PYRAMIDINE IMIDAZOLE

AS POTENT DRUGS FOR

TREATING GUILLAIN BARRE

SYNDROME - GB004

Chythra Rani Chandregowda - Under the Guidance of Prof.

Evegeny Vulfson

1

TABLE OF CONTENTS:

Sl No. Content Page No.

1 Introduction 4

2 Drug, Medications and Treatments for GB Syndrome 6

3 Drug Target/ Medical Need 7

4 Lead Molecule 10

5 Structure Activity Relations 12

6 In-Vitro Pharmacological Profiling 16

7 On Target Toxicity 19

8 Biomarkers 23

9 Pre-Clinical Studies 26

10 Investigational New Drug Application 30

11 Phase I Clinical Trials 32

12 Phase II Clinical Trials 34

13 Phase III Clinical Trials 37

14 New Drug Application 39

15 References 43

2

List of Tables

TABLE CONTENT PAGE

NUMBER

1 List of Immunoglobulins for treatment of GB Syndrome 7

2 Properties of Lead Molecule 10

3 Different NCE’s with structure 12

4 Structure Activity Relations table 13

5 NCE-Enzyme Interactions 15

6 In-vitro studies results for different NCE’s 19

7 Summary of all assay values of NCE’s 21

8 Efficacy Studies on animal models 25

9 DRF studies on rat 26-27

10 Toxicology Data for rat 28

11 Toxicology Data for dogs 28

12 Phase I results 33

13 Phase II results 36

14 Phase III results 39

3

A BRIEF LOOK INSIDE

Guillain Barre Syndrome is a reversible peripheral poly neuropathy, which is an

autoimmune disorder caused due to infection by the C.jejuni bacteria. Gangliosides on

the myelin sheath of the neurons are molecular mimics of the cell wall LPS layer

components of C.jejuni bacteria and this triggers the auto immune attack. The cascade

of immune reaction is triggered by p-38 MAP Kinase which is responsible for the

production of cytokines which in turn is responsible for T cell differentiation and

immune attack on myelin of neurons.

GB004 are pyrimidine imidazole compounds, these bind irreversibly to the ATP binding

site ofp-38 MAP Kinase blocking its activity of phosphorylation which blocks

differentiation of the T cells hence the auto immune reaction against neurons is

aborted and the damage caused to myelin is reduced.

4

INTRODUCTION:

uillaine-Barre syndrome also called as Landry’s paralysis is an auto-immune post infection

polyneuropathy [1] which is a reversible nerve damage disorder. This disorder was first identified by Jean

Baptiste Octave Landry (1859) [2]. An infection by a bacteria (Campylobacter jejuni) or virus is a trigger

factor, due to which the body’s immune system gets provoked. It is mainly an auto immune disorder where the

body’s immune system starts acting against the peripheral nervous system. There are various forms of GB

syndrome, the most common of which is the damaged caused to the peripheral nervous system also called as the

Miller Fischer variant [3]. In this type of GB syndrome the patient’s body starts producing antibodies against the

nerve gangliosides as a result of which there is neuromuscular block which leads to partial or complete paralysis

of the body.

There are two main subtypes of this disorder:

- Acute inflammatory demyelinating polyneuropathy (AIDP).

- Acute motor axonal neuropathy (AMAN) [4].

Of these the second type is more prone to be preceded by an infection in any part of the body. Sometimes this

disorder is seen in association with AIDS, food poisoning also.

CAUSES:

Occurs after the body is exposed to an infection like gastroenteritis or respiratory tract infection [1]. The link

between occurrences of GB syndrome is very much higher when it is a Campylobacter jejuni diarrhea. Research

shows that in AMAN type occurrence the antibodies are targeted against gangliosides on the myelin sheath of the

neurons, this in turn triggers macrophages to attack the neuron at the node of Ranvier [5]. This auto immune attack

is triggered after an infection by C.Jejuni, reason being the lipopolysaccharide structure of the cell membrane in

the microorganism has similar structure of gangliosides like epitopes on their surface. As a result of this even

after the infection is cured the anti-ganglioside antibodies will target the gangliosides present on the neurons in

humans. This auto immune response is mainly triggered against the following ganglioside complexes being

GM1b, GM1 and GD1a and in the AMAN type it is GQ1b which is the major target. In some other forms of GBS

T cell mediated response is observed against myelin proteins like P2, P0, PMP2 [6] etc. Ultimately this damage

caused to the myelin sheath proteins or to the gangliosides leads to damaged nerves and hence person suffers

from partial or complete paralysis.

Fig 1: Illustration of how damaged myelin can slow down the message transfer which leads to weakness. [1].

G

5

SYMPTOMS:

The symptoms that appear are due to damage caused to the 3, 4 & 6 cranial nerves (gangliosides damage). The

major symptoms are:

1) Weakness, numbness and tingling sensation in limbs with pain.

2) Weakness in the facial muscles leads to difficulty in swallowing (Dysphagia) [7].

3) Opthalmoplegia – Loss of activity in ocular muscles.

4) Weakness of heart muscles lead to heart related problems like tachycardia, bradycardia etc.

5) 25% of the people suffer from respiratory failure hence they need to be intubated and mechanical

ventilation needs to be provided.

6) Organs start to fail in the later stages.

DIAGNOSIS:

There are various diagnostic methods available for detecting GB syndrome. Few of them are as follows:

1) Cerebrospinal fluid analysis: The cerebrospinal fluid is extracted using a needle from the vertebral

column. Elevated levels of protein and reduction in WBC’s is a positive indication [1].

2) Nerve conduction studies

3) Testing for the presence of antiganglioside antibodies in the blood.

4) MRI scanning: for detection of abnormal sizes of nerve roots.

TREATMENTS:

There are majorly two types of treatments (Immunotherapy) which are either provided separately or as a

combination. Research shows that with combined effect of the treatment patients tend to recover faster.

1) Immunotherapy: This treatment mainly aims at reducing the immune system attack on the nerve cells [1].

The two ways of immunotherapy are

Plasmapheresis, in which blood from the patient is drained out and antibodies are filtered out, the

anti-gangliosides antibodies are now administered back into the patient [8].

Intravenous administration of Immunoglobulins to diminish the effect of the anti-ganglioside

antibodies on the nerve.

During the entire course of treatment the patient is kept in intensive care unit with mechanical ventilation and

various other life supporting devices. After a long term course of treatment the patient needs to undergo

physiotherapy in order to regain full muscular activity [9].

EPIDEMIOLOGY:

Average annual findings of this disorder includes 0.4 to 1.7 per 100,000 in the world population. The

occurrence was found to be higher in females than in males and also more in older people as compared to

younger population [10]. Also the percentage rate of occurrence of different subtypes of the GB syndrome varies

in different countries for example in western countries ADIP subtype is seen in 60 to 80 % of the people

whereas AMAN subtype is 6 to 7 % only. On the contrary AMAN sub type is seen 30 to 65 % in Asian

countries [1].

6

DRUGS, MEDICATION AND TREATMENT:

As mentioned earlier Guillain-Barre syndrome is an autoimmune disorder triggered by an infection [1]. The

treatment to this disorder is mainly Immuno-modulatory therapy which includes Plasmapheresis and

administration of immunoglobulins (against anti-ganglioside antibodies) to reduce the effect of attack on the

nerves [11].

Plasmapheresis: Usually this course of treatment is considered when it is still an early course of the disease [12].

This is also called as blood plasma exchange. In this procedure patient’s blood Is withdrawn and is filtered to

separate the different components of the blood (basically different blood cells are separated and isolated from the

original blood plasma) later they are suspended in a synthetic plasma and administered back into the patient. This

way the anti-ganglioside antibodies is eliminated from the patient’s body. This procedure only works for initial

stages of the disorders and has its own disadvantages.

Immunoglobulins: Immunoglobulins are obtained by purifying plasma collected from many donors, these plasma

contain antibodies against the anti-ganglioside antibodies which neutralizes the effect. The different

Immunoglobulins drugs used are listed below in the table:

Sl. No.

DRUG MANUFACTURER ROUTE EFFICACY SIDE EFFECTS DOSAGE

1 Carimune NF CSL IV Reacts efficiently with any complexes formed by antibodies without affecting IgG’s [13].

Thrombosis, Renal dysfunction or acute renal failure.

400-800 mg/kg every 3-4 weeks.

2 Gammagard Liquid

Baxter IV or Subcutaneous

Passive transfer of antibodies (antibodies usually obtained from healthy humans).

Headache, Fatigue, Thrombosis, Renal dysfunction or acute renal failure [14].

300-600 mg/kg every 3-4 weeks.

3 Bivigam Biotest Pharmaceuticals Corporation.

IV Treats muscle weakness in auto immune disorders by transfer of antibodies.

Acute renal dysfunction and acute renal failure.

300-800 mg/kg every 3-4 weeks.

4 Octagem Medispan Limited

IV Effectively transfers the antibodies and reduces weakness of limbs.

Headache, Minor chest pain, dizziness, also harmful to the kidneys.

300-600 mg/kg for every 3-4 weeks.

Table 1: List of Immunoglobulin based drugs used to treat Guillain-Barre syndrome.

7

MEDICAL NEED FOR A NEW DRUG:

It is explained before that GB syndrome has two treatments which are provided to the patients either separately

or in a combination for a long period of time after which the patient needs to undergo physiotherapy for complete

recovery. It is noticed that the only medication provided is either in the form of Immunoglobulins or treatments

like Plasmapheresis. Instead why not discover a drug that can act on the anti-ganglioside antibodies or any factors

involved in the pathology of the syndrome and the medical need for this is as follows:

1) When a patient is diagnosed with the disorder in the initial stages, when the concentration of the anti-

ganglioside antibodies is still not alarmingly high, it is better to treat it by using a drug rather than to

subject the patient to a tedious and complicated process like Plasmapheresis which has its own

disadvantages like formation of blood clots etc.

2) All immunoglobulins provided are through Intra venous administration, instead if a drug is discovered

against the anti-ganglioside antibodies then this can be administered in the form of a pill.

This way the treatment process, the route of administration can be simplified and time required for the effective

eradication of the anti-ganglioside antibodies can be reduced.

DRUG TARGET:

My target for treating GBS is p-38 MAP Kinase, which are the enzymes involved in production of cytokines

involved in proliferation of T helper cells during immune response [15].

IMPORTANCE OF IFN- γ IN GBS

The gangliosides on myelin sheath in the neurons are molecular mimics of certain structures on the

lipopolysaccharide layer of the C.jejuni bacteria. During the duration of the infection human body produces

antibodies against the bacteria whose memory is stored by the body’s immune system. Since the gangliosides

present on the myelin sheath share similar structures to the LPS of the bacteria an auto immune response is

triggered against them. IFN- γ and IL-12 are cytokines produced by the Th1 cells during an immune response, of

these IFN- γ is majorly involved in the induction of cell mediated autoimmune response [16]. Once the APC cells

interact with the CD4+ T cells the T cells gets activated and become naïve T helper cells [17], due to further

interactions between the CD28 on the CD4+ T cells and proteins on APC’s Th cells proliferate into Th0 cells.

The Th0 cells produce IFN- γ, IL-2 and IL-4 (cytokines) which promotes conversion of Th0 cells to Th1 cells

which further produces cytokines to produce Th2 cells. The Th2 cells further differentiate into effector Th cells,

memory Th cells and regulatory Th cells. Effector Th cells will produce more of the IFN- γ cytokines to increase

the BNB permeability, and to activate macrophages to attack on the compliment formed by the Anti-ganglioside

antibody and the gangliosides on the myelin sheath resulting in myelin sheath damage. Figure 2 gives a brief

picture of the immune mechanism in GBS. In this overall process we notice that IFN- γ is mainly involved in the

following events during the immune response:

1) Induces the differentiation of Th0 cells to Th1 leading to production of more cytokines.

2) Activates and enhances the expression of MHC complex II and increases the antigen binding capacity of

the macrophages [18] (Activating macrophages).

3) Increasing the permeability of BNB by allowing more macrophages to migrate towards the myelin sheath.

8

Mainly in the immune response of the human body against GBS, IFN- γ has immuno stimulatory and immuno

modulatory responses [19].

ROLE OF p-38 MAP KINASE

The CD4+ Th cells (Naïve) recognize specific MHC peptide complexes on APC through T cell receptor

complexes [15]. This is called the TcR mediated signal for T cell differentiation. Apart from this there is another

signal that plays an important role in the differentiation of naïve Th cells, being the co stimulatory signal which

is produced by the APC. A combination of these two signals induces the clonal expansion of T cells to produce

armed Th cells which triggers the immune response. This is a brief description of the T cell differentiation process.

Although there is selective differentiation of precursor CD4+ T cells into Th1 and Th2 cells. This process occurs

due to certain factors like:

1. Cytokine environment

2. Concentration of antigen – the costimulatory signal.

Off the two the first factor plays a major role and cytokines like IFN- γ which are produced by the NK and the

Th1 cells require p-38 MAP Kinase for its production. The p-38 MAP kinase is involved in regulation of

expression of specific cytokine genes. So it is involved in altering the production of these cytokines.

Fig2: Role of p-38 MAP Kinase in the differentiation of Th cells [15].

9

Fig3: The immune response against the gangliosides and the roles and functions of Th cells in GBS (T cell

mediated auto immune response) [19].

HYPOTHESIS:

By inhibiting p-38 MAP kinase the following therapeutic effects can be obtained:

1) Prevent differentiation of Th0 cells to Th1 cells, which is a major step in the T cell mediated auto immune

response.

2) Preventing the activation of macrophages which attack on the Anti-ganglioside antibody and the

compliment formed on the myelin sheath.

3) Can also prevent the increase in the permeability if the BNB.

Technically by preventing the p-38 MAP Kinase we prevent the production of cytokines like IFN- γ and hence

prevent the differentiation of T cells which mediate the auto-immune response.

LINK TO THE MEDICAL NEED:

It was mentioned in the medical need that instead of only immunotherapy for the patient which involves prolonged

tedious procedures, a drug against the anti-ganglioside antibodies or any factors involved in the pathology of GBS

can be discovered. From the available resources it is evident that IFN- γ and other cytokines plays a major role in

triggering the immune response in GBS, hence drugs targeting the factors necessary for the production of these

cytokines might give positive results and serve the purpose of avoiding immune suppression as a treatment for

GBS. Research are still going on discovering drugs against p-38 MAP Kinase for treating auto immune disorders

and there are not much drugs that are released into the market [17], hence competition in the market is also less.

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LEAD MOLECULE

The lead molecule of my choice for inhibiting the cytokine production is Benzylsulfanyl Imidazole [20]. Imidazole

pyrimidines are new class of chemical molecules that exhibit cytokine release inhibitory role. Some of the

important properties of the lead molecule is listed below.

Sl no. Property Value

1 Structure

2 Chemical name 2-benzylsulfanyl-4,5-dihydro-1H-imidazole

3 Chemical formula C10H12N2S

3 Molecular Weight 192.28068 g/mol

4 Hydrogen Bond Donor Count 1

5 Hydrogen Bond Acceptor Count 2

6 Log P 1.7

7 N+O count 2

Table 2: Properties of the drug molecule. [21]

NCE (New Chemical Entity):

The lead molecule being 2-benzylsulfanyl-4,5-dihydro-1H-imidazole has a core imidazole ring [23] through which

hydrogen bond is formed with the lysine amino acid residue of one of the active site pockets of the JNK (p-38

MAP kinase) enzyme and the benzene side chain makes its way into the phosphate binding pocket of the enzyme.

Although these interaction do bring about certain amount of the changes required in the enzyme (since the

phosphate binding site itself is blocked) we still need to optimize the lead molecule to make these bonds stronger.

The lead molecule structure is as below:

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Fig4: structure of the lead molecule indicating the positions for addition of functional groups for optimization.

The different NCE’s designed are tabulated below:

COMPOUND R1 R2 R3 STRUCTURE [24].

NCE(1) F-Phe 4-Pyr - Addition of F-Phe to fit the hydrophobic pocket and 4-Pyr

to interact with methionine.

NCE(2) F-Phe 4-Pyr S-CH3 S-CH3 is added to extend the link between the glycine

amino acid and the lead.

12

Table3: The different NCE’s and their structures.

SAR (Structure Activity Relationship):

COMPOUND LogP [24]. MW (g/mol) Assay data, IC50 nM

Lead 1.99 192.28 11

NCE(1) 4.028 363.46 9

NCE(2) 4.430 409 5.5

NCE(3) 4.692 437.56 40 nM

NCE(4) 3.873 428.55 10 nM

Table4: Assay data of the lead and the different NCE’s.

NCE(3) F-Phe 4-Pyr S-O-

CH3

O is bonded to S-CH3 because there is a stronger bond

between O and H of the glycine.

NCE(4) F-Phe Imidazole

ring

S-O-

CH3

4-phe is replaced by an imidazole ring so that there are

two bonds formed in the methionine binding pocket which

is stronger.

13

The table mainly indicates the assay data obtained for different NCE’s. The assay chosen for determining the

inhibition capacity of the NCE’s was the p-38 MAP kinase assay [25]. According to which the assay data was

interpreted based on the OD obtained after ELISA. The OD was directly proportional to the cytokine produced

and also the OD is used to calculate the IC50 value, if the p-38 MAP kinase (JNK) was to be inhibited then there

is less or no color developed which in turn means no or less cytokine produced and the OD will be less as a result

the IC50 also reduces. Hence from the tabular column of assay data it is evident that OD produced by NCE (4) is

way less than those produced by the rest of the compounds hence the IC50 value of NCE (4) is lesser than the

rest. Taking this into consideration we can arrange the kinase inhibition activity of these compounds in the

increasing order as below:

Lead < NCE (1) < NCE (2) < NCE (3) < NCE (4).

ACTIVE SITE POCKETS:

Fig5: Active site pockets in the enzyme [23].

There are four important binding site pockets available in the kinase enzyme and they are:

1) Lysine pocket: The drug compounds will react with the hydrogen of the amide group of Lys 55.

2) Phosphate binding pocket: This is the main active site where the phosphate group or ATP binds when the

kinase is involved in transfer of phosphate from ATP to the compound involved. This site has a glycine

amino acid residue. The lead molecule is to react with the H on the alpha carbon of glycine.

3) The hydrophobic pocket: mainly contains a Threonine residue.

4) Another additional binding pocket available which has Methionine residue which provides additional

interaction.

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INTERACTION OF THE LEAD AND NCE’s WITH THE ACTIVE SITE POCKETS:

Compo

und

SAR and Active site interaction Explanation

Lead

Here we can see two interactions:

1) The N of the imidazole ring

reacting with the H of the

amide group in the Lys 55

residue.

2) The benzene ring of the lead

molecule fits into the

phosphate binding site of the

enzyme.

Although this interaction serves the

main purpose of blocking the

phosphate binding pocket we can

notice that there are still two pockets

left unfilled and also the interaction of

the benzene and the phosphate binding

pocket is not strong enough.

NCE(1)

In addition to the interaction between

the imidazole and the Lys 55 pocket

and the Benzene – phosphate binding

pocket, in this we notice that the 4-

Pyridine group added interacts and

fills in the Met 109 pocket and the F-

Phenyl ring added fills the

hydrophobic pocket. This additional

interaction stabilizes the remaining

two active site pockets as well hence

the increase in affinity compared to the

lead molecule.

15

NCE(2)

The S-CH3 group added to the

benzene of the imidazole lead atom

provides an additional interaction of

the H of glycine and the CH3 of the

extension. Hydrogen bond interaction

mainly causes the increased affinity

since now the pocket is not only

occupied but there is some chemical

interaction.

NCE(3)

In this compound the S-CH3 is

replaced by S-O-CH3. This extension

of the bond provides a better leverage

to the binding site by occupying it

completely and also the O of the S-O-

CH3 interacts with the hydrogen of the

H of glycine residue which is a much

stronger linkage that H-H. Hence the

increased affinity compared to the

previous compound.

16

NCE(4)

This is by far the most strongly

binding compound and the reasons for

this is:

The 4-Pyridine ring had only one

interaction with the Met pocket (N-H).

By replacing it with another imidazole

ring we achieve two strong

interactions as depicted in the picture,

one being the N-H and the other being

O-H.

This along with the rest of the

interactions makes NCE (4) the most

strongly binding compound of all the

four NCE compounds.

Table 5: The interaction between NCE compounds and the Active site of the enzyme kinase

From the above table it is clearly evident that our NCE (4) has the most binding affinity and based on the

interactions and by the assay binding information from the table 4 NCE(4) has the least OD which indicates that

it has the most kinase inhibitory activity compared to the other NCE compounds.

Note: All chemical structures are drawn using molinspiration website and all the interaction was manually done using MS Office.

IN VITRO PHARMACOLOGICAL PROFILING:

In order to study the efficiency with which these molecules bind to the target and bring about the required result,

various assays were conducted in-vitro. The different assays conducted and their description is as follows:

1) CELL BASED BINDING ASSAY [26]:

Mononuclear cells are used for this assay. Dilutions of the inhibitor molecules are prepared. The

mononuclear cell suspensions are mixed with the inhibitor dilutions and incubated for 15 minutes under

room temperature. Lipopolysaccharide layer obtained from bacteria is added to the incubated samples and

incubated for 4 hours. This stimulates the mononuclear cells to produce cytokines as an immune response

against the bacterial LPS.

Expected result:

The p-38 MAP Kinase enzymes are involved in production of cytokines to trigger immune response

against the bacterial LPS layer. Hence if the inhibitor molecule have the capacity to attack the target

And inhibit the enzyme then there will be less or no production of the cytokines. This can imply that more

the concentration of the cytokines lesser is the binding affinity of the inhibitor molecule. The concentration

of the cytokine produced is then converted into IC50 values for calculating the best binding inhibitor.

17

2) KINASE ASSAY [22]:

As discussed earlier in this assay the direct binding ability of the inhibitor molecule to the enzyme can be

studied. In this assay the inhibitor dilutions are directly incubated with enzyme solution and the activity

is measured by ELISA by using ATF-2 as substrate. The color developed is measured as OD at 405 nm.

Mechanism of assay:

ATF-2 is a substrate of p38 MAP Kinase enzymes. Kinase phosphorylates ATF-2 to phospho-ATF-2.

Hence when ELISA is conducted, anti-phospho-ATP-2 antibody (primary antibody) binds to Phospho

ATP and in turn alkaline phosphatase conjugated GAR antibody (secondary antibody) binds to the

previous complex and when the substrate (4-Nitrophenolphosphate) is added there is color development

which can be measured as OD (proportional to the concentration of the cytokines produced). When the p-

38 MAP Kinase is inhibited then there is no color development since ATF-2 is not phosphorylated.

Fig 6: pictorial representation of assay

Expected result:

If the p-38 MAP kinase is blocked by the inhibitor then there is no or less color developed and hence OD

appears to be less. The OD obtained is then converted to IC50 values using certain calculations.

3) CYP INHIBITION ASSAY [27].

CYP3A4 plays an important role in drug metabolism [28], hence in this assay we concentrate on the

inhibition capacity of CYP3A4 by the inhibitor molecules. CYP3A4 enzymes are members of cytochrome

P450 family of oxidizing enzymes. These enzymes reside in the liver and are involved in metabolizing

the drug molecules. In this assay we use fluorescent substrates of CYP3A4, and these substrates have

blocked fluorescent dyes which means that they yield fluorescence signals only if the substrates are

cleaved else no. The inhibitor molecule dilutions are prepared and incubated with the enzyme (CYP3A4)

solution. To this known concentration of the fluorescent substrates are added and the amount of the

fluorescent signal obtained is recorded.

Expected Result:

If the inhibitor molecule inhibits the CYP3A4 activity then the enzyme will fail to cleave the fluorescent

substrate hence no signal will be generated, on the contrary if the inhibitor molecule does not block the

CYP3A4 enzyme then the enzyme is capable of cleaving the substrate leading to signal generation. Hence

18

the intensity of the signal generated is indirectly proportional to the CYP inhibition capacity of the

inhibitor molecule.

4) hERG INHIBITION ASSAY [29]:

For this assay human embryonic kidney cells (HEK 293) are used. These cells are then transfected with

hERG cDNA to produce hERG recombinant gene. These hERG recombinants are now expressed in HEK

293 cells which are very identical to the human cardiac cell Ikr channels. A patch clamp amplifier is used

to record the membrane current generated. This is the main parameter used in this assay to determine the

inhibition capacity. A hERG containing bath solution is prepared and this is used to generate and record

the steady state current.

hERG expressing cells are placed in a glass bath chamber into which the bath solution is supplied

continuously and in order to record the steady state current simultaneously repetitive test pulses of 0.05Hz

is applied to the set up. Now once the steady state current is recorded the bath solution is replaced by

diluted concentration of the inhibitor test molecule one after the other and the membrane current generated

is recorded.

Expected result:

Through the amount of membrane potential generated the % inhibition can be calculated. More the

inhibition lesser the membrane potential. The % inhibition value can be used to calculate the IC50 by the

following equation:

𝐼𝐶50 =100 − %𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛

% 𝐼𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 𝑥 𝐶𝑜𝑛𝑐.

Conc. = is the concentration of the inhibitor used in the solution.

MW

(g/mol)

Log P Cell

assay

[IC50, µM]

Kinase assay

[IC50, µM]

CYP Inhibition assay

[IC50, µM]

hERG assay

[IC50, µM]

LEAD 192.28 1.99 11 18 2 2

NCE(1) 363.46 4.028 9 15 6 8

NCE(2) 409 4.430 5 8 9 10

NCE(3) 437.56 4.692 40 nM 35 nM 15 15

NCE(4) 428.55 3.873 10 nM 10 nM 20 22

Table 6: in- vitro studies results for different NCE’s

19

Chart1: Graphical representation of the assay data.

RESULT:

From the various assays conducted and the results obtained from the same it is evident that NCE (4) molecule is

selected as a potent inhibitor of the other compounds. The reasons being:

1) NCE (4) fits the Lipinski’s rule of four since its log P is lesser than 4, Molecular weight is very slightly

above 400 etc.

2) Its IC50 value in both the cell assay and the kinase assay is lesser than that of the remaining molecules

indicating NCE (4) to be a potent inhibitor having the best binding capacity compared to the other

molecules.

3) NCE (4) has maximum percentage inhibition of both CYP and hERG. This conclusion is drawn based on

the IC50 value obtained for the same.

ON TARGET TOXICITY

The enzyme p-38 MAP kinase is involved in many biological functions in the human body. Based on them the

various side effects (on target toxicity) can be explained as follows:

1) Immune-suppression:

The main function associated with this enzyme is initiation of cytokine production during an immune

response which leads to T cell differentiation and the further steps. If our inhibitor molecule suppresses

the activity of the enzyme, the resulting adverse effect would be Immune-suppression. Hence the patient

will be prone to minor infections like cold, cough etc. But still since these patients are usually kept in ICU

during the course of treatment, even though the person’s immune response is delayed or suppressed

chances of catching an infection is low.

0

50

100

150

200

250

300

Lead NCE(1) NCE(2) NCE(3) NCE(4)

hERG assay(%inhibition)

CYP assay (%inhibition)

Target assay (nM)

Cell assay (nM)

20

2) Hyperglycemia:

P-38 MAPK is known to inhibit Glucocorticoid Receptors (GR) and reduce its availability for the

glucocorticoids. The enzyme phosphorylates the GR and inhibits its transcription activity [30]. For

glucocorticoids (GC) to perform its function it needs to first bind to the GR. One of GC’s main functions

are maintaining glucose metabolism i.e. enhancing gluconeogenesis. Hence by inhibiting p-38 MAPK

GR’s are made available for GC and hence there is over expression of the same. This means that the

gluconeogenesis process is enhanced and hence high glucose level can be observed in the blood, which is

termed as Hyperglycemia.

3) Hypertension:

Also GCs are released by the adrenaline glands as a response to stress, during which the enzyme prepares

the body for fight or flight. The major changes observed in the body during a stress response are

hypertension [31]. Hence by inhibiting the enzyme p-38 MAPK, GC expression is enhanced and hence

chances of the patient acquiring hypertension are high.

4) Cardiovascular effects:

As we all know, Ischemia one of the major causes for damage of myocardium. Research work explains

that during ischemic damage caused to the heart -38 MAPK pathway is stimulated which positively

prevents the damage caused to the heart [32]. Hence by inhibiting the enzyme patients might be prone to

cardiovascular disorders caused due to damage of myocardium.

OFF TARGET:

One of the isoforms of p-38 MAPK is p-38 β [33]. It is involved in few important biological functions and the

related side effects are listed below:

1) Hypoglycemia:

P-38 β is involved in metabolism i.e. inhibition of GS (Glycogen synthase) activity by inhibiting its

activity by phosphorylating it [34]. GS is involved in conversion of glucose to glycogen for storage. Hence

by inhibiting the activity p-38 MAPK, GS will not be inhibited as a result of which there will be less

concentration of glucose in the blood and hence Hypoglycemia.

2) P-38 β are involved in keratinocyte differentiation. These cells are involved in functions like formation of

new skin layer, hair growth. Based on these the probable adverse side effects can be as follows:

Delayed wound healing.

Cessation of hair growth.

ADRENERGIC ALPHA 2C RECEPTORS [35]:

I choose the above mentioned receptor for pharmacological profiling of my drug because this receptor is very

closely associated with majority of the side effects caused by the drug molecule as explained before such as

hypertension and cardiac ischemia, hyperglycemia etc. Since the drug molecule has high chances of leading to

21

these safety concerns and Aα2c receptors are highly associated with these side effects I choose this receptor from

the research paper provided [36].

ASSAY FOR PROFILING:

Fluorescence Based Binding assay:

Assay involves binding a fluorescent molecule [fluorophore] to the drug molecule to be tested. This fluorescently

conjugated drug molecule is incubated with Aα2c expressing cells. If the molecule effectively bind to the receptor

and bring about its effect of inhibiting the receptor then the fluorescence intensity is reduced. Intensity of

fluorescence is measured confocal microscope through which confocal images. The intensity of fluorescence is

used to calculate IC50 values.

Cell assay

[IC50, µM]

Kinase

assay

[IC50,

µM]

CYP Inhibition

[IC50, µM]

hERG

Inhibition

[IC50, µM]

Aα2c

Inhibition

[IC50, µM]

p-38 β

[IC50, µM]

LEAD 11 18 2 2 2 17

NCE(1) 9 15 6 8 5 15

NCE(2) 5 8 9 10 10 13

NCE(3) 40 nM 35 nM 15 15 13 12

NCE(4) 10 nM 10 nM 20 22 21 9

Table 7: Summary of all assay values for different NCE’s.

As explained earlier the IC50 values of the NCE (4) molecule has way more positive implications in all the assays

than the rest.

ANIMAL MODELS:

GBS being a poly neuropathy disorder is associated with sensory and motor impairment related symptoms. For

clinical trials there have been two models which replicate the symptoms of GBS and they are as follows:

1) RABBIT MODELS WITH T CELLS SENSITIZED AGAINST GM1 GANGLIOSIDES [37]:

GBS is an autoimmune disorder triggered by C.jejuni bacterial infection, due to the molecular mimicry

between the LPS of the bacteria and the gangliosides the myelin sheath of the nerve cells, due to which

IGg auto antibodies are developed against the gangliosides resulting in the degeneration of the myelin.

Keeping this concept in mind, in order to create diseased rabbit models, the rabbits were sensitized with

bovine brain gangliosides mixture that included GM1 [38] (since GM1 are the auto antigens in this

disorder). After the administration of the GM1 into the rabbit’s system IGg anti-GM1 antibodies were

observed in the animal. The production of the same led to the onset of symptoms in the animal like flaccid

limb, weakness with a monophasic course. On investigating for the reason behind the symptoms it was

found that the peripheral nerves had Wallerian like degeneration.

These pathological findings were very similar to those of the human AMAN type GBS.

22

Fig7: a) Symptoms of flaccid limb due to motor axonal neuropathy;

b) Electron micrograph of the nerve indicating macrophage infiltration.

2) L31/CD4-/- MICE MODEL:

B7.2 is a type of peripheral protein found in APC’s that trigger immune response due to the co stimulatory

signal generation. Transgene derived constitutive expression of co stimulator signal of B7.2 on antigen

presenting cells leads to neurological disorders [39]. It was observed that depletion of CD4-/- cells in L31

mice accelerated the symptoms associated with GBS. According to this research C.jejuni infection leads

to autoimmunity disorders in mice through co stimulatory activity of APC which triggers the expansion

of the T cells to act against the neurons. L31 mice were genetically modified to deplete the CD4+ Tcells

which led to the onset of the disease due to constitutive expression of B7.2 protein [40]. 2-6 month old mice

were used for this study. 49 L31/CD4-/- mice and 18 control mice were tested and it was observed that

there was an increased expression of B7.2 in macrophages of L31/CD4-/-. These mice in the study showed

the following symptoms during the test are motor impairment, weakness in the limb due to which the

mice were not able to walk properly, numbness in the limb observed as lack of pain, myelin loss

were observed in symptomatic L31/CD4-/- leading to axonal damage in peripheral nerves.

In conclusions these genetically modified mice shows over expression B7.2 proteins on APC as a result

of which these mice show mimics of clinical and pathological aspects on GBS in humans. Based on this

research a cascade of events was proposed to the following mouse model:

1) Transgene derived over expression of B7.2 on APC’s sensitizes APC’s in nervous system.

2) T cells which are specific to PNS recognize the B7.2 on APC as antigens and gets activated.

3) This is followed by infiltration of macrophages, expansion of the T cells due to production of

cytokines.

4) This immune reaction is directed against the neurons leading to demyelination and hence the

symptoms.

Of the two models described above I choose L31/CD4-/- mice model for efficacy testing trials due to the following

reasons:

23

1) This model has a clear explanation of the cascade of events which occurs during the auto immune reaction

which is similar to those observed in GBS.

2) It emphasizes on the fact that the symptoms are seen due to the T cell activation which is important in our

trials since my drug molecule is targeted towards the p-38 MAPK required for expansion of T cells.

3) Mice are easy to handle.

BIOMARKERS:

The major source of biomarkers associated with Guillain Barre Syndrome is the Cerebrospinal Fluid (CSF).

During GB syndrome, when the peripheral nerves get damaged during auto immune attack, this leads to swelling

of nerve root which in turn leads to disturbance of micro circulation hence plasma proteins and other factors are

released into the CSF [41]. These factors act as biomarkers for this disorder. Two of these are listed below:

1) Axonal damage marker: Axonal damage can be indicated by the neurofilaments, these are axoskeletal

proteins which are heteropolymers with four subunits. They are abbreviated as Nfs. Research shows that

during GB syndrome due to axonal damage the Nfs concentration is increased in the CSF. Hence elevated

level of Nfs in the CSF indicates damage to proximal axon parts. Study shows that patients with GBS

have > 0.73 ng/ml concentration of Nfs in the CSF [42]. Through high throughput analysis of Nfs is possible

using ELISA technique. These act as efficacy biomarkers since the subsequent decrease in the

concentration of Nfs in the CSF indicate the efficacy of the drug.

2) Chemokines: These are small cytokines or signaling protein that are secreted by the macrophages and T

cells during an immune attack [43]. CSF levels of these chemokines seems to be increased in GBS

indicating their role in the neural inflammatory process [44]. Since my drug molecule aims at targeting the

enzyme p-38 MAPK which prevents the production of cytokines, hence the decrease in the concentration

of the chemokines like IP-10 indicates that the drug is effectively inhibiting the enzyme form producing

cytokines. Hence this can be termed as a target biomarker.

CSF is extracted from the animals through the lumbar region of the spinal cord.

ENDPOINT ASSAYS:

Since the main symptom associated with GBS is weakness in the limb which leads to movement impairment the

main endpoint for this would be ability of the mice to walk which can be measured by the following assay:

3) Rotorod Assay [39]:

This assay mainly measures the motor coordination of the symptomatic mice. In this task the mouse was

made to walk on a speed ramp of speed 0 to 30 rpm, followed by a maximal speed for 240s. The time

taken by each animal is used to relate ant amount of motor impairment caused. An increase in walking

mice is suggestive of repair of the motor impairment.

4) Neuropathy impairment score [45]:

NIS is a complete clinical scoring system that is used to assess the severity of peripheral neuropathy. It

mainly quantifies the findings of various neurological examination that include testing of components like

24

sensation, reflexes and muscle weakness. The scoring system starts from 0(normal) to 88(total

impairment).

DOSE SELECTION:

Previously I had presented the assay information of all the NCE molecules. Considering the data from this we

have the following information about NCE (4)

IC50 = 10 nM

Molecular Weight= 428.55 g/mol.

Calculation:

IC50 = 10 nM

Hence IC90= 100 nM (Approximately 10x of IC50)

Assuming the bioavailability of the drug to be 20% (ignoring protein binding)

We get IC90 = 100 / 0.2 = 500 nM

Drug dose= Drug available x MW of NCE.

= (500 x 428.55) nM*g/mol

= 214275 x 10-6 mg/kg.

= 0.214 mg/kg.

Similarly dose at IC50 = 0.02 mg/kg and dose at IC10 = 0.002 mg/kg.

The dose range selected for this study was 0 – 1.0 mg/kg. The reading obtained in various tests conducted and the

biomarkers were observed and tabulated as follows:

Dose (mg/kg) No. Of Animals

used in testing.

Rotorod

Assay

(sec)

NIS

Score

IP-10

ng/ml

Nfs

ng/ml

0 10 0 60 50 1.0

0.002 10 5 50 48 0.9

0.01 10 10 40 35 0.8

0.02 10 30 30 20 0.75

0.1 10 45 20 15 0.6

0.21 10 50 0 8 0.5

0.5 10 50 0 8 0.5

1.0 10 48 10 10 0.7

Table 8: Efficacy studies on animal models

Description:

25

Since we are using diseased mouse models to check for the efficacy of the drug it is expected that the mouse with

no dose provided (control) would fail in the rotorod assay and it’s NIS due to motor impairment. As the drug is

administered in the doses tabulated due to its effect we see a progressive repair of the impairment which concludes

that the drug is efficient in stopping the nerve damage caused due to the auto immune reaction, by aborting the T

cell action against the neurons. Of all the doses the most efficient dose is 0.21 mg/kg since at this dose we find

NIS to be 0 which implies that the motor impairment is completely cured. We also observe that the animal seems

to be efficiently performing in the rotorod assay as well. Even though the mouse shows improvement in motor

functions at doses above 0.21 we observe side effects and hence I prefer to select 0.21 mg/kg as the best dose for

further studies.

The concentration of biomarkers in the CSF was tabulated for the respective doses. It is seen that the concentration

of both IP-10 and Nfs is decreasing with the increase in the dose administered indicating that the drug is working

effectively in the animal model. And it decreases to the normal level at an efficacious dose of 0.21 mg/kg.

Graphical representation of the tabulated column:

Chart2: x-axis: Dose in mg/kg; y-axis: rotorod assay values in sec.

The performance of animals at 0.21 mg/kg is at its peak and it decreases thereafter due to side effects of the drug

in the mouse.

Hence with this study I conclude that the efficacious dose is 0.21 mg/kg.

PRE-CLINICAL STUDIES:

Pre-clinical studies were conducted to understand the major toxicities of the test item and to set up margins of

doses, before we can move forward to the clinical studies on humans [46]. Through this, an initial safety dose for

the human trials is calculated by first testing the drug on animals. Before starting the actual toxicology study an

initial DRF is conducted to set the margins of dose as MTD and NOAEL.

0

10

20

30

40

50

60

0 0.2 0.4 0.6 0.8 1 1.2

Y-Values

26

DOSE RANGE FINDING

DRF studies are conducted to establish a dose response and to provide the appropriate dose margins for the

following regulatory studies. I have used rat as the animal model for DRF studies. The starting dose and the side

effects associated with it are represented in table 9

DOSE

mg/kg

No. of animals Adverse effects

0.21 10 No

0.6 10 No

2 10 No

5.61 10 <10% weight loss

17.01 10 >10% weight loss

Less food consumption

51 10 >20% weight loss, anorexia (complete absence of

food intake)

150.0 10 2 animals had to be euthanized.

Table 9: Dose Range Finding Studies

Illustration of the data:

The starting dose 0.21 mg/kg is taken from the previous efficacy studies since at this level no side effects were

observed. The subsequent doses are taken at half log intervals. From the above data we see that at dose = 1.89

mg/kg there is still no adverse effects observed, but immediately after this, at the administration of the next dose

AE’s are observed. Hence this dose is taken as the NOAEL (No Adverse Effect Level). As we increase the dose

beyond this level at the maximum dose of 150 mg/kg 2 animals needed to be euthanized. Hence the dose

immediately before it is selected as the MTD (Maximum Tolerated dose), since at this level we do find adverse

effects but it is tolerable.

27

REGULATORY TOXICOLOGY STUDIES

The two animal species used for the safety studies are:

1) Rat

2) Adult Beagle Dogs.

DRF studies provided us with the NOAEL and MTD using which we select intermediate doses between the two

for our regulatory studies for both rat and dogs.

ANIMAL MODEL 1 : RAT

DOSE

mg/kg/day

No. OF ANIMALS ADEVRSE EFECTS

0 (Control) 10 No

2 10 No

7 10 <10% weight loss

18 10 >10% weight loss

Less food consumption

50 10 >20% weight loss, anorexia (complete absence of

food intake)

Table 10: Toxicology data for rat

Table illustration:

We select the first dose based on the DRF studies to be 2 mg/kg and the MTD to be 50 mg/kg (lesser than the

actual MTD). Two intermediate doses are administered. All doses are administered to the rat once a day

throughout the month and the adverse effects are also tabulated.

Conclusion of DRF Studies:

NOAEL: 2 mg/kg

MTD: 51 mg/kg

28

ANIMAL MODEL 2 : ADULT BEAGLE DOG

DOSE

mg/kg/day

No. OF ANIMALS ADEVRSE EFECTS

0 (Control) 4 No

8 4 No

24 4 <10% weight loss

65 4 >15% weight loss

Less food consumption with vomiting

Table 11: Regulatory toxicity studies in dogs.

The NOAEL being 8 mg/kg and the MTD being 65 mg/kg is used as margins between which two intermediate

doses are selected to be administered to dogs.

Note: All doses are administered once a day for a period of one month.

HUMAN EQUIVALENT DOSE:

The NOAEL of each of the species used is converted into a dose equivalent to be administered in humans based

on a scaling factor formulated by FDA as indicated below:

This conversion is based on the normalization of doses in relation to the body surface area which is indicated as

a Body Surface Area-Conversion Factor (BSA-CF) [47], this is obtained from the guidelines and standards set by

FDA. Calculation of the HED is indicated below:

SPECIES BSA-CF

HUMANS 37

RAT 6

DOGS 20

29

For rat:

As per prior results we have NOAEL as 1.89 mg/kg

Hence HED = 2 x (6/37)

= 0.324 mg/kg

For dogs

As per prior results we have NOAEL for dogs as 15 mg/kg

Hence HED = 8 x (20/37)

= 4.32 mg/kg.

Although the HED of rat is lesser, I chose the HED value of dogs for further dose calculations because of the

following reasons:

1) The HED dose for dogs is higher than that of rats hence it would provide a much better Therapeutic index

for further calculations.

2) The ADME routes of dogs are very well known and is more similar to humans

3) Dogs show more similar symptoms when they are induced with peripheral neuropathy, unlike rats which

shows only limb flaccidity.

MAXIMUM RECOMMENDED STARTING DOSE:

MRSD can be defined as the highest amount of the test item that can be safely administered in humans without

complications to the patient while it is maintaining its maximum efficiency. For calculations of MRSD a safety

factor of atleast 10 is used in order to provide better protection to humans. Hence

MRSD ≤ 0.1 (HED)

RESULTS OF REGULATORY TOXICOLOGY STUDIES

FOR RAT: FOR DOGS:

NOAEL: 1.89 mg/kg NOAEL: 8 mg/kg

MTD: 50 mg/kg MTD: 65 mg/kg

HED: 0.324 mg/kg HED: 4.32 mg/kg

30

≤ 0.1 x 4.32 mg/kg

≤ 0.432 mg/kg

THERAPEUTIC INDEX

TI can be defined as a comparision of the amount of a therapeutic agent that can cause the therapeutic effect to

the amount that causes toxicity. It can be calculated as follows:

TI = NOAEL/ Efficacious dose

Efficacious dose (from efficacy studies) = 0.21 mg/kg

Hence TI = 8/0.21

= 38.09

The entire pre-clinical studies was conducted for a duration of one year.

INVESTIGATIONAL NEW DRUG APPLICATION:

Drug GB004 [NCE (4)]

MW 428.55 g/mol

Log P 3.873

Mechanism of Action The S-OCH3 extension in the molecule binds to the

phosphate binding pocket of the enzyme making the space unavailable for the phosphate, hence the

enzyme will not be able to phosphorylate ant substrates that are involved in cytokine production. The two

hydrophobic pockets being Imidazole ring and F-phenyl bind to the other regions on the enzyme making

the binding strong.

Route of administration Oral administration

SUMMARY OF PRE-CLINICAL STUDIES:

1) Human Equivalent Dose: 4.32 mg/kg

2) MRSD ≤ 0.432.

3) Therapeutic Index: 38.09

31

Clinical Protocol and investigator’s information The protocols for clinical studies were documented and is submitted along with this and also the

information of the investigator was also provided.

Chemistry, Manufacturing, Control The drug substance with its properties, name and address of the manufacturer, manufacturing protocols is

submitted. Stability tests was conducted for one year. The ADME characteristics of the drug was also

tested and information is provided.

Pharmacokinetics and Pharmacodynamics study Efficacy tests were conducted and two animal models being rats and adult beagle dogs and the efficacious

dose from this was determined to be 0.21 mg/kg.

During these tests it was also tested and determined that the drug remains in the animals system for 12

hours and reaches Cmax at the 6 to 8 hours of administration.

In-vitro studies Various assays were conducted in-vitro to determine how efficiently the drug binds to the target and to

determine its inhibitory property. The two in-vitro assays conducted were cell binding assay and p-38 map

kinase assays. The tabulated information of these two assays is provided.

Toxicology studies In order to study the toxicology of the drug safety studies was conducted on healthy mouse models and

the results obtained is provided.

Regulatory toxicology studies were conducted on both rat and adult beagle dogs for a period of one year

and an MTD of 50 mg/kg for rats and 65 mg/kg for dogs was determined.

These pre-clinical data predicts the drug’s safety in using them on humans also all the pre-clinical activities

are conducted under GLP.

Safety Pharmacology Studies Various safety tests were conducted in-vitro and in-vivo and are as follows:

- CYP inhibition assay: The drug was found to be minimally inhibiting CYP.

- hERG inhibition assay: It was found that the drug caused negligible side effects on the heart and

the hERG inhibition assay values are also provided.

- On target toxicity studies and off target toxicity studies were conducted.

- Genotoxicity studies were conducted by in-vitro AMES test and the drug was proved to not to be

a mutagen.

First In Human Dose Through regulatory toxicology studies the Human Equivalent Dose was calculated to be: 4.32 mg/kg and

the Maximum Recommended Starting Dose calculated to be: 0.432 mg/kg which will used as the

initial dose in Phase I clinical trials.

The IND application was submitted to the F.D.A for approval.

32

INVESTIGATOR’S BROCHURE:

The document was submitted to Institutional Review Board (IRB) for review. A copy of this as per requirement

was submitted to FDA with IND application. This includes all clinical, pharmacological information, toxicology

studies of the drug GB004 [NCE (4)].

PHASE I CLINICAL TRIALS IN HUMANS:

Purpose: To determine the safe dose range, clinical safety signals and to determine the hMTD.

Condition: Guillain-Barre syndrome

Primary purpose: To formulate a safe dose range and to test toxicity of the drug in humans.

Drug: GB004 (NCE4)

Starting Dose: Concentration of 0.432 mg/kg/day.

STUDY DESIGN

Primary purpose: Toxicology study

Allocation: Randomized [48]

Endpoint: Safety and efficacy study

Intervention model: Parallel assignment

Masking: Single Blind

Number of participants: 112 in cohorts of 16.

Healthy Volunteers: Yes

Title: Phase1, randomized, single blind, single ascending dose study to investigate the safety and efficacy of

GB004 (NCE4).

Inclusion criteria:

Healthy volunteers of age group 18 to 65 years old.

Both male and females can participate.

Exclusion criteria:

Should not have a previous record of cardiovascular disorders.

Should not be pregnant or lactating women.

Volunteers under medication for other disorders are not allowed.

Clinical trials were conducted for a period of 6 months. Volunteers were administered with a dose every day and

the various adverse effects were observed. This was conducted for a period of one week before the next dose

could be administered. The dose was incremented by 2X, 4X etc. for the consecutive weeks. The results of the

phase one trials are as indicated in table 12:

33

Drug dose mg/kg No. of subjects No. of subjects with

adverse effects

Adverse effects

0 (Control Placebo) 16 0 None

0.43 (MRSD) 16 0 None

0.86 16 0 None

1.72 16 0 None

3.44 16 0 None

6.88 16 3 Gastrointestinal problems,

Hair fall

13.76 16 9 Gastrointestinal Issues, Kidney issues,

Hair fall

Table 12: Doses and its adverse effects in the phase 1 trial.

DISCUSSION:

From the information available in the table we can see that starting from the MRSD 0.43 mg/kg till 3.4 mg/kg

there are no adverse effects observed. At 6.8 mg/kg 3 out of 16 patients showed signs of AE but since it is optimal

and not as bad as the AE indicated in the drug doses afterwards. The last dose administered shows that 9 of 16

volunteers (more than 33%) showed adverse effects. This is way higher than the number of subjects showing

adverse effects in the dose range 6.8 mg/kg (approximately equal to 408 mg as per the avg. weight of human

beings taken as 60 kg) hence this dose can be considered as hMTD for further studies since less than 33%

of volunteers show adverse effects.

IND UPDATE

Phase I Clinical Trial Summary:

Phase I clinical trials was conducted on 112 volunteers in cohorts of 16.

Initial dose of 0.43 mg/kg (MRSD) was given.

No fatal toxicological side effects observed and the hMTD was determined to be 6.8 mg/kg

Major side effects were GI problems and hyperglycemia which could be controlled by insulin injection

(prevailed only during course of drug administration).

The entire study was conducted for a duration of 6 months.

Pharmacokinetic & Pharmacodynamics Studies

During Phase I trials on collecting the blood samples of patients and observed for the concentration of

the drug, it was noticed that the drug remained in the human system for a period of 6 hours and the Cmax

was observed at 2 to 3 hours of administration, hence for further studies drug needs to be administered

twice a day.

QT prolongation did not prevail in volunteers.

34

Non-Clinical Activities:

Further testing of toxicity was conducted on animals, for a period of one year the drug was safe for further

usage.

Single and repeat dose toxicity studies were conducted for a duration of 6 months

Chemistry, Manufacturing, Control

Drug Manufacturing protocols have been provided.

Stability tests for the drug were conducted for a period of one year and the drug is stable for clinical use.

The physical, chemical and biological characteristics of the drug was provided.

All assays and methods used to detect the purity, quality of the drug is provided.

INVESTIGATOR’S BROCHURE UPDATE:

An updated copy of the document was submitted to Institutional Review Board (IRB) for review. A copy of this

as per requirement was submitted to FDA with IND application. This includes all clinical, pharmacological

information, toxicology studies of the drug GB004 [NCE (4)].

PHASE II CLINICAL TRIALS

Purpose: To study the efficacy and safety of GB004 drug in the treatment of GB syndrome on diseased patients

and to find an optimal dose for Phase III.

Study Type: Interventional

Study Design:

Allocation Randomized

Endpoint Classification Efficacy/Safety study

Intervention mode Crossover Assignment

Masking Double masking

Enrollment 240

35

Primary Endpoints Measured:

Neuropathy Impairment Score (NIS) [49]: The main symptom associated with GB syndrome is weakness,

numbness in the limbs of the patients due to the damage caused to peripheral nerves. NIS is a clinical

scoring system which is used to study the severity of the peripheral neuropathy. Scoring is provided as a

cumulative score which includes various components like sensation, reflexes, limb movement and muscle

weakness. The scoring system lies between 0(total recovery) to 80(total impairment).

Secondary Endpoints Measured:

Cytokine concentration in CSF: The drug mainly is designed to act on p-38 MAPK which is involved in

cytokine production which in turn reduces the differentiation of T cells. Hence the efficacy of drug action

can be measured by the concentration of cytokines in the CSF i.e. lesser the concentration more efficient

the drug.

Cerebrospinal Fluid biomarker concentration as explained earlier- the CSF is extracted from patients

through lumbar puncture from the spinal cord.

Patient Eligibility:

Age group: 19 – 75 years.

Genders: Both

Healthy Volunteers: No

Inclusion Criteria:

Patients must be of age group 19 to 75 years only.

Patient should have neurological impairment due to GBS only which has been stable for less than 12

months.

Patient must comply with the therapy and protocols provided during the study.

Exclusion Criteria:

Subjects must not be involved in any other medication.

Pregnant and lactating women are not allowed to participate.

Heart patients are not allowed to participate.

Subjects must not be enrolled in any other trial.

Study Title [50]:

Assessment of efficacy/safety of GB004 in GB syndrome patients.

This trial was conducted on a total of 240 patients suffering from GB Syndrome. Patients were orally administered

with the GB004 drug in cohorts of 80. Initial dose was 50 mg twice a day the dose was increased in an order for

the subsequent cohorts. The dose range selected does not exceed the hMTD deduced in the previous phase. The

different endpoints and the biomarker assays were conducted every week to check the progress of treatment in

patients. The results obtained were tabulated as follows. Placebo was used as a control to measure the results

36

against. This trial was conducted for a time period of a year. The different values obtained for respective doses

and the results are shown in table 13.

Dose

(mg)

No. Of

Patients

No. Of

Patients

with AE

NIS Score

Cytokine

Conc.

(ng/ml)

Biomarkers

Nfs (ng/ml) IP-10 (ng/ml)

Placebo=0 60 1 78 200 0.73 11

50 60 10 70 180 0.60 9

150 60 15 30 100 0.30 5

250 60 21 15 80 0.18 3

Table 13: Phase II trial results

Adverse effects:

The adverse effects were minimal and tolerable. The two main adverse effects observed with these patients were

Hyperglycemia: Normal blood glucose levels in humans before food needs to be 70-99 mg/dL. The

patients in the clinical trials who were administered with the drug showed slight elevation in the glucose

level. This elevation in glucose level was constant with increase in drug doses. Precautions were taken to

administer insulin injections if glucose level exceeded alarmingly.

Weight Loss

CONCLUSION OF PHASE II TRIAL:

From the study it was determined that dose of 150 mg twice a day for the following reasons:

- The number of patients showing adverse effects are less than 33% unlike the very next dose where it

exceeds 33%.

- NIS score and cytokine concentration drops to optimum levels.

- Decrease in concentration of the biomarkers Nfs and IP-10 indicate the effectiveness of the drug.

- Even though the very next dose shows decrease in biomarker concentration in the CSF this dose cannot

be selected for further calculations since more than 33% of the patients show adverse effects.

IND UPDATE

- PHASE II Clinical Trial Summary

An optimum dose of 150 mg twice a day was concluded for the further phase III trials in humans.

Phase II trials was conducted for a duration of 2 years.

- Product Release information:

The dose of administration and the route of administration and other information related to the drug

release in provided.

- Chemistry, Manufacture and cost:

Stability tests are conducted for a time period of 2 years and the drug is stable for clinical use.

37

Details about protocol and materials required for manufacturing is provided.

Chemical, physical and biological properties of the drug is provided.

Labeling information and packaging information has been updated and the information is

provided.

Cost margin for the manufacture of the drug is provided.

- All impurities are identified, qualified and quantified and the information is provided.

- Further non clinical studies on animals were conducted for a period of one year, to test the toxicology of

the drug before entering phase III trials.

INVESTIGATOR’S BROCHURE UPDATE:

An updated copy of the document was submitted to Institutional Review Board (IRB) for review. A copy of this

as per requirement was submitted to FDA with IND application. This includes all clinical, pharmacological

information, toxicology studies of the drug GB004 [NCE (4)].

PHASE III CLINICAL TRIALS:

Study Objective: To test for the safety and effectiveness of GB004 drug in treating patients suffering from

Guillain Barre Syndrome.

Study Type: Intervention

Study Design [51]:

Allocation Randomized

Intervention Model Single group assignment

Masking Open label

Endpoint Classification Efficacy/Safety study

Purpose Treatment

Enrollment 2000

Eligibility:

Age group: 18 years and above.

Gender: Both

Healthy Volunteers: No

38

Inclusion Criteria:

It must have been atleast 2 weeks since the start of symptoms associated with GB Syndrome.

Patients must have an NIS score greater than or equal to 70.

Exclusion Criteria:

Patients with complete paralysis are not allowed to participate

Should not be involved in any other trials or in any other medications

Pregnant and lactating women are not allowed to participate.

Patients should not be treated with Immunoglobulin therapy prior to this study.

Patients with liver, cardiovascular diseases are not allowed.

Primary and Secondary endpoints mentioned is as same as the one performed in phase II.

Title: A randomized, open label study for testing safety and effectiveness of GB004 drug in treating patients with

GB Syndrome.

The study involved 2000 patients divided into cohorts of 1000 each. Placebo was selected as the Gold standard

against which the safety and efficacy of the drug would be measured. The first cohort was administered with the

placebo and the second cohort was administered with 150 mgs of the drug twice a day. The study was conducted

for a duration of one year. The trial outcome was tabulated as follows:

Trial Arm

Number

of

Patients

Number of

patients with

AE

NIS Score Cytokine Concentration

ng/ml

Placebo 1000 50 70 190

GB004

150 mg

1000 100 5 80

Table 14: Phase III trial outcomes.

Adverse Effects

The major side effects seen were fever, headache and slight increase in blood glucose level (not alarming). These

side effects were optimal and tolerable. Paracetemol tablets could be administered if fever persisted for more than

24 hours. Headache also if persistent can be treated with pain meds.

CONCLUSION:

In the first 1000 patients treated with Placebo it was seen that more than 700 people had NIS of 75 and

cytokine concentration of 190 ng/ml indicating the ineffectiveness of the placebo in treating the disease.

In the next set of 1000 patients treated with GB004 900 subjects showed a drop in the NIS score from 75

to 5, which seemed to be a drastic change in the motor functions of these patients. Also the cytokine

concentration in the blood dropped to 80 ng/ml indicating the effectiveness of drug.

39

This proves that the drug is positively inhibiting p-38 MAPK (cytokine concentration drop) as a result of

which the autoimmune attack on the nerves are inhibited which is reflected by the drop in NIS indicating

patients regaining their motor functions.

The number of people showing adverse effects when the dose of 150 mg is lesser than 33% hence it shows

the safety of the drug.

The points indicated above shows that the drug at 150 mg twice a day is effective in treating the disease and is

also safe for patients.

The total duration on phase III trials was 2 years

NEW DRUG APPLICATION (NDA)

Pyramidine Imidazole molecules are used as potent drugs to treat GB Syndrome.

Drug: GB004

Mechanism of Action: These are oral drugs that are p-38 MAP Kinase inhibitors. P-38 MAPK are responsible

for cytokine production which leads to T cell differentiation which leads to immune response against the neurons.

GB004 bind irreversibly to the enzyme, blocks the active site of the enzyme for substrate binding and hence

inhibits the immune reaction.

All three phases of the clinical trial show positive results and is proof that the drug is safe, effective and tolerable

with minimal side effects. An efficacious dose of 150 mg twice a day has been deduced from phase 2 n 3 and the

results for the same is provided with the trial details. 408 mg was deduced as the hMTD during phase 1 clinical

trial, hence administration of 150 mg twice a day (making it 300 mg) is way less than the hMTD value this

indicates the safety of the drug.

The medical need for the discovery of GB004 was that till date there has been no such oral drugs used for treating

GB Syndrome patients, IV administration of antibodies and Plasmapheresis were the only two treatments

available. GB004 is an oral drug which is proved to treat GB Syndrome with a dose of 150 mg twice a day. With

this research it is evident that the drug full fills the medical need it was built on.

Hence the approval of this drug would avoid the painful and tedious treatments like Plasmapheresis or

Immunoglobulin administration, instead patients with GB Syndrome can just orally consume GB004 at specified

dose. Patients will not have to be bed ridden for the entire course of treatment.

40

A Summary of the entire clinical trial activities is provided below in the form of a flow chart indicated

below.

IND APPLICATION

DRUG GB004 [NCE (4)]

Route of Administration Oral

CMC Stability tests for 1 year

Manufacturer’s information

Manufacturing protocol

PK-PD Studies Efficacious dose: 0.21 mg/kg

Cmax at 6 to 8 hours after administration

Safety Pharmacology and Toxicology Studies CYP and hERG inhibition assay

On target and off target toxicity studies

Genotoxicity

Regulatory toxicology studies

First In Human Dose MSRD: 0.432 mg/kg for human trials

PHASE I CLINICAL TRIALS:

Purpose Safety & Efficacy study

Number of patients 112 in cohorts of 16

Healthy Volunteers Yes

Total duration 6 months

hMTD Determined 6.88 mg/kg = 408 mg

IB PROVIDED

41

IND UPDATE:

Non clinical toxicity studies for one year.

Stability tests for 1year.

CMC information provided

IB provided.

PHASE II CLINICAL TRIALS:

Purpose Safety and initial efficacy studies

Enrollment 240 in cohorts of 60

Healthy Volunteers No

Starting dose 50 mg twice a day

Adverse Effects Hyperglycemia, weight loss

Efficacious dose 150 mg twice a day

Total duration of study 1 year

IB

IND UPDATE:

Optimum dose for next trial 150 mg twice a day

Non clinical toxicity studies for one year.

Stability tests for 2year.

CMC

IB provided.

IB

42

FILING NDA

NEW DRUG APPLICATION was filed and submitted to the FDA for approval.

PHASE III CLINICAL TRIALS:

Purpose To Test the Effectiveness of the drug

Enrollment 2000 in cohorts of 1000

Healthy Volunteers No

Gold Standard Placebo

Dose administered 150 mg twice a day

Total Duration 2 years

43

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