,

but do not affect NS1 protein gene expression. NS1 protein inhibitors typically have a molecular weight of about

500g/mol or less [42].

The inventors screened a number of compounds of which Compound 8.3 was found to be the most potent with

Compound 8.6 being the second most effective in the infection assay (Figure 23 and 24). The library that they used

for high-throughput screening constituted about 200,000 small molecules which is owned by the University of

Texas, Southwestern [42].

Figure 23 : Data taken from [42]. The table depicts the compounds that were screened. Compound 8 was the most effective.

24

Figure 24: Data taken from [42]. Compound 8.3 was found to be the most potent followed by Compound 8.6

SELECTED LEAD COMPOUND CANDIDATE

This is the compound that I have selected as the lead compound. Since it shows efficacy against NS1 and since

NS1 and VP35 domains are strikingly similar, I hypothesize that this organopharmaceutical would also be effective

in inhibiting VP35 and thus allowing the initiation of the IFN pathway and subsequent activation of various other

immune responses thus stopping the progression of the infection.

Figure 25 : My lead compound

25

LEAD OPTIMIZATION

The 3D structure of the target VP35 Interferon Inhibitory Domain is given below. The yellow portion indicates

the 40 amino acids essential for VP35 activity-residues from 300-340 [39]. The red portion indicates the three basic

residues – ARG305, LYS109 and ARG312 , which are critical for VP35 to effectively block activation of an IRF-3

responsive promoter [39]. The following image (Figure 26) is taken from the RCSB Protein Database; Key word

3FKE [32].

The following is a 3D structure of VP35 inhibitory domain (shown in pink, red, blue and green showing the three

essential residues) complexed with dsRNA (shown in cyan). The PDB file is taken from RCSB Protein database;

Keyword- 3L25. [43] Figure 27:

26

LEAD OPTIMIZATION & CREATION OF NCEs BY SAR STUDIES FOR AN ANTI-LEAD OPTIMIZATION RESULTS

The lead compound that I had selected is given below

Table 1 : Structural modifications of the lead:

27

Since lysine and arginine are basic amino acids I have included a lot of Hydrogen bond forming groups in the

structures.

Table 2 : The R1 and R2 groups of the different structures are given below:

COMPOUND R1 R2

LEAD -piperdine -(Ch3)5 - COOH

NCE (1) -piperdine - CH3-C4H9NO

NCE (2) -NO-OH -CH3-C4H9NO

NCE (3) -NO-OH -(Ch3)5 - COOH

NCE (4) - -CH3-COOH

NCE (5) -COOH -CH3-COOH

NCE (6) -NO-OH -CH3-NO-OH

NCE (7) -NO-OH -CH3-CH3

NCE (8) - -CH3-NO-OH

NCE (9) -(CH3)3-C4H9NO -CH3-NO-OH

NCE (10) -NO-OH -methylmaleic anhydride

In the figure to the left I have shown the side chains of the three residue protruding outside into the active site [32].

In the figure to the right I have shown the amino acids in the active site and how they are configured/oriented. I got

this image by using the RCSB PDB Ligand Explorer 3.7 for VP35 (Key word-3L25).

Figure 28 Figure 29

28

IN VITRO SAFETY PHARMACOLOGY PROFILING

Anti-viral drugs are difficult to develop and the two broad-spectrums anti-viral compounds in the market-

Ribavirin and Interferon alpha have several side effects such as anemia, headache, irritability, anxiety, alopecia,

itchiness, insomnia, arthralgia, myalgia, anorexia, neutropenia, nausea, vomiting, fever, chills and fatigue [44].

My drug is going to be administered by intravenous injection; therefore I can hypothesize that those

problems of nausea and vomiting can be avoided. The adverse effects of my lead compound could therefore be

associated with hematology, musculoskeletal, dermatology and to certain extent maybe neurology as well.

Therefore the in vitro SPP assays that are used to screen the NCEs are as follows:

• Luciferase Reporter based gene assay [39] – Measure the binding affinity

This assay measures the inhibition of VP35 by NCE compounds in

Sendai virus (SeV) infected 293 T cells, a potent inducer of the type I

interferon system, which activates the IFN-responsive promotor ISG56 in an

IRF-3-dependent manner, and this activation is significantly inhibited by the

presence of VP35 from Ebola virus. In this transfection-based reporter gene

assay, 293T cells were transfected with

ISG56-firefly luciferase plasmid (pISG56/pGL3b)

• A VP35 expression plasmid (pCEZ-VP35), and

• a constitutively expressed Renilla luciferase plasmid (phRG-TK)

After induction of the interferon pathway by SeV infection, firefly luciferase activity was measured and

normalized relative to Renilla expression. SeV infection induced approximately a 10-fold increase in firefly luciferase

activity in control wells, and this expression was significantly inhibited by the presence of VP35 but not the

ebolavirus NP protein. So if VP35 is inhibited by a particular compound in EBOV-infected cells then fluorescence

emitted will be the same as that of SeV infected cells.

Figure 30 : SeV induction of ISG56-luciferase expression in the presence of an empty vector control plasmid, ebolaviruss VP35, OR ebolavirus NP.[39]

29

• Plaque assay for Ebola virus [36] – Confirmatory assay for VP35 inhibtion by NCE

This assay is also referred to as the Neutral Red Uptake

Assay. Confluent Vero cells are infected with an EBOV strain.

After absorption of the viral innoculum for 1h, 30 ml of EBM

containing 5% FBS, antibiotics and HEPES buffer are incubated

in flasks for 10 days. Harvested medium is centrifuged and the

supernatant fluid is made into dilutions using L-15 medium and

inoculated into monolayers of cell lines being tested. They are

then treated with the different NCEs possibly in different

concentrations as well. After various incubation periods, cell

monolayers are stained using an agarose overlay containing

neutral red. Plates are then examined for plaques 24 and 48

hours after adding the stain overlay. The viral origin of the

plaques is verified by inoculating one of the plaques mixed with L-

15 medium into suckling mice. After death of the mice, the livers

are removed, processed for virus isolation, and examined for viral

antigens with the IFA procedure. Liver tissue collected from mice

which had died postinoculation will show EV-specific fluorescence

by IFA and from this the PFUs can be counted.

• In vitro absorption, distribution, metabolism and e xcretion (ADME) assays:

ADME-Tox assays define or investigate a lead candidate’s pharmacokinetic and metabolic stability/toxicity. They

help in flagging adverse clinical effects at low cost and can prevent high attrition rates during clinical trials.[50] The

ADME-Tox assays that I have selected for screening my NCEs for are as follows:

� Cytochrome P450 (CYP450) inhibition [45][46]

Anti-viral drugs have been shown to have an effect on this family of enzymes [50]. It is a

luminescence based method to measure cytochrome P450 (CYP) activity. The assay is designed to

measure the activities of CYP enzymes and test the effects of the NCEs on CYP activitiesThe CYP

enzyme substrate which are usually derivatives of luciferin is converted by CYP enzymes to a

luciferin product that is detected in a second reaction with the Luciferin Detection Reagent. The

amount of light produced in the second reaction is proportional to CYP activity.

Figure 31: From [36]

30

A.

P450-Glo™ Substrate

(proluciferin)

CYP Enzyme

O

R B.

N N

O

S

HO

Luciferin Detection Reagent

Light

� Human Liver Microsome Assay [47]

HLM assays are carried for investigating whether the lead compounds have any effect on as

well as for measuring UDP glucuronosyltransferase (UGT) activity. The UGT family of enzymes

are involved in the metabolism of various compounds in the body by transferring a hydrophilic

glucuronic acid moiety to their substrates, rendering them more water soluble and suitable for

excretion.

To measure UGT activity, two glucuronidation reactions are set up in parallel. Both reactions

contain a source of UGT and the proluciferin substrate, but only one of them contains the

uridine 5´-diphosphoglucuronic acid (UDPGA) cofactor. During the incubation period with the

UGT enzyme or enzymes, a portion of the proluciferin substrate is glucuronidated in the

reaction containing UDPGA. None of the proluciferin is glucuronidated in the reaction lacking

UDPGA. In the second step of the assay, incubation of the proluciferin substrates with D-

Cysteine in the Luciferin Detection Reagent results in conversion of the proluciferins into

luciferin molecules.

Luciferin produced from the unmodified proluciferin will give light in the Luciferin Detection

Reagent, but the luciferin produced from the glucuronidated proluciferin will not give light.

Therefore, the decrease in light output when comparing the plus-UDPGA reaction to the minus-

UDPGA reaction is proportional to the glucuronidation activity in the first step [47].

Figure 32: taken from [46]

31

O

OH

UGT HO O N

N

C N C N

HO S

S

HO

O

UDPGA UDP OH

Plus D-Cysteine

O O

OH

O

HO

N

O

N OH

N

N OH

HO S S

O S S

HO

OH

Plus Luciferin Detection Reagent

Light No Light

7984

MA

Figure 33: . Conversion of UGT Multienzyme Substrate by UGT enzymes. UGT enzymes attach a glucuronic acid

moiety to the proluciferin substrate. During the detection step, the proluciferin is simultaneously converted to a luciferin

by cyclization with D-Cysteine. Luciferase uses the luciferin analog of the initial substrate to produce light but does not

produce light with the glucuronidated luciferin. Light output is inversely proportional to UGT enzymatic activity.[47]

� hERG Potassium Channel Screening

The protein product of the human ether-a-go-go gene

(hERG) is a potassium channel that when inhibited may lead

to cardiac arrhythmia [48]. Prediction of the hERG affinity

value, QT prolongation, and action potential parameters are

recommended as test criteria by the FDA [49]. RLB assays

are commonly used as primary screens for hERG inhibition,

whereas patch clamp analysis remains the gold standard

[50].

Planar patch clamp is the one that I have selected for

measuring hERG liability because it allows for a multiwall

format and automation, which is much faster and less labor

intensive [49].

Patch clamp technique which is a refinement of the voltage clamp allows the study of ion channels in

cells. Patch clamp recording uses, as an electrode, a glass micropipette , a size enclosing a membrane

surface area or "patch" that often contains just one or a few ion channel molecules. The interior of the

Figure 33 taken from [47]

Figure 34: The cell-attached patch clamp uses a micropipette attached to the cell membrane to allow recording from a single ion channel [51].

32

pipette is filled with a solution matching the ionic composition of the bath solution, as in the case of cell-

attached recording, or the cytoplasm for whole-cell recording. A chlorided silver wire is placed in contact

with this solution and conducts electrical current to the amplifier. The investigator can change the

composition of this solution or add drugs to study the ion channels under different conditions. The

micropipette is pressed against a cell membrane and suction is applied to assist in the formation of a

high resistance seal between the glass and the cell membrane [51].

In case Planar patch clamp assay , cell suspension

(containing a single cell randomly chosen by

application of suction) is pipetted on a chip

containing a microstructured aperture. A single cell

is then positioned on the hole by suction and a tight

connection (Gigaseal) is formed. The planar

geometry offers a variety of advantages compared to

the classical experiment:

• it allows for integration of microfluidics, which

enables automatic compound application for ion

channel screening.

• the system is accessible for optical or scanning

probe techniques

• perfusion of the intracellular side can be performed

[51, 52].

Figure 35 . The patch pipette is moved to the cell using a micromanipulator under optical control. The cell is then positioned by suction. [51]

Figure 36 : (A)Procedure of cell contacting. (B)Measured current response to a voltage pulse and after a cell is sealed onto the aperture by suction (C) Close-up view of the mechanically and electrically tight contact of the cell membrane and the chip in cell attached mode [51]

33

IN VITRO SPP PROFILE RESULTS

NCE ST 1: VP35 INHIBITION, LUCIFERASE BASED GENE ASSAY

IC50 (µM)

ST2: PLAQUE ASSAY(confirmatory assay for VP35 inhibition)

PFU

ST3: hERG INHIBITION (Planar patch clamp assay)

IC50 (µM)

ST4: CYP 450 INHIBITION ASSAY

IC50 (µM)

ST5: UGT ACTIVITY ASSAY (Human Liver Microsome assay)

IC50 (µM)

NCE (1)

5.3 3.8x103

6.8 7.6 2.2

NCE (2)

3.4 103 8.9 8.2 7.0

NCE (3)

2.3 100 >10 >10 9.1

NCE (4)

9.1 5.4x103 3.0 2.9 2.2

NCE (5)

4.1 1.2x103 7.2 7.6 8.1

NCE (6)

4.2 2x103 5.6 5.2 5.0

NCE (7)

7.0 4.5x103 4.1 4.7 4.9

NCE (8)

7.5 5x103 4.6 5.0 5.1

NCE (9)

2.1 75 >10 >10 >10

NCE (10) 3.9 3.1x103 6.2 5.6 6.7

VP35 Inhibition Assays ADME-Tox Assays Table 3:

34

CONCLUSION

I hypothesize that NCE (9) is the best as:

• It is functionalized with hydrogen bond forming groups that could engage the basic amino acids ARG305, LYS109 and ARG312, which are critical for VP35 to effectively block activation of an IRF-3 responsive promoter.

• It has got good inhibitory activity that is evident from the IC50 values of the luciferase reporter based gene assay and plaque assay.

• It has got a safe ADME-pharmacokinetic profile that is evident from the ADME-tox (In vitro SPP) assays. The second best is NCE (3). NCE (9) is going to be referred as NCT 1087 in futu re studies.

35

PATENT APPLICATION

A standard patent application for NCT1087, a VP35 protein inhibitor with the potential to act as an anti-ebola small

molecule drug will be filed with the United States Patent and Trademark Office (USPTO).

ABSTRACT

Compounds in the present invention inhibit the activity of VP35 protein, thereby mitigating Ebola Viral infection as

VP35 is critically involved in its pathogenesis. The compounds, the method of use and formulation is included in this

patent.

Compound being patented:

• NCE-9 also known as NCT1087

• NCE-3

• NCE-2

• NCE-5

NCT1087 shows the maximum potency as a VP35 inhibitor. The other compounds show comparable potency.

Therefore they will also be patented to slow down potential competition.

Background of the invention:

Ebola hemorrhagic fever is a severe, often-fatal viral hemorrhagic disease in humans and nonhuman primates 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]. 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].

Summary of the invention:

The present invention has identified small organopharmaceuticals that can inhibit VP35, a major virulence factor in

EBOV infection. It can be hypothesized that EBOV infection can be treated by administering an effective amount of

compound NCT1087.

36

SELECTION OF APPROPRIATE ANIMAL MODELS FOR EVALUATI NG THE

EFFICACY OF THE ANTI-EBOLA DRUG

In order to effectively evaluate the efficiency of the lead compounds in halting the progression of the disease

through inhibition of VP35, we need an animal model that can accurately represent the pathogenic mechanisms

that underlie EHF.

The three widely used animal models in Ebola research are mice, guinea pigs and NHPs with EHF being

best reproduced in NHPs. There are several disadvantages associated with the use of rodent models and these

are: [27]

• Wild type EBOV is not lethal in them.

• EBOV strains have to be adapted to these models through specific mutations in NP and VP24.

• Some aspects of the human form of the disease cannot be authentically replicated such as the

haemorrhagic manifestations.

• There is considerable difference in rodent and human immunology.

• Bystander apoptosis of lymphocytes, which is observed in humans, is not prominent in rodents.

• a number of antiviral therapies and vaccines that were effective in mice failed to protect nonhuman

primates. [53]

Table 4: Comparison of animal models with the human disease [27][54]

Mouse Guinea pig NHP Human

Adaptation required

Yes Yes No No

Macular rash No No Yes Yes

Haemorrhagic manifestations

Not profound No Yes Yes

Coagulation abnormalities

Not profound Conflicting data Yes Yes

37

Mouse Guinea pig NHP Human

Liver enzymes Elevated Elevated Elevated Elevated

Thrombocytopenia

Yes Yes Yes Yes

Bystander apoptosis

No No Yes Yes

Cytokine response

Yes Yes Yes Yes

Time to death 4–6 days 6–9 days 6–9 days 6–16 days

Required infrastructure and/or costs

Moderate Moderate Very high NA

Ethical concerns Moderate Moderate High NA

Permissive host cells

Monocytes, macrophages, dendritic cells, fibroblasts, hepatocytes, adrenal cortical cells, endothelial cells, epithelial

Monocytes, macrophages, dendritic cells, fibroblasts, hepatocytes, adrenal cortical cells, endothelial cells, epithelial

Monocytes, macrophages, dendritic cells, fibroblasts, hepatocytes, adrenal cortical cells, endothelial cells, epithelial

Monocytes, macrophages, dendritic cells, fibroblasts, hepatocytes, endothelial cells, epithelial cells

Cytokines/chemokines (increased circulating levels)

MCP-1, TNF-a NE IFN-a, IL-6, IL-18, MIP-1a, MIP-1b, MCP-1, TNF-a

IFN-a, IL-2, IL-6, IL-10, TNF-a

The only drawback with using NHPs is that the typical time of death is 6-9 days after exposure to ZEBOV infection,

whereas in humans the time of death is prolonged which might be due to an adaptive immune response which is

not seen in NHPs. However this doesn’t matter much in the drug that I will be testing as its primary function is to

activate the innate immune system through initiation of the type I IFN pathway. In addition, experimentally infected

NHPs are normally exposed to higher doses of EBOV than humans would be during a natural exposure [27].

38

Because of the ethical issues associated with the use of NHPs for research, I would first like to evaluate the efficacy

of the NCEs on a mouse model and then proceed testing the promising candidates using a primate model.

MOUSE MODEL USED FOR PRECLINICAL STUDIES (Efficacy evaluation)

The most commonly used mouse strains for EHF research are:

• BALB/c mice [55]

• SCID mice [56]

• ICR (CD-1) outbred mice [57]

The Ebola VP35 protein is multifunctional, acting as a component of the viral RNA polymerase complex, a viral

assembly factor, and an inhibitor of host interferon (IFN) production [31].

Therefore in my initial study with mouse models, SCID mice will be used in initial drug evaluation studies to gauge

the lead compound’s ability to inhibit viral replication independent of immunological factors.

Then in the second phase of the Mouse preclinical studies, adult, immunocompetent BALB/c mice will be used to

evaluate the efficacy of the lead compound in inhibiting the VP35 antagonism of Type 1 IFN pathway.and thus see

if this leads to increased expression of antiviral genes and subsequent immune cell clearance of the virus.

Figure 37 - Image taken from [58] **Image taken from [59]

39

The adaptation of EBO-Z to mice passaging intracerebrally in suckling mice, then in Vero cells and finally by

subcutaneous or i.p. inoculation in progressively older suckling BALB/c mice. Virus recovered from the liver of a

moribund ninth-passage mouse was plaque-purified twice and amplified. This ‘mouse-adapted virus’ is lethal for

adult BALB/c and other mouse strains when inoculated i.p. The 50% lethal dose (LD50) is 0.03 plaque-forming units

(pfu), or approximately one virion [60].

Adult SCID and BALB/c mice will be divided into four groups:

• Control

• EBOV infected (Negative control group)

• EBOV infected treated with NCE at one concentration

• EBOV infected treated with NCE at a different concentration.

Infection will be done by inoculatiion (i.p.) with 1000 pfu (30 000 LD50) of mouse-adapted EBO-Z. Negative control

groups can be treated with a placebo (PBS) or left as such [61].

Parameters checked for in the animal models:

In case of the SCID mice group:[60]

• serum viral titer levels will be examined every day postinfection.

In case of BALB/c mice group: [60][61]

• Serum cytokine concentrations of IFN-α, macrophage chemotactic protein-1 (MCP-1), interferon-gamma

(IFN-γ) and tumor necrosis factor-alpha (TNF-α) will be measured using commercial kits.

• Liver and spleen of infected and uninfected mice will be examined by Immunohistochemistry and Electron

microscopy so that we can study the effect of the NCE on the progression of the infection in major target

organs.

An experiment we can do to support my hypothesis that the NCE elicits its effect by way of the type I IFN pathway

is by co-administering antibodies to interferon alpha/beta. This will eliminate the efficacy of the drug that it mediates

via the IFN pathway. This will prove beyond doubt that the NCE acts by inhibiting VP35 and subsequent activation

of the IFN pathway. We can again confirm this by comparing the production of IFN-α in Ebola virus-infected mice

treated with the NCE or with placebo. [61]

40

NON-HUMAN PRIMATE MODEL USED FOR PRECLINICAL STUDIE S

EBOV has been shown to cause fatal hemorrhagic manifestations in: [57][53]

• Rhesus macaques ( Macaca rhesus ) • Cynomolgus macaques ( Macaca fascicularis ) • African green monkeys ( Cercopithecus aethiops) • Baboons ( Papio hamadryas ) • Vervet monkeys ( Cercopithecus aethiops )

These are the NHP models most commonly used for Ebola research as they accurately replicate the clinical

manifestations of EHF in humans. I will be choosing a combination of Rhesus and Cynomolgus macaques for the

preclinical testing of my NCEs as these two models have been the most widely used NHP model for EBOV

research.

Figure 37 -Representative cutaneous

rashes from cynomolgus monkeys

experimentally infected with EBOV-

Zaire. A: Characteristic petechial rash

of the right arm at day 4.B: Petechial

rash of the inguinal region at day 5.

Image taken from [62].

Figure 38 . Rhesus monkey

infected with Ebola virus showing

an extensive rash on the face and

anterior aspects of the arms [63].

41

As in the case of mouse models the NHPs will be divided into three groups:

• Control

• EBOV infected (Negative control group)

• EBOV infected treated with NCE at a concentration defined in the mouse model study.

Infection will be done by inoculation (i.p.) with 1000 pfu (30 000 LD50) of EBO-Z. Adaptation of the EBOV strain is

not required. EBOV from a fatally infected patient can be directly used. [62] Negative control groups can be treated

with a placebo (PBS) or left as such.

Parameters checked for in the animal models:

• Cytokine/chemokine levels of interleukin (IL)-2, IL-4, IL-10, IL-12, interferon (IFN), tumor necrosis factor

(TNF), IL-6, IFN-α, IFN-ß, MIP-1, MIP-1ß, IL-1ß, IL-8, IL-18, and MCP-1 in monkey sera/plasma can be

assayed using commercially available ELISA kits. (generally their levels should have increased in treated

models) [62][64].

• Nitrate levels were determined using a colorimetric assay. (reduction should be seen in treated models)

[62][64].

• Viral titer levels will be determined every day post infection. (reduction should be seen in treated models).

• Necropsy will be performed on the models. Tissue samples of the liver, spleen and the lung were collected

from each monkey for histopathological, immunohistochemical, in situ hybridization examination as well as

electron microscopy examination [62][64].

• Total white blood cell counts, white blood cell differentials, red blood cell counts, platelet counts,

total hemoglobin concentration will be determined to account for bystander apoptosis which is a common

manifestation of EBOV infection [64].

• Concentrations of albumin (ALB), amylase (AMY), alanine aminotransferase (ALT), aspartate

aminotransferase (AST) will be analyzed as AST and ALT levels are found to be elevated during EBOV

infection. So there should be a reduction in their levels in treated models [64][65].

• Plasma levels of fibrin degradation products (D-dimers) will be analyzed using ELISA as they are found to

decrease during the infection [64],

42

BIOMARKERS

Biomarkers in the context of a viral infection are biological molecules associated with a disease caused by

an infectious viral agent. They are essential in diagnostic and therapeutic processes as they: [66][76]

• Enable early diagnosis

• Reduce risk in drug development

• Improve patient outcomes

• Patient stratification

• Assessment of drug toxicity and efficacy

• Disease staging

• Disease prognosis

• Guide molecularly targeted therapy

• Monitor the activity and therapeutic responses across a variety of diseases.

Figure 39 : Left: Disease pathway and potential impact of biomarkers. Image taken from [67] Right: .Biomarker

science is a multifaceted discipline which impacts all aspects of drug development and modern medicine. Image

taken from [81]

43

Figure 40: Biomarker discovery and application as an integral component of drug development. (a) Disease

program approaches should yield biomarker candidates. (b) Parallelism of biomarker development in the drug

development pipeline. Biomarkers can be applied to many steps in the drug-discovery pipeline. They can also be

evaluated for use in both a pre- and post-market follow-up study. Image taken from [82].

DISEASE BIOMARKERS

There are essentially three kinds of disease biomarkers:

1. Disease Trait- is essentially a biomarker of exposure that is used to calculate the risk of exposure,

that is the likelihood that an individual is susceptible to a particular disease. [67] The likelihood of

getting Ebola hemorrhagic fever is not governed by any genetic factors. Therefore this disease doesn’t

have a disease trait biomarker as every segment of the population is equally susceptible to it.

44

2. Disease State- is a biormarker used for diagnostic purposes. The potential uses of this biomarker is

the [67]

• identification of individuals who will be or are in the preclinical stages of the disease.

• reduction in disease heterogeneity in clinical trials.

• reflection of a disease pathogenesis that is the phase of induction, latency and

detection.

• target for a clinical trial.

Table 5: Biomarker for diagnosis and typing of Ebola Hemorrhagic Fever [68]

Validated Potential

Detection/Diagnosis Glycoprotein (GP)

Nucleoprotein

GP gene

NP gene

VP24

VP30(minor

nucleoprotein)

VP35 (P-like protein)

VP40 (Matrix Protein)

VP24 gene

VP30 gene

VP35 gene

VP40 gene

Pathogen Typing GP gene

Virulence Factor Glycoprotein (GP)

3. Disease Rate - This biomarker is used to indicate or evaluate the stage of the disease. It can also be

referred to as a prognostic biomarker [84]. In case of ebola hemorrhagic fever it will be the viral titer

that is copies of EBOV/ml of blood or serum.

45

SURROGATE BIOMARKER

“A surrogate biomarker is defined as a biomarker intended to substitute for a clinical endpoint, the latter

being ‘a characteristic or variable that reflects how a patient feels, functions, or survives” [69].

In case of Ebola Hemorrhagic Fever, hypotension, generalized fluid distribution problems, lymphopenia,

coagulative disorders, and hemorrhages are its characteristic features [70]. In Ebola-infected patients,

there is extensive apoptosis of leukocytes which is associated with the fatal outcome of the disease. This

observation appears consistent with considerable lymphopenia and severe damage to lymphoid tissues,

such as spleen, lymph node and bone marrow, seen in patients and experimentally infected monkeys

[71].

Therefore the surrogate marker that I have selected is the level of lymphocytes in the blood which will

progressively decrease with disease pathogenesis and will increase with a therapeutic intervention.

TOXICITY BIOMARKERS

These biomarkers are used to monitor the adverse effects of a specific drug on any unintended cellular

processes, cells, tissues or organs [84].

RENAL TOXICITY

Due to the possible renal side effects of my NCE, the following nephrotoxic biomarkers which has been

approved by the FDA will be used to monitor the patients during the treatment period [72][73].

• Serum creatinine

• Blood urea nitrogen

• Glomerular filtration rate

HEPATIC TOXICITY

Pro-inflammatory cytokines such as:

• tumor necrosis factor alpha (TNFalpha),

• interleukin 1beta(1L-1beta)

• and interleukin 6 (IL6)

are involved in acute and chronic liver damage. They are released both from the liver and from distal sites

during hepatic toxic injury [74]. These cytokines are also involved in disseminated intravascular

46

coagulation (DIC), which is a prominent manifestation of Ebola virus (EBOV) infection. DIC is a syndrome

characterized by coagulation abnormalities including systemic intravascular activation of coagulation

leading to widespread deposition of fibrin in the circulation, which contributes to the multiple organ failure

and high mortality rates characteristic of EBOV infections [70].

Alternatively alanine transaminase and bilirubun could be used as a measure of liver toxicity [75].

TARGET BIOMARKER

A target biomarker is used to report the interaction of the NCE with the target. The Ebola VP35 protein is

multifunctional, acting as a component of the viral RNA polymerase complex, a viral assembly factor, and

an inhibitor of host interferon (IFN) production [77]. The EBOV virus VP35 protein, which plays an

essential role in viral RNA synthesis, acts as a type 1 IFN antagonist by blocking phosphorylation of the

IFN-regulatory factor 3 (IRF3), which acts as a transcription factor for IFN production and turns on a

number of anti-viral genes such as IFN-α/β, IL-15, ISG15, and ISG56 [78][79].

Inhibition of VP35 by the NCE will result in the phosphorylation of IRF3 that forms a complex with

CREBBP that translocates to the nucleus and activates the transcription of the different anti-viral genes;

therefore IRF3 or phosphorylation of IRF3 will be the target biomarker [86].

MECHANISM BIOMARKER

Mechanism biomarker is a biomolecule associated with a specific pathway that gets affected by the drug

and brings about the desired clinical effect. In case of Ebola hemorrhagic fever, the NCE will inhibit VP35

bringing about the phosphorylation of IRF3. This leads to the transcriptional up-regulation of type I IFN,

IFN-α, IFN- γ and IFN-β, genes. This signaling stimulates transcription of IFN-sensitive genes, including a

number that encode antiviral proteins, and leads to the induction of an antiviral state. Among the antiviral

proteins induced in response to type I IFN are dsRNA-dependent protein kinase R (PKR), 2′,5′-

oligoadenylate synthetase (OAS), and the Mx proteins [80].

So the mechanism biomarker for the EHF will be IFN- α, IFN- γ and IFN-β. IFN- γ is indicative of the

adaptive immune system getting activated [85].

47

EFFICACY BIOMARKER

Efficacy biomarkers are used to predict a patient’s response to a drug [81]. They essentially monitor the

beneficial effects of a specific drug on the intended drug target or medical condition [84]. In case of EHF,

the efficacy biomarker will be the viral load in the patient’s blood. It should decrease if the drug is effective

which is indicative of its efficacy.

TRANSLATIONAL BIOMARKER

Translational biomarker is a bridging biomarker that can be used in multiple species and can be used to

translate the results from preclinical animal studies to clinical human studies i.e. directly link responses

between species and follow such injury in both preclinical and clinical settings [83].

In case of EHF, translational biomarkers can be several things such as:

• viral titer

• interferon (IFN-α and IFN-ß) levels

• interleukin (IL-2, IL-4, IL-10, IL-12) level

• tumor necrosis factor levels

STRATIFICATION BIOMARKER

Stratification biomarker is used to identify patients likely to respond to a specific drug or suffer from its

side-effects prior to administration of the drug [84]. Since the NCE targets VP35, therefore one of the

stratification biomarkers as follows:

• presence of EBOV infection so that the drug can target VP35

• the patient must be immunocompetent as the drugs works by removing the block imposed by the

virus on the initiation of the innate and adaptive immune system. Therefore the second stratification

biomarker would be the presence of functional type I IFN genes as well as IRF3.

48

EFFICACY STUDIES FOR MINIMUM EFFECTIVE DOSE

ANIMAL

MODELS

NO. OF

ANIMA

LS

NCT108

7

CONC.

(mg/kg)

EFFICAC

Y BM-

VIRAL

TITER

(pfu/ml)

SURROG

ATE BM-

LYMPHO

CYTES

CONC. (%

total

cells)

TARGET

BM- IRF3

CONC.

(%

activation

)

MECHANISM

BM- IFN- γ

CONC.

(p

g/

ml

)

EFFICACY

ENDPOINT (

No. of

animals

recovered

from EBOV

infection)

SCID 2 Placebo 1.3 x 109 5% 0.1 <100 0/2

SCID 4 1mg/kg 4.8 x 105 10% 11 800 1/4

4 2 mg/kg 5.2 x 104 18% 45 1200 3/4

4 4 mg/kg 2.2 x 102 35% 65 2000 4/4

BALB/c 2 Placebo 4.1 x 1010 3.5% 1 <100 0/2

BALB/c 4 1 mg/kg 2.3 x 105 12% 12 900 2/4

4 2 mg/kg 1.4 x 103 22% 56 1300 4/4

4 4mg/kg 102 40% 75 2500 4/4

Rhesus

Monkeys 2 Placebo 2 x 109 6% 1.2 <100

0/2

Rhesus

Monkeys 4 1 mg/kg 3.4 x 105 13% 14 780

2/4

4 2 mg/kg 1.3 x 104 20% 57 1500 3/4

Table 6:

49

4 4 mg/kg 1.2 x 102 38% 81 3000 4/4

Cynomolgus

Monkeys 2 Placebo 3.2 x 109 6% 1.1 <100 0/2

Cynomolgus

Monkeys 4 1 mg/kg 1.9 x 104 12% 20 860 1/4

4 2 mg/kg 0.7 x 103 22% 78 1400 4/4

4 4 mg/kg 0.9 x 102 38% 89 2500 4/4

• *All values are measured 3 days post-infection.

• *Treatment with NCT1087 was done 1 day post-infection and continued till the 7th day.

• *Normal lymphocyte concentration is 25-33% of the WBC count. [101]

MINIMUM EFFECTIVE DOSE

• The lower bound of the dosing range is referred to as the minimum effective dose (MED). The MED

is then defined as the lowest dose level of a pharmaceutical product that provides a clinically

significant response in average efficacy, which is also statistically significantly superior to the

response provided by the placebo. [99]

• The minimum effective dose of NCT1087 required to elicit the desired clinical effect is 1 mg/kg

given once daily. However the optimal dose is 4mg/kg given once daily.

PRECLINICAL STUDIES IN ANIMAL MODELS WITH NCT1087

Preclinical studies may be defined as animal studies that support Phase I safety and tolerance studies

[87]. The goals of the nonclinical safety evaluation generally include a characterization of toxic effects with

respect to target organs, dose dependence, relationship to exposure, and when appropriate, potential

reversibility. This information is helpful for the estimation of an initial safe starting dose and dose range for

the human trials and the identification of parameters for clinical monitoring for potential adverse effects

[88]. Because many animals have much shorter life spans than humans, preclinical studies can provide

valuable information about a drug’s possible toxic effects over an animal’s life cycle and on its offspring

[87].

50

FDA requirement for nonclinical safety studies are [88]:

• Safety pharmacology studies

• Repeated dose toxicity studies

• Toxicokinetic and nonclinical pharmacokinetic studies

• Reproduction toxicity studies

• Genotoxicity studies

• Assessment of carcinogenic potential for long term use.

• Phototoxicity studies

• Immunotoxicity studies

• Juvenile animal toxicity studies

ANIMAL MODELS IN SAFETY STUDIES

Typically preclinical safety studies require a rodent and non-rodent model.The same models that have

been used for efficacy studies is used for toxicity studies that is:

Rodent models:

• SCID mice- 5 in number (2- control)

• BALB/c mice- 5 in number (2- control)

Non-rodent models:

• Rhesus macaques- 5 in number (2- control)

• Cynomolgus macaques- 5 in number (2- control)

51

Figure 41 : Fundamental principles applied to the selection of animal models for conducting preclinical toxicology

studies of pharmaceuticals. (a) In selecting the preclinical species for toxicology studies of small-molecule

pharmaceuticals, it is crucial to consider three important criteria: (i) the presence of the desired pharmacodynamics

(whenever this is possible); (ii) the presence of all potential major human metabolites; (iii) adequate bioavailability

and systemic exposure. Image taken from [90].

ROUTE OF ADMINISTRATION OF THE DRUG

It will be intravenously injected for sufficient exposure of the animal to the NCE & its metabolites. It will be

administered once a day.

SAFETY PHARMACOLOGY STUDIES

Safety pharmacology studies are defined as those studies that investigate the potential undesirable

pharmacodynamic effects of a substance on physiological functions in relation to exposure in the

therapeutic range and above [89].

OBJECTIVES:

• to identify undesirable pharmacodynamic properties of a substance that may have relevance to its

human safety

• to evaluate adverse pharmacodynamic and/or pathophysiological effects of a substance observed

in toxicology and/or clinical studies

• to investigate the mechanism of the adverse pharmacodynamic effects observed and/or suspected.

52

Investigation of the test substance on vital functions of the cardiovascular, respiratory and central nervous

systems will be done in addition to the in vitro ADME-TOX studies and they will be done as follows: [89]

a) Central Nervous System - Evaluation of motor activity, behavioral changes, coordination,

sensory/motor reflex responses and body temperature will be evaluated to gauge any possible

effect of the NCE on the CNS [89].

A Functional Observational Battery (FOB) will be performed on the animal models which is a

neurotoxicity screening process that is used ti identify chemicals that are neurotoxic hazards. In

addition neuropathologic examination following euthanization will be done [91].

Endpoints of the Functional Observational Battery b y Domain of Neurological Function [91]

b) Cardiovascular System- Effects on the cardiovascular system is assessed by evaluating: [89]

• Blood pressure

• Electrocardiogram – will show if there is any possible Qt prolongation problems.

• Heart rate

This is done in addition to the invtro hERG channel inhibition assay.

c) Respiratory Sytem- Effects on the respiratory system is assessed by measuring: [89]

• Tidal volume

• Hemoglobin oxygen saturation

d) Renal system - Renal parameters have to be assessed such as : [89]

• Urinary volume

• Specific gravity

1)Autonomic Pupil response (Q)° Defecation (Q Urination (Q Salivation (R) Lacrimation (R) Palpebral closure (D)

2)Convulsions Clonic movements (D)

3)Neuromuscular Gait score (R) Forelimb grip strength (C) Hindlimb grip strength (C) Landing foot splay (C) Righting reflex (R)

4)Sensorimotor Approach response (R) Click response (R) Touch response (R) Tail pinch response (R)

5)Activity Home cage posture (D) Motor activity—counts (C)* Rearing (C) 6)Excitability Arousal (R) Ease of removal (R) Handling reactivity (R)

53

• Osmolality

• pH and fluid/electrolyte balance

• proteins

• blood urea nitrogen

• creatinine

• plasma proteins

• cytology

ACUTE TOXICITY STUDY

Two rodent (one SCID and one BALB/C) and two non-rodent models (two Rhesus Macaques) will be

used for this study.

In all cases a limit dose of 2000 mg/kg/day in rodents and 1000 mg/kg/day in non-rodents is considered

appropriate for acute, subchronic, and chronic toxicity studies [88]. Their respiratory, central nervous

system, cardiovascular system, renal system and hepatic system will be analyzed.

REPEATED DOSE TOXICITY STUDY

The primary goal of repeated dose toxicity studies is to characterize the toxicological profile of the test

compound following repeated administration. [92] The recommended duration of the repeated dose

toxicity studies is usually related to the duration, therapeutic indication and scope of the proposed clinical

trial.[88] Two rodent (one SCID and one BALB/C) and two non-rodent models(two Rhesus Macaques)

will be used for this study.

7

54

Table taken from [88]. Since the antiviral therapy duration would be for 2 weeks at the maximum.

Therefore the repeated dose toxicity study in animal models will done for 2 weeks.

Table taken from [88]. As shown in the table, for marketing purposes the repeated dose toxicity study will

be carried out for one month.

The following will be monitored during the course of the study: [92]

• food intake

• general behavior

• body weight

• hematological parameters

• clinical chemistry

• urinalysis

• ophthalmology

• Electrocardiographic recordings in non-rodent species.

The parameters will be determined at relevant time points, taking the pharmacodynamic/pharmacokinetic

profiles into account. Animals that die or are sacrificed during the study should be autopsied and if

feasible, subjected to microscopic examination [92].

GENOTOXICITY STUDIES

Genotoxicity tests can be defined as in vitro and in vivo tests designed to detect compounds that induce

genetic damage by various mechanisms. These tests enable hazard identification with respect to damage

to DNA and its fixation [93].

8

55

In vitro assessment of mutagenicity can be done by means of: [93]

• Ames test (Bacterial reverse mutation test)

• Other in vitro mammalian cell systems such as

• in vitro metaphase chromosome aberration assay

• In vitro micronucleus assay

• mouse lymphoma L5178Y cell tk gene mutation assay.

In vivo testing for chromosomal damage in animal models is done by either an analysis of micronuclei in

erythrocytes in blood or bone marrow, or of chromosomal aberrations at metaphase in bone marrow cells.

Lymphocytes cultured from treated animals can also be used for cytogenetic analysis, although

experience with such analyses is less widespread [93].

In case of genotoxicity studies for NCT1087 the following will be done. FDA requires two in vitro tests and

one in vivo study to be done before filing an IND :

1. Ames Test - Salmonella typhimurium strains TA100 and TA98 have point mutations that make

them incapable of growing in medium unless histidine is supplied. Treatment with a genotoxic

chemical causes a mutagenic event to occur, during which base substitutions or frameshifts within

the His gene may cause a reversion to histidine prototrophy, and the mutated organisms become

able to grow in histidine-deficient medium [94].

Ames Assay Protocol:[94]

• Approximately 107 TA100 or TA98 organisms are exposed to 6 serial dilutions of test agent at 37ºC

for 90 minutes in medium containing sufficient histidine to support approximately two cell divisions.

• The cultures are diluted in pH indicator medium lacking histidine and aliquoted into 48 wells of a

384-well plate.

• The plates are incubated for 48 hours at 37ºC, then wells containing cells that have undergone the

reversion to histidine prototrophy and have grown into colonies are counted for each dose and

compared to a zero-dose (solvent) control. Each dose is done in triplicate to allow for statistical

analysis of the data.

• A two-fold increase in the number of revertant colonies upon exposure to test chemical relative to

the zero-dose controls indicates that the chemical is mutagenic in the Ames MPF™ 98/100 assay.

• The maximum dose level recommended is 5000 µg/plate when not limited by solubility or

cytotoxicity [93].

56

2. In vitro Metaphase chromosome aberration assay- The purpose of the in vitro chromosome

aberration test is to identify agents that cause structural chromosome aberrations in cultured

mammalian cells.

Cell cultures are exposed to the test substance both with and

without metabolic activation. At predetermined intervals after

exposure of cell cultures to the test substance, they are

treated with a metaphase-arresting substance (e.g.,

Colcemid or colchicine), harvested, stained, and metaphase

cells are analysed microscopically for the presence of

chromosome aberrations [30]. The maximum top

concentration recommended is 1 mM or 0.5 mg/ml,

whichever is lower, when not limited by solubility or

cytotoxicity [93].

2. In vivo genetoxicity studies- Since NCT1087 will be intravenously injected, it will be systemically

absorbed. Therefore analysis of bone marrow, blood and liver will be done [28]. Analysis can be

done by means of:[93]

• the DNA strand break assays such as the single cell gel electrophoresis (“Comet”) assay

and alkaline elution assay

• the in vivo transgenic mouse mutation assays

• DNA covalent binding assays,

• to the liver unscheduled DNA synthesis (UDS) assay.

Typically three dose levels are used with a limit of 2000 mg/kg [93]. Animal models of both

sexes will be used for this purpose [93].

RESULTS OF GENETOXICITY STUDIES:

Mutagenic and carcinogenic effects of NCT1087 was found to be nil in both in vitro as well as in vitro

studies.

Image taken from [31]

Figure 42 : taken from [96]

57

CARCINOGENICITY STUDIES

Since NCT1087 doesn’t have any genotoxic properties and plus since the intended duration of treatment

is within 2 weeks, therefore there is little need for conducting long term carcinogenicity studies.

REPRODUCTION TOXICITY STUDIES

The aim of reproduction toxicity studies is to reveal any effect of one or more active substance(s) on

mammalian reproduction [97]. Reproduction toxicity studies must be completed prior to inclusion of

women of child bearing potential in the clinical studies. Men can be included in the Phase 1 and 2 trials

prior to completing the male fertility studies [88]. The study will be continued through one complete life

cycle, i.e., from conception in one generation through conception in the following generation. It can be

subdivided into the following stages: [97]

• Premating to conception (adult male and female reproductive functions, development and

maturation of gametes, mating behavior, fertilization).

• Conception to implantation (adult female reproductive functions, preimplantation development,

implantation).

• Implantation to closure of the hard palate (adult female reproductive functions, embryonic

development, major organ formation).

• Closure of the hard palate to the end of pregnancy (adult female reproductive functions, fetal

development and growth,organ development and growth).

• Birth to weaning (adult female reproductive functions, neonate adaption to extrauterine life,

preweaning development and growth).

• Weaning to sexual maturity (postweaning development and growth, adaption to independent life,

attainment of full sexual function)

Animals used:

In case of reproduction toxicity studies, both rodent and non-rodent models are required. For rodents,

BALB/c mice will be used. In case non-rodents, rabbits will be used [97].

The animal models on regular exposure to NCT1087 will be analyzed for:

• Fertility and early embryonic development,

58

• Prenatal and postnatal development, including maternal function, and

• Embryo-fetal development.

Results for Reproduction toxicity studies:

No detectable effect on the fertility, embryonic development, prenatal and postnatal development and

embryo fetal development has been observed in both rodent and non-rodent models when treated with

NCT1087.

IMMUNOTOXICITY STUDIES:

Toxicity to the immune system encompasses a variety of adverse effects. These include suppression or

enhancement of the immune response. Suppression of the immune response can lead to decreased host

resistance to infectious agents or tumor cells. Enhancing the immune response can exaggerate

autoimmune diseases or hypersensitivity [98].

Immunotoxicity studies are conducted by taking into account the following factors: [98]

• findings from STS

• the pharmacological properties of the drug

• intended patient population

• structural similarities to known immunomodulators

• disposition of the drug

• clinical information.

Assessment of immunotoxicity will include the following: [98]

• Hematological changes such as leukocytopenia/leukocytosis, granulocytopenia/ granulocytosis, or

lymphopenia/ lymphocytosis

• Alterations in immune system organ weights and/or histology (e.g., changes in thymus, spleen,

lymph nodes, and/or bone marrow)

• Changes in serum globulins that occur without a plausible explanation, such as effects on the liver

or kidney, can be an indication that there are changes in serum immunoglobulins

• Increased incidence of infections

• Increased occurrence of tumors can be viewed as a sign of immunosuppression in the absence of

other plausible causes such as genotoxicity, hormonal effects, or liver enzyme induction.

59

The following table lists that parameters that should be evaluated in standard toxicity studies for signs

of immunotoxicity [98].

Additional assays such as T-cell dependent Antibody Response assays (TDAR) can be done [98].

Results of Immunotoxicity Studies:

Since NCT1087 works by removing the roadblock imposed by the Ebola virus on the natural course of

the body’s immune system, all immunotoxicology studies indicate that NCT1087 will have no

detectable effects on the immune system of the patient.

PHOTOTOXICITY STUDIES:

Phototoxicity studies will be influenced by:

1) the photochemical properties (photoabsorption and photostability) of the molecule,

2) information on the phototoxic potential of chemically-related compounds,

3) tissue distribution, and

4) clinical or nonclinical findings indicative of phototoxicity.

An initial assessment of phototoxic potential based on a drug’s physical/chemical properties for

photoreactivity, spectral absorption properties, and pharmacologic class / SAR should be performed.

Thereafter, an evaluation of the nonclinical drug distribution to skin and eye should be undertaken to

further inform on the human risk [88].

Results from phototoxicity studies: The study indicates that NCT1087 has no phototoxicity side effects.

Table 9:

60

MAXIMUM TOLERATED DOSE

Maximum Tolerated Dose (MTD) is defined as the high dose used in chronic toxicity testing that is

expected on the basis of an adequate subchronic study to produce limited toxicity when administered for

the duration of the test period. It should not induce: [100]

• overt toxicity, for example appreciable death of cells or organ dysfunction, or

• toxic manifestations that are predicted materially to reduce the life span of the animals

except as the result of neoplastic development or

• 10% or greater retardation of body weight gain as compared with control animals. In some

studies, toxicity that could interfere with a carcinogenic effect is specifically excluded from

consideration.

TOXICITY STUDIES FOR FINDING THE MAXIMUM TOLERATED DOSE

In case of the toxicity study all animals are healthy prior to be treating with NCT1087. Rodent and Non-

rodent species is used as required by the FDA.

• Dosage is done once daily.

• Route of administration is Intravenous injection.

• Duration of the study - one month

At the end of one month, two animals in each group will be sacrificed and autopsy of all the organs will be

done to analyze any possible organ toxicities. During the study, Kidney toxicity will be analyzed by

measuring the level of: [72][73]

• Serum creatinine

• Blood urea nitrogen

• Glomerular filtration rate

Liver toxicity will be analyzed by measuring the level of: [9]

• tumor necrosis factor alpha (TNFalpha),

• and interleukin 6 (IL6)

61

GROUPS NCT1087

DOSE

ANIMAL

TYPE

NO. OF

ANIMAL

S

BODY

WEIGHT

(avg)

MORT

ALITY

MORBU

NDITY

LIVER

TOXICITY

KIDNEY

TOXICITY

Control BALB/c 5 21g Nil Nil Nil Nil

100mg/kg BALB/c 5 20.2 g 0 0/5 0/5 0/5

Control RHM 5 3.62 kg Nil Nil Nil Nil

1

100mg/kg RHM 5 3.2 kg 0 0/5 0/5 0/5

Control BALB/c 5 20g Nil Nil Nil Nil

200mg/kg BALB/c 5 18.9g 0 2/5 1/5 2/5

Control RHM 5 3.68 kg Nil Nil Nil Nil

2

200mg/kg RHM 5 2.9 kg 0 2/5 2/5 1/5

Control BALB/c 5 22g Nil Nil Nil Nil

400mg/kg BALB/c 5 17.9g 2 5/5 3/5 3/5

Control RHM 5 3.7 kg Nil Nil Nil Nil

3

400mg/kg RHM 5 2.5 kg 3 5/5 3/5 3/5

*RHM- Rhesus Macaques

The Maximum Tolerated Dose in the animal studies that did not cause mortality, but caused liver and

kidney toxicity was found to be 200 mg/kg. Therefore the Maximum Tolerated Dose for estimating the

dosage in Phase I human clinical trials will be taken as 100 mg/kg as it has been shown not to cause liver

or kidney toxicity.

Maximum Tolerated Dose – 100 mg/kg

Table 10:

62

REPEATED DOSE TOXICTY STUDY WITH 2mg/kg & 4mg/kg FO R TWO MONTHS

The study is done in healthy animals (a rodent and non-rodent species is used). The dosage is 2mg/kg

and 4 mg/kg. Route of administration is intravenous injection.

At the end of two months, two animals in each group will be sacrificed and autopsy of all the organs will

be done to analyze any possible organ toxicities. During the study, Kidney toxicity will be analyzed by

measuring the level of: [7][8]

• Serum creatinine

• Blood urea nitrogen

• Glomerular filtration rate

Liver toxicity will be analyzed by measuring the level of: [9]

• tumor necrosis factor alpha (TNFalpha),

• and interleukin 6 (IL6)

GROUPS NCT1087

DOSE

ANIMAL

TYPE

NO. OF

ANIMAL

S

BODY

WEIGHT

(avg)

MORT

ALITY

MORBU

NDITY

LIVER

TOXICITY

KIDNEY

TOXICITY

Control BALB/c 5 21g Nil Nil Nil Nil

2 mg/kg

(after 2

weeks)

BALB/c 5 21 g 0 0/5 0/5 0/5

2 mg/kg

(after 4

weeks)

BALB/c 5 21 g 0 0/5 0/5 0/5

1

Control RHM 5 3.62 kg Nil Nil Nil Nil

Table 11:

63

2 mg/kg

(after 2

weeks)

RHM 5 3.62kg 0 0/5 0/5 0/5

2 mg/kg

(after 4

weeks)

RHM 5 3.61 kg 0 0/5 0/5 0/5

Control BALB/c 5 20g Nil Nil Nil Nil

4 mg/kg

(after 2

weeks)

BALB/c 5 20g 0 0/5 0/5 0/5

4 mg/kg

(after 4

weeks)

BALB/c 5 20 g 0 0/5 0/5 0/5

Control RHM 5 3.68 kg Nil Nil Nil Nil

2

4 mg/kg

(after 2

weeks)

RHM 5 3.67 kg 0 0/5 0/5 0/5

4 mg/kg

(after 4

weeks)

RHM 5 3.67 kg 0 0/5 0/5 0/5

There is no observed liver or kidney toxicity when dosed repeatedly for two months. Since the intended

duration of treatment in humans is estimated to be for 5 days, two months of repeated toxicity studies will

be sufficient for filing an IND.

64

ESTIMATION OF THE FIRST DOSE IN HUMANS

From the preclinical animal studies, following are the results:

• Minimum Effective Dose- 1mg/kg

• Maximum Tolerated Dose- 100mg/kg

• Estimated human Maximum Tolerated Dose – 10mg/kg (i.e. 10 times lower than the MTD in animal

studies)

Figure 43: Whole blood concentration–time profiles of NCT1087 following a single 2 mg/kg i.v. dose administration

in animal models.

From the Pharmacokinetic studies done in the Rhesus Macaques animal models, the bioavailability of

NCT1087 upon intravenous injection was found to be 100% upon administration and was found to be

present in the systemic circulation for up to 24 hours. Therefore the dosage will be once in human clinical

trials and then modified if necessary.

The initial dose in humans for Phase I clinical trials will be taken as 2mg/kg and then Dose Acceleration

studies will be done to find out the most effective dose in humans.

NCT1087 Blood Concentration (ng/ml)

65

IND APPLICATION

The primary goal in filing an IND is to show that the product is reasonably safe for initial use in early stage

human clinical trials, and that the compound exhibits pharmacological activity that justifies commercial

development [113].

A Commercial Sponsor IND will be filed for NCT1087 with the FDA. The IND application must contain

information in three broad areas: [113]

• Animal Pharmacology and Toxicology Studies - Preclinical data to permit an assessment as to

whether the product is reasonably safe for initial testing in humans

• Manufacturing Information - Information pertaining to the composition, manufacturer, stability,

and controls used for manufacturing the drug substance and the drug product is assessed to ensure

that the company can adequately produce and supply consistent batches of the drug.

• Clinical Protocols and Investigator Information - Detailed protocols for proposed clinical studies

to assess whether the initial-phase trials will expose subjects to unnecessary risks.

Figure 44:

66

PHASE I CLINICAL TRIALS [40]

PURPOSE:

The purpose of this study is to determine the safety and tolerability of NCT1087 when administered via

intravenous injection and how it affects the amount of EBOV viral load in patients.

PRODUCT NAME:

• NCT1087

PRIMARY ENDPOINTS:

• Determine change in EBOV viral load in patients

• Determine clinical safety and tolerability

• Determine safe dose range

SECONDARY ENDPOINTS:

• Analysis of human pharmacokinetic profile and ADME profile

• Bioavailability

STUDY TYPE:

• Interventional

STUDY DESIGN:

• Allocation: Non-randomized

• Control: Uncontrolled

• Endpoint Classification: Safety and Efficacy study

• Primary Purpose: Treatment

• Masking: Open Label

ENROLLMENT : 20-30

ELIGIBILITY:

• Ages Eligible for Study: 18 Years and older

• Genders Eligible for Study: Both

• Accepts Healthy Volunteers: No

67

INCLUSION CRITERIA:

• Clinical or laboratory evidence of EBOV infection

• Patients with suspected EBOV infection

• Patients with “pure disease state” preferred.

• Female patients of childbearing potential must not be pregnant at the start of the study

• All patients of reproductive potential must be willing to use contraception or abstain from

intercourse.

• Adequate renal function

• Adequate hematologic function

EXCLUSION CRITERIA:

• History of autoimmune disorder and immunodeficiency

• Potential effect on lactating and pregnant woman is not known, so care should be taken while

recruiting them.

• Use of any drugs or vaccines within a month of enrollment or atleast two weeks before enrollment.

• Patients with chronic kidney and liver problems.

• Known HIV or active hepatitis B virus (HBV) infection

• Antiviral therapy within 3 months before study

• Investigational therapy within a month before study

• Major surgery within a month before study

FACTORS THAT WILL BE MONITORED:

• Routine Blood, Stool and Urine analysis

• Blood Pressure

• ECG if required

• Body Temperature

• Body weight

ANTICIPATED SIDE EFFECTS

Potential side effects of the drug can be:

68

• Possible kidney problems

• Possible liver toxicity

Monitoring kidney problems: [101]

Blood and urine tests such as the following can be used to evaluate acute renal failure:

• Serum creatinine – An increase in blood creatinine levels is indicative of acute renal failure.

• Blood urea nitrogen (BUN) – BUN level increases if the kidney is not able to remove urea from the

blood normally.

• Blood electrolyte tests, such as calcium, phosphate, potassium and sodium.

• Complete blood cell count .

Monitoring liver toxicity: [102]

• Liver function tests have to be performed prior to starting the trial.

• The following are the cut off levels for liver dysfunction when the treatment for the particular patient

has to be stopped or reduced:

� Alanine transaminase level rising to three times or above the upper limit of normal.

� Bilirubin level rising to two times or above the upper limit of normal.

� Alkaline phosphatase

�

INITIAL DOSE:

The initial dose for Phase I clinical trials will be 2mg/kg given once.

ROUTE OF ADMINISTRATION:

Administration of the drug is via intravenous injection. Bioavailability will therefore be 100%.

DURATION OF THE STUDY:

The treatment will last for a maximum of two weeks. The patients will be followed for 3 months after

treatment for any potential relapse or side effects.

69

DESCRIPTION OF THE STUDY:

Informed consent will be obtained from the patient prior to starting the trial. Body Temperature, Blood

pressure and other initial parameters will be recorded in the patient. An initial dose of 2mg/kg will be given

to the patients and observed for 24 hours for any adverse events. Then an accelerated dose of 4mg /kg

will be tried followed by observation for 24 hours. Dose acceleration will be done until an optimal dose

with efficient pharmacokinetic profile is obtained.

RESULTS OF PHASE I CLINICAL TRIALS OF NCT1087

NCT1087

CONC.

NO. OF

SUBJETCS

EFFECACY

BM- viral titer

(pfu/ml)

RENAL

TOXICITY

BM-Serum

Creatinine

level

(mg/dL)

HEPATIC

TOXICITY

BM- Alkaline

Phoshatase

Level (IU/L)

NO. OF

SERIOUS

ADVERSE

EVENTS

2 mg/kg 3 7.3 x 1010 1.0 100 0

4 mg/kg 4 5.4 x 107 1.2 110 0

8 mg/kg 8 3.2 x 106 1.2 105 1

16 mg/kg 11 6.5 x 104 1.1 106 2

32mg/kg 16 2 x 102 1.2 111 4

64 mg/kg 20 1.5 x 101 1.4 139 6

Normal Serum Creatinine level in the blood is 0.8 to 1.4 mg/dL [103].

Normal Alkaline Phosphatase level is 20 to 140 IU/L [104].

ADVERSE EVENT OBSERVED:

� Slight liver toxicity

Table 12:

70

� Slight kidney toxicity

� Other serious AE at the highest dose

CONCLUSION:

� From Phase 1 study, it can be inferred that the Dose Limiting Toxicity (DLT) dose is 64mg/kg.

The reason behind this conclusion is that, although serum creatinine levels and Alkaline

Phosphatase levels are just within the normal range, there are other serious AEs. Overall NCT1087

was well tolerated.

� However, 32mg/kg dosage has been found to bring down the viral load significantly without any

associated organ toxicity and manageable AEs.

� Phase II studies will therefore be carried out with the dose of 32mg/kg and 50mg/kg. As can be

seen there is good availability for 24 hours at the former dose concentration in human patients.

Bioavailability of NCT1087 (12mg/kg)

0

2000

4000

6000

8000

10000

12000

0hrs 5hrs 10hrs 15hrs 20hrs 25hrs 30hrs 35hrs 40hrs

Bioavailability of NCT1087

(12mg/kg)

Figure 45 . Whole blood concentration–time profiles of NCT1087 following a single 32 mg/kg i.v. dose

administration in patients.

Time (hrs)

NCT1087 Blood Concentration (ng/ml)

71

PHASE II CLINICAL TRIALS

Since NCT1087 is a “first in class” Ebola anti-viral therapeutic, the studies can neither be labeled as

Superiority or Non-inferiority studies.

Non-clinical toxicology studies such as:

• Single and repeated dose studies (carried out for two months)

• Genetoxicity studies

• Reproductive Toxicity studies

• Safety Pharmacology studies (which encompasses study on the Qt prolongation as well as on

other organs such as the respiratory, renal and hepatic system)

have been completed.

PURPOSE:

The purpose of Phase II trials to investigate the anti-viral efficacy, safety and tolerability of NCT1087 in

a larger patient population. This study is also aimed at fine-tuning the dose concentration to be

administered in the patients through a sequential dose escalation study.

PRODUCT NAME:

• NCT1087

PRIMARY ENDPOINTS:

• Determine change in EBOV viral load in patients

• Determine clinical safety and tolerability

• Fine tune the dose range

• Validating clinical efficacy end points and mechanism of action of the drug.

SECONDARY ENDPOINTS:

• Analysis of human pharmacokinetic profile and ADME profile

• Bioavailability

STUDY TYPE:

• Interventional

72

STUDY DESIGN:

• Allocation: Non-randomized

• Control: Uncontrolled

• Endpoint Classification: Safety and Efficacy study

• Primary Purpose: Treatment

• Masking: Open Label

ENROLLMENT : 150-200

ELIGIBILITY:

• Ages Eligible for Study: 18 Years and older

• Genders Eligible for Study: Both

• Accepts Healthy Volunteers: No

INCLUSION CRITERIA:

• Clinical or laboratory evidence of EBOV infection

• Patients with suspected EBOV infection

• Patients with “pure disease state” preferred.

• Female patients of childbearing potential must not be pregnant at the start of the study

• All patients of reproductive potential must be willing to use contraception or abstain from

intercourse.

• Adequate renal function

• Adequate hematologic function

EXCLUSION CRITERIA:

• History of autoimmune disorder and immunodeficiency

• Potential effect on lactating and pregnant woman is not known, so care should be taken while

recruiting them.

• Use of any drugs or vaccines within a month of enrollment or atleast two weeks before enrollment.

• Patients with chronic kidney and liver problems.

• Known HIV or active hepatitis B virus (HBV) infection

• Antiviral therapy within 3 months before study

73

• Investigational therapy within a month before study

• Major surgery within a month before study

FACTORS THAT WILL BE MONITORED:

• Routine Blood, Stool and Urine analysis

• Blood Pressure

• ECG if required

• Body Temperature

• Body weight

ANTICIPATED SIDE EFFECTS

Potential side effects of the drug can be:

• Possible kidney problems

• Possible liver toxicity

Monitoring kidney problems: [101]

Blood and urine tests such as the following can be used to evaluate acute renal failure:

• Serum creatinine – An increase in blood creatinine levels is indicative of acute renal failure.

• Blood urea nitrogen (BUN) – BUN level increases if the kidney is not able to remove urea from the

blood normally.

• Blood electrolyte tests, such as calcium, phosphate, potassium and sodium.

• Complete blood cell count .

Monitoring liver toxicity: [102]

• Liver function tests have to be performed prior to starting the trial.

• The following are the cut off levels for liver dysfunction when the treatment for the particular patient

has to be stopped or reduced:

74

� Alanine transaminase level rising to three times or above the upper limit of normal.

� Bilirubin level rising to two times or above the upper limit of normal.

� Alkaline phosphatase

DOSE:

32 and 50 mg/kg given once.

ROUTE OF ADMINISTRATION:

Administration of the drug is via intravenous injection. Bioavailability will therefore be near 100%.

DURATION OF THE STUDY:

The treatment will last for a maximum of two weeks. The patients will be followed for 3 months after

treatment for any potential relapse or side effects.

DESCRIPTION OF THE STUDY:

Informed consent will be obtained from the patient prior to starting the trial. Body Temperature, Blood

pressure and other initial parameters will be recorded in the patient. Patients will be given a dose of 32 or

50 mg/kg and treated for the duration of 5 days after, which the viral load will be accessed.

If the viral load hasn’t decreased below 102 pfu/ml; treatment will be continued for another five days.

75

RESULTS OF PHASE II CLINICAL TRIALS OF NCT1087

Table 13; Determining the most effective dosage:

NCT1087

CONC.

NO. OF

SUBJETCS

EFFECACY

BM- viral titer

(pfu/ml)

RENAL

TOXICITY

BM-Serum

Creatinine

level (mg/dL)

HEPATIC

TOXICITY

BM- Alkaline

Phoshatase

Level (IU/L)

NO. OF

SERIOUS

ADVERSE

EVENTS

32 mg/kg 45 1.3 x 102 1.2 111 5

50 mg/kg 65 0.6 x 102 1.25 130 10

Normal Serum Creatinine level in the blood is 0.8 to 1.4 mg/dL. [103]

Normal Alkaline Phosphatase level is 20 to 140 IU/L. [104]

From the studies, 50mg/kg seems to be more of an effective dose without resulting in any organ toxicity or adverse

events. Therefore further studies in Phase III will be done with this dose.

Table 14: Clinical Efficacy Results (Only 30 patien ts were evaluated daily)

DAYS NO. OF

PATIENTS

NCT1087

CONC.

(mg/kg)

EFFICACY

BM-VIRAL

TITER

(pfu/ml)

SURROGATE

BM-

LYMPHOCYTE

S CONC. (%

total cells)

TARGET

BM- IRF3

CONC.

% acti-

vation)

MECHANISM

BM- IFN- γ

CONC.

(pg/ml)

0 150 0mg/kg 3.2 x 109 5% 0.1 <100

1 150 50 mg/kg 4.8 x 106 8% 11 500

2 150 50 mg/kg 5.2 x 104 15% 33 900

3 150 50 mg/kg 4.1 x 103 20% 49 1000

4 150 50 mg/kg 1.2 x 102 25% 58 1500

5 150 50 mg/kg 0.5 x 102 31% 75 2400

76

� Lymphocyte concentration is estimated by conducting the WBC count and then subsequently analyzing the

lymphocyte composition in them. Normal Lymphocyte concentration is 25-33% [41].

� IRF3 concentration is analyzed by estimating IRF3 mRNA concentration by means of Real Time PCR.

� IFN- γ concentration is estimated by means of commercially available ELISA kits.

From the studies and results above, the drug – NCT1087 has an efficient pharmacokinetic profile and

elicits the desired clinical effect within 5 days when the patients are dosed with a drug concentration of 50

mg/kg.

Proof that the drug elicits its desired clinical effect by inhibiting VP35 and allowing the activation of IRF3

gene and subsequent initiation of IFN Type I pathway is shown by the increase in IRF3 activation and

IFN- γ concentration in the blood of the patients as the treatment progresses.

CONCLUSION:

Therefore it can be concluded that Phase II trials with NCT1087 has been successful in showing that:

� A concentration of 50mg/kg is sufficient in eliciting the desired clinical effect.

� The desired clinical effect is elicited within 5 days with a significant decrease in viral load.

� The recommended dose concentration has been found to have safe pharmacokinetic profile as well

as a safe ADME profile.

� The recommended dose concentration has a good bioavailability for 24 hours, therefore dosage is

only required every 24 hours.

77

PHASE III CLINICAL STUDY DESIGN

Ebola virus (EBOV) causes lethal hemorrhagic fever in humans and no-human primates and these

viruses can be pursued as a potential biological weapon. The current drug under investigation is a small

molecule drug that inhibits a critical protein involved in disease pathogenesis.

PURPOSE:

The present study aims to evaluate the efficacy and safety of the drug in a larger patient population and to

gauge the overall benefit-risk relationship of the drug. There is no standard treatment for Ebola

hemorrhagic fever except for supportive care.

TRIAL DESIGN:

The study will be conducted with no placebo or other control comparison therapeutics as no FDA

approved drugs exist. It will also be an open-labeled study.

PRODUCT NAME:

• NCT1087

PRIMARY ENDPOINTS:

• Determine change in EBOV viral load in patients

• Inhibition of disease progression and disease fatality.

• Confirm clinical safety and tolerability

• Validating clinical efficacy end points and mechanism of action of the drug.

SECONDARY ENDPOINTS:

• Analysis of human pharmacokinetic profile and ADME profile

• Bioavailability

• Immunogenecity (cellular and humoral immune function assays)

78

STUDY TYPE:

• Interventional

STUDY DESIGN:

• Allocation: Non-randomized

• Control: Uncontrolled

• Endpoint Classification: Safety and Efficacy study

• Primary Purpose: Treatment

• Masking: Open Label

ENROLLMENT : 500-1000

ELIGIBILITY:

• Ages Eligible for Study: 18 Years and older

• Genders Eligible for Study: Both

• Accepts Healthy Volunteers: No

INCLUSION CRITERIA:

• Clinical or laboratory evidence of EBOV infection

• Patients with suspected EBOV infection

• Patients with “pure disease state” preferred.

• Female patients of childbearing potential must not be pregnant at the start of the study

• All patients of reproductive potential must be willing to use contraception or abstain from

intercourse.

• Adequate renal function

• Adequate hematologic function

79

EXCLUSION CRITERIA:

• History of autoimmune disorder and immunodeficiency

• Potential effect on lactating and pregnant woman is not known, so care should be taken while

recruiting them.

• Use of any drugs or vaccines within a month of enrollment or at least two weeks before enrollment.

• Patients with chronic kidney and liver problems.

• Known HIV or active hepatitis B virus (HBV) infection

• Antiviral therapy within 3 months before study

• Investigational therapy within a month before study

• Major surgery within a month before study

FACTORS THAT WILL BE MONITORED:

• Routine Blood, Stool and Urine analysis

• Blood Pressure

• ECG if required

• Body Temperature

• Body weight

ANTICIPATED SIDE EFFECTS

Potential side effects of the drug can be:

• Possible kidney problems

• Possible liver toxicity

Monitoring kidney problems: [101]

Blood and urine tests such as the following can be used to evaluate acute renal failure:

• Serum creatinine – An increase in blood creatinine levels is indicative of acute renal failure.

• Blood urea nitrogen (BUN) – BUN level increases if the kidney is not able to remove urea from the

blood normally.

• Blood electrolyte tests, such as calcium, phosphate, potassium and sodium.

• Complete blood cell count.

80

Monitoring liver toxicity: [102]

• Liver function tests have to be performed prior to starting the trial.

• The following are the cut off levels for liver dysfunction when the treatment for the particular patient

has to be stopped or reduced:

� Alanine transaminase level rising to three times or above the upper limit of normal.

� Bilirubin level rising to two times or above the upper limit of normal.

� Alkaline phosphatase

DOSE:

The dosage for Phase III clinical trials will be 50 mg/kg given once. Taking the average human weight to

be 60 kg, the amount of drug to be administered is taken as 3 grams delivered via a single injection.

ROUTE OF ADMINISTRATION:

Administration of the drug is via intravenous injection. Bioavailability will therefore be near 100%.

DURATION OF THE STUDY:

The treatment will last for a maximum of two weeks. The patients will be followed for 3 months after

treatment for any potential relapse or side effects.

DESCRIPTION OF THE STUDY:

Informed consent will be obtained from the patient prior to starting the trial. Body Temperature, Blood

pressure and other initial parameters will be recorded in the patient. The patients will be dosed with 3g of

NCT1087 once daily via intravenous injection for 5 days. Blood analysis will be done for the first set of 50

patients daily to assess the decrease in viral load and subsequent increase in IRF3 expression as well as

increase in IFN concentrations. After treatment they will be followed for 3 months to keep track of any

possible side effects or relapse.

81

RESULTS FOR PHASE III STUDIES

DAYS NO. OF

PATIENTS

NCT1087

CONC.

(grams)

EFFICACY

BM-VIRAL

TITER

(pfu/ml)

SURROG

ATE BM-

LYMPHO

CYTES

CONC. (%

total

cells)

TARGET

BM- IRF3

CONC.

(%

activation

)

MECHANISM

BM- IFN- γ

CONC.

(pg/

ml)

0 750 3 6.2 x 109 4% 0.2 <100

1 750 3 5.6 x 106 9% 13 400

2 750 3 4.9 x 105 16% 45 850

3 750 3 3.8 x 103 20% 55 1100

4 750 3 1.2 x 102 26% 68 1400

5 750 3 0.3 x 102 33% 82 2300

� Lymphocyte concentration is estimated by conducting the WBC count and then subsequently analyzing the

lymphocyte composition in them. Normal Lymphocyte concentration is 25-33% [106].

� IRF3 concentration is analyzed by estimating IRF3 mRNA concentration by means of Real Time PCR.

� IFN- γ concentration is estimated by means of commercially available ELISA kits.

From the studies and results above, the drug – NCT1087 has an efficient pharmacokinetic profile and

elicits the desired clinical effect within 5 days when the patients are dosed with a drug concentration of 50

mg/kg or 3g. This has been proven to be true when studies were conducted in a larger population.

Proof that the drug elicits its desired clinical effect by inhibiting VP35 and allowing the activation of IRF3

gene and subsequent initiation of IFN Type I pathway is shown by the increase in IRF3 activation and

IFN- γ concentration in the blood of the patients as the treatment progresses. Again the mechanism of

action of NCT1087has been proven in a larger population.

Table 14: Efficacy of NCT1078 (BMs reading was determined in a subset of 100 patients)

82

Table 15: Potential toxicity study on a larger popu lation for the dose intended for marketing:

NCT1087

CONC.

NO. OF

SUBJETCS

DAYS RENAL

TOXICITY

BM-Serum

Creatinine

level (mg/dL)

HEPATIC

TOXICITY

BM- Alkaline

Phoshatase

Level (IU/L)

NO. OF

ADVERSE

EVENTS

3 grams 750 1 1.1 111 20

3 grams 750

2 1.2 120 35

3 grams

750 3 1.1 130 30

3 grams 750 4 1.1 128 30

3 grams 750 5 1.2 131 35

Normal Serum Creatinine level in the blood is 0.8 to 1.4 mg/dL. [103]

Normal Alkaline Phosphatase level is 20 to 140 IU/L. [104]

From the studies, 3g dose of NCT1087 is reasonably well tolerated and efficacious.

83

CONCLUSION

Therefore it can be concluded that Phase III trials with NCT1087 has been successful in showing that:

� A concentration of 50 mg/kg or 3 grams is sufficient in eliciting the desired clinical effect.

� The desired clinical effect is elicited within 5 days with a significant decrease in viral load.

� The recommended dose concentration has been found to have safe pharmacokinetic profile as well

as a safe ADME profile.

� The recommended dose concentration has a good bioavailability for 24 hours, therefore dosage is

only required every 24 hours.

All these conclusions have been validated in a larger patient population giving statistical significance to

the results.

ROUTE OF DELIVERY, ADMINISTRATION REGIMEN & DOSE CO NC:

Route of administration is by intravenous injection using a Hypodermic needle through the median cubital

vein. The reason behind this being that, EHF is a very fatal disease progressing at a very high rate,

therefore immediate exposure to an anti-Ebola drug with a very good bioavailability profile is required.

Administraion regimen is 3 grams of the drug given once daily.

84

NDA APPLICATION

“The NDA application is the vehicle through which drug sponsors formally propose that the FDA approve

a new pharmaceutical for sale and marketing in the U.S. The data gathered during the animal studies and

human clinical trials of an Investigational New Drug (IND) become part of the NDA.” [114]

The goals of the NDA are to provide enough information to permit FDA reviewer to reach the following key

decisions:[114]

• Whether the drug is safe and effective in its proposed use

• Whether the benefits of the drug outweigh the risks.

• Whether the drug’s proposed labeling (package insert) is appropriate

• what it should contain.

• Whether the methods used in manufacturing the drug and the controls used to maintain the

drug's quality are adequate to preserve the drug's identity, strength, quality, and purity.

Figure 46:

85

LIFE CYCLE MANAGEMENT OF NCT1087 or EBONAVIR ®

Three strategies for managing the life cycle of NCT1087 to be marketed as Ebonavir® is as follows:

• Legal/sales and marketing defense

• Active chemical modification

• Reformulation to improve efficacy

There are several technologies that can aid in the last two such as:

• Chiral switching

• Targeting the compound to specific cells

• Pharmacokinetic modification through development of sustained-release technology (reducing the

number of dosages)

• Changing the route of administration

• Improving bioavailability by increasing solubility or permeability

• Increasing purity of API

Reformulation is an advantageous avenue as they will require only a shorter approval route that will

include new preclinical, but less clinical, trial data and reduces the development process to a period as

short as 2-5 years.

With Ebonavir® I would probably try to make the drug orally bioavailable. Plus more research would be

done to design and synthesize more stable polymorphs.

In terms of patents, the following will be filed in the following order to extend exclusivity:

• Composition of matter patent

• Polymorph patent

• Method of Manufacture patent

• Improved Formulation patent

• Additional therapeutic use patent will be filed for other types of EBOV infections such as REBOV,

SEBOV and CIEBOV Infections after suitable preclinical and clinical trials. This will be spread out

over the twenty year exclusivity period of the first Composition of matter patent.

In addition, since EBONAVIR® is an orphan drug, extra 7 years of exclusivity will be granted by FDA

86

2-3 years

3-4 years

Figure 47

87

2-3 years

3-7 years

10-18 years

88

[108][110][111][112]

Figure 48

89

[107][108][109]

Figure 49

90

POSSIBLE EMERGENCE OF RESISTANCE:

The Ebola virus has a single negative stranded RNA for a genome. Therefore there are three ways in

which genetic changes can occur: [115]

1. nucleotide substitutions resulting from purportedly high error rates during RNA synthesis

2. reassortment of the RNA segments of multipartite genomic viruses

3. RNA-RNA recombination between non-segmented RNAs

In case of EBOV only the first and third is possible since it only has one negative stranded RNA strand. In

order to gain an insight into the genetic stability of EBOV, the CDC researchers compared the Ebola strain

captured from the 1976 outbreak in Kikwit, Zaire, to one taken from the 1995 outbreak in Yambuku, Zaire.

Even though, the two epidemics occurred more than 1,000 kilometers apart, and the virus had 18 years to

mutate, the genetic sequences of the two isolates of Ebola-Zaire are virtually identical. In addition,

Bernard LeGuenno at the Pasteur Institute found that the recent 1996 outbreak of the Ebola-Zaire strain in

Gabon is also nearly identical to the 1976 isolate. Something seems to be restraining the natural tendency

of filoviruses toward genetic divergence [116].

Plus Ebonavir® targets VP35-a critical protein required for pathogenesis. Mutation of select basic

residues within the C-terminal half of VP35 inhibits its dsRNA-binding activity, impairs VP35-mediated

IFN antagonism, and attenuates EBOV growth in vitro and in vivo [31]. A functional VP35 is required for

efficient viral replication and pathogenesis; knockdown of VP35 leads to reduced viral amplification and

reduced lethality in infected mice [31]. Data suggest that the dsRNA binding activity mediated by the C

terminus of VP35 is critical for viral suppression of innate immunity and for virulence. There are 2 basic

patches that are highly conserved among members of the Filoviridae family (identical among EBOV

isolates). Therefore the virus cannot induce a mutation in VP35 active site because if it does it will also

affect its ability to bind to the anti-viral ds RNA that the host cell generates on being infected. Therefore

by targeting VP35, it offers a possibility of circumventing the problem of resistance.

91

REFERENCE:

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