Summary And Conclusion -...

1

Summary

And

Conclusion

2

Summary:

1.0 Imatinib Mesylate

A robust and sensitive HPLC method was developed for the determination and

quantitation of the following process impurities in the drug substance, Imanitinib

mesylate, viz., Impurity A and Impurity B, whRG1 are potential genotoxic impurities.

The LHASA predictions of Derek Nexus Report support the potential genotoxicity of

Impurity A and Impurity B.

1.1 LHASA predictions for Impurity A:

N N

N

NH

CH3

NH2

Impurity A

REASONING SUMMARY ALERTS FOUND

Carcinogenicity in mammals is plausible 587 aromatic amine or amide

Methaemoglobinaemia in mammals is

plausible

006 Simple aoine or precursor

Mutagenicity in vitro in bacterium is

plausible

352 aromatic amine or amide

Rapid prototypes: hepatotoxicity in

mammals is equivocal

Rapid prototype: 008 2-Aminopyridine

Skin senisitisation in mammals is

plausible

427 Aromatic primary or secondary amine

The predictions have been proved in bacterium and mammals.

Based on the predictions, the impurity is characterized as Carcinogenic, Genotoxic,

Chrosomal damaging, HERG Channel Inhibitory, Hepatotoxic, Irritant, Mutagenic,

3

Ocular toxicant and also to cause blood in urine, blader disorders, bone marrow toxicity,

chromosome damage in vitro, bradycardia, , kidney disorders, mitochondrial dysfunction,

nephrotoxic, splenotoxic, thyroid toxicity, reproductive toxicity, respiratory senisitisation,

skin senisitation.etc.

1.2 LHASA predictions for Impurity B:

N N

N

NH

CH3

NH

Cl

O

Impurity B


Carcinogenicity in mammal is

PLAUSIBLE

073 alkylating agent

Chromosome damage in vitro in

mammal is Plausible


Irritation (of the eye) in mammal is

plausible

228 Benzyl halide

Irritation (of the respiratory tract) in

mammal is plausible

228 Benzyl halide

Irritation (of the skin) in mammal is

plausible

228 Benzyl halide

Methaemoglobinaemia in mammals is

plausible

006 Simple aoine or precursor


plausible


Rapid prototypes: Hepatoxicity in

mammal is equivocal

Rapid prototype: 008 2-Aminopyridine

Skin senisitisation in mammals is

plausible

413 Haloalkane

4

The predictions have been proved in bacterium and mammals.



Ocular toxicant and also to blader disorders, bone marrow toxicity, chromosome damage

in vitro, bradycardia, kidney disorders mitochondrial dysfunction, nephrotoxic,

splenotoxic, thyroid toxicity, reproductive toxicity, respiratory senisitisation, skin

senisitation.etc.

The maximum daily dose of Imanitinib is 800 mg.

Impurity A and Impurity B bears structural alerts and is positive in several genotoxic

systems as well as yielded toxicological findings (hyperplasia, necrosis) in various organs

in 28days study in rats.

However, the drug substance batches spiked with upto 280 ppm yielded negative

genotoxicity test results (could have been proved by calculation).

The Limit for the impurities was set as 20 ppm based on technical feasibility. This limit

was set considering highest human daily intake/exposure would be 8 and 16 µg, whRG1

is equivalent to 1.6 and 3.2 ng/ml blood assuming the total absorption of the same.

Human exposure is also clearly below in vitro genotoxic concentrations. (Data source:

Novartis Case Study)

Impurity A is formed as a major metabolite in mice and rabbits, however, no major

pathway was found in rats, dogs and humans.

The challenge lies here: Are analytical methods capable of excluding the Impurities as a

minor human metabolite?

It does not make sense to be very strict with applying low ppm limits for impurities, but

to be not able to exclude formation by metabolism at higher levels.

Therefore, a robust and sensitive analytical method for the determination and quantitation

of the impurities, viz., Impurity A and Impurity B in Imanitinib mesylate by High

Performance Liquid Chromatography was developed and subsequently validated. The

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method was validated as per RG1 (RG1) guidelines in terms of Specificity, RV2

(RL1), Limit of Quantitation (RL2), Linearity, RV6, RV5 and RV7.

1.2.3 Method Summary:

Instruments Used : Name : HPLC Make : Agilent Model : 1200 Series

Column used : Inertsil ODS 3V column (150 mm x 4.6 mm x 5 µm)

Mobile phase

Solution A : 0.1% Triethyl amine in water and pH adjusted to 2.9

with glacial acetic acid

Solution B : RR2 and RR1in the ratio of 10:90 (v/v)

Diluent : Solution A: Solution B (50:50) (v/v)

Flow rate : 1.5 ml/ minute

Column oven temperature : 35°C

Detection wavelength : 268 nm

Injection volume : 20 µl

Mode : Gradient

Time % Solution A % Solution B

0.01 90 10

10.0 90 10

20.0 50 50

28.0 50 50

32.0 10 90

38.0 10 90

38.1 90 10

45.0 90 10

Sample Concentration 10mg /ml

Standard concentration of 0.02mg/ml Imp- A and Imp-B

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Imatinib Imp-A and Imp-

B

1.2.4 Validation Results Summary:

Validation Parameters

Acceptance Criteria Results obtained

RV1

Specificity - Blank interference

The blank solution should not show

interference at the retention time of Impurity A and

ImpurityB.

There should not be any

interference of Impurity A

and ImpurityB among

themselves.

No interference seen from blank.

No interference

System Suitability for Impurity A

- Theoretical plates - Symmetry factor

- RSD for retention time - RSD for peak area

RM2 2000 RM2 0.8 and RM1 2.0

RM1 1.00 % RM1 5.00 %

5452 1.1 0.3 1.8

System Suitability for Impurity B



RM2 2000 RM2 0.8 and RM1 2.0

RM1 1.00 % RM1 5.00 %

6784 1.0 0.2 1.2

7

Validation Parameter Acceptance Criteria Results obtained

RV2

RS3 of Impurity A

RS3 Impurity B

Should be equal to or more

than 3.0


than 3.0

5.0

4.0

% concentration of

Impurity A w.r.t. sample % concentration of

Impurity B w.r.t. sample

For information

For information

0.00024 %

0.00024%

Limit of Quantitation

RS3 of Impurity A RS3 of Impurity B


than 10.0

Should be equal to or more than 10.0

15

12

% concentration of

Impurity A w.r.t. sample % concentration of

Impurity B w.r.t. sample

For information

For information

0.0008 %

0.0008 %

RS2 of Area for Impurity A

RS2 of Area for Impurity B

RM1 5.0 %

RM1 5.0 %

4.39%

1.7 %

RS2 of Retention time for

Impurity A RS2 of Retention time for

Impurity B

RM1 1.0 %

RM1 1.0 %

0.0 %

0.2 %

8


RV4 %RSD of retention time

for RH1 (Impurity A)

%RSD of retention time for Last RH4 (Impurity A) %RSD of retention time for RH1 (Impurity B)

%RSD of retention time

for Last RH4 (Impurity B)

RM1 1.0 %

RM1 1.0 %

RM1 1.0 %

RM1 1.0 %

0.1%

0.0%

0.0%

0.1%

%RSD of peak area for RH1 (Impurity A)

%RSD of peak area for Last RH4 (Impurity A)

%RSD of peak area for

RH1 (Impurity B)

%RSD of peak area for Last RH4 (Impurity B)

RM1 5.0 %

RM1 5.0 %

RM1 5.0 %

RM1 5.0 %

3.1 %

2.4%

4.1%

3.9%

Correlation coefficient (Impurity A)

Correlation coefficient

(Impurity B)

RM2 0. 999

RM2 0. 999

1.000

0.9999

% y intercept (Impurity A)

% y intercept (Impurity B)

Within + 3

Within + 3

0.9

-2.1


9

RV6

% Recovery in RH1 RV6 solution

% Recovery of Impurity A in RH1 RV6 solution

% Recovery of Impurity B

in RH1 RV6 solution

RM2 85.0 % and RM1 115.0 %

RM2 85.0 % and RM1 115.0 %

108.13 -113.44%

91.56 -97.50 %


% Recovery of Impurity A

in RH2 RV6 solution

% Recovery of Impurity B in RH2 RV6 solution

RM2 90.0 % and RM1 110.0 %

RM2 90.0 % and RM1 110.0 %

102.60-110.00%

91.00 - 92.60 %.



in RH3 RV6 solution


RM2 90.0 % and RM1 110.0 %

RM2 90.0 % and RM1 110.0 %

108.15-109.85 %

91.80- 93.85 %



in RH4 RV6 solution


RM2 90.0 % and RM1 110.0 %

RM2 90.0 % and RM1 110.0 %

106.60 -112.07 %

91.70-91.80 %

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Trend data

Compound name Impurity-A Impurity-B B.No. A O O B.No. B O O B.No. C O O

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2.0 Sorafenib Tosylate

2.1 Methyl Tosylate



Ocular toxicant and also to blood in urine, blader disorders, bone marrow toxicity,


nephrotoxic, splenotoxic, thyroid toxicity, reproductive toxicity, respiratory sensitization,

skin sensitization.etc.

LHASA predictions for Methyl Tosylate



PLAUSIBLE


Chromosome damage in vitro in mammal

is Plausible



Plausible


Skin sensitisaiton in mammal is Plausible 414 Alkyl Sulphate or sulphonate

Tetratogenicity in mammal is plausible 516 Alkyl sulphonate

2.2 Ethyl Tosylate


Chromosomal damaging, HERG Channel Inhibitory, Hepatotoxic, Irritant, Mutagenic,

Ocular toxicant and also to cause, blood in urine, blader disorders, bone marrow toxicity,

chromosome damage in vitro, bradycardia, , kidney disorders, mitochondrial

dysfunction, nephrotoxic, splenotoxic, thyroid toxicity, reproductive toxicity, respiratory

sensitization, skin sensitization.etc.

12

LHASA predictions for Ethyl Tosylate



PLAUSIBLE


Chromosome damage in vitro in

mammal is PLAUSIBLE



PLAUSIBLE


Skin sensitisaiton in mammal is

PLAUSIBLE

414 Alkyl Sulphate or sulphonate

Tetratogenicity in mammal is

PLAUSIBLE

516 Alkyl sulphonate

2.3 Isopropyl Tosylate


Chromosomal damaging, HERG Channel Inhibitory, Hepatotoxic, Irritant, Mutagenic,

Ocular toxicant and also to cause blood in urine, blader disorders, bone marrow toxicity,


nephrotoxic, splenotoxic, thyroid toxicity, reproductive toxicity, respiratory sensitization,

skin sensitization.etc.

LHASA predictions for Isopropyl Tosylate:



PLAUSIBLE


13

Chromosome damage in vitro in mammal

is PLAUSIBLE



PLAUSIBLE


Skin sensitisaiton in mammal is

PLAUSIBLE

414 Alkyl Sulphate or sulphonate

Tetratogenicity in mammal is

PLAUSIBLE

516 Alkyl sulphonate

The above mentioned genotoxic impurities are characterized under process impurities.

These impurities are formed in the synthesis process of Sorafenib tosylate.

e.g

CH3OHS

O

O

OHCH3S

O

O

OCH3

CH3

4-Methyl Benzenesulphonic acid Methyl Tosylate

S

O

O

OHCH3S

O

O

OCH3 CH3

C2H5OH

4-Methyl Benzenesulphonic acid Ethyl Tosylate

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S

O

O

OHCH3S

O

O

OCH3 CH3

CH3

C3H7OH

4-Methyl Benzenesulphonic acid Isopropyl Tosylate

The maximum daily dose of Sorafenib is 800 mg.

Methyl Tosylate, Ethyl Tosylate and Isopropyl Tosylate bears structural alerts and is

positive in several Genotoxic systems. It also yielded toxicological findings in various

studies.

2.4 Method Summary:

Instruments Used : Name : UPLC Make : Waters Model : AcquityTM

Column used : RRHD (50 mm x 2.1 mm , 1.8 µm)

Mobile phase

Solution A : 50mm Sodium Perchlorate pH-3 with glacial acetic

acid

Solution B : RR1

Mobile Phase : Solution A: Solution B(60:40) (v/v)

Diluent : Mobile Phase

Flow rate : 0.5 ml/ minute

Column oven temperature : 40°C

Detection wavelength : 226 nm

Injection volume : 10 µl

Mode : Isocratic

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Sample Concentration 15mg /ml

Standard concentration of

Methyl Tosylate,

Ethyl Tosylate

and Isopropyl Tosylate

0.2 µg/ml

0.2 µg/ml

0.2 µg/ml

2.5 Validation Results Summary:

Validation Parameters

Acceptance Criteria Results obtained

RV1

Specificity - Blank interference

The blank solution should not show

interference at the retention time of Methyl Tosylate, Ethyl Tosylate and Isopropyl Tosylate.

There should not be any

interference of Methyl

Tosylate, Ethyl Tosylate

and Isopropyl Tosylate

among themselves.

No interference seen from blank.

No interference

System Suitability for Methyl Tosylate - Theoretical plates - Symmetry factor


RM2 2000 RM2 0.8 and RM1 2.0

RM1 1.00 % RM1 5.00 %

39402 1.53 0.1 1.2

System Suitability for Ethyl Tosylate

16



RM2 2000 RM2 0.8 and RM1 2.0

RM1 1.00 % RM1 5.00 %

53040 1.38 0.4 0.67

System Suitability for Isopropyl Tosylate - Theoretical plates - Symmetry factor


RM2 2000 RM2 0.8 and RM1 2.0

RM1 1.00 % RM1 5.00 %

83568 1.32 0.2 0.89


RV2

RS3 Methyl Tosylate

RS3

Ethyl Tosylate

RS3

Isopropyl Tosylate


than 3.0


than 3.0


than 3.0

4 5

4

% concentration of

Methyl Tosylate w.r.t. sample

% concentration of

Ethyl Tosylate w.r.t. sample

% concentration of

Isopropyl Tosylate w.r.t. sample

For information

For information

For information

0.0003 %

0.0003 %

0.0003 %

Limit of Quantitation

RS3 of Methyl Tosylate


than 10.0

12

17

RS3

Ethyl Tosylate

RS3

Isopropyl Tosylate

Should be equal to or

more than 10.0

Should be equal to or more than 10.0

11

12

% concentration of

Methyl Tosylate w.r.t. sample

% concentration of Ethyl Tosylate w.r.t.

sample

% concentration of Isopropyl Tosylate

w.r.t. sample

For information

For information

For information

0.0006 %

0.0006 %

0.0006 %

RS2 of Area for

Methyl Tosylate

RS2 of Area for Ethyl Tosylate

RS2 of Area for

Isopropyl Tosylate

RM1 5.0 %

RM1 5.0 %

RM1 5.0 %

3.67%

3.89 %

2.98%

RS2 of Retention time for Methyl Tosylate

RS2 of Retention time

for Ethyl Tosylate

RS2 of Retention time for Isopropyl Tosylate

RM1 1.0 %

RM1 1.0 %

RM1 1.0 %

0.0 %

0.1 %

0.1%

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RV4 %RSD of retention time

for RH1 (Methyl Tosylate)


for Last RH4 (Methyl Tosylate)


for RH1 (Ethyl Tosylate)


for Last RH4 (Ethyl Tosylate)


for RH1 (Isopropyl Tosylate)


for Last RH4 (Isopropyl Tosylate)

RM1 1.0 %

RM1 1.0 %

RM1 1.0 %

RM1 1.0 %

RM1 1.0 %

RM1 1.0 %

0.0%

0.0%

0.0%

0.04%

0.0%

0.0%

%RSD of Area

for RH1 (Methyl Tosylate)

%RSD of Area for Last RH4

(Methyl Tosylate)

%RSD of Area for RH1

(Ethyl Tosylate)


(Ethyl Tosylate)

%RSD of Area for RH1

(Isopropyl Tosylate)

RM1 5.0 %

RM1 5.0 %

RM1 5.0 %

RM1 5.0 %

RM1 5.0 %

2.1 %

1.1%

2.0%

1.9%

0.7%

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(Isopropyl Tosylate))

RM1 5.0 %

0.8%

Correlation coefficient (Methyl Tosylate)

Correlation coefficient

(Ethyl Tosylate)

Correlation coefficient (Isopropyl Tosylate)

RM2 0. 999

RM2 0. 999

RM2 0. 999

0.9996

0.9999

1.0000

% y intercept (Methyl Tosylate)

% y intercept

(Ethyl Tosylate)

% y intercept (Isopropyl Tosylate)

Within + 3

Within + 3

Within + 3

2.64

-0.97

0.37

20


RV6


% Recovery of Methyl Tosylate in RH1 RV6

solution

% Recovery of Ethyl Tosylate in RH1 RV6

solution

% Recovery of Isopropyl Tosylate in RH1 RV6

solution

RM2 85.0 % and RM1 115.0 %

RM2 85.0 % and RM1 115.0 %

RM2 85.0 % and RM1 115.0 %

96.6-101.1%

113.4-114.6 %

101.9-109.9%



solution


solution


solution

RM2 90.0 % and RM1 110.0 %

RM2 90.0 % and RM1 110.0 %

RM2 90.0 %

and RM1 110.0 %

99.0-105.0%

99.0 – 100.0 %.

104.0-109.1%



solution


solution


solution

RM2 90.0 % and RM1 110.0 %

RM2 90.0 % and RM1 110.0 %

RM2 90.0 % and RM1 110.0 %

106.6-107.6%

102.0-106.6 %

100.5-106.6%

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solution


solution


solution

RM2 90.0 % and RM1 110.0 %

RM2 90.0 % and RM1 110.0 %

RM2 90.0 % and RM1 110.0 %

100.7-108.1 %

100.3-108.4 %

97.9-104.3%

Trend data

Compound name Methyl Tosylate Ethyl Tosylate Isopropyl Tosylate

B.No. 1 O O O

B.No. 2 O O O

B.No. 3 O O O

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Conclusion:

Control of impurities in DS and DP. In the regulated market as per current trends the

sponsor of NDA needs to monitor and control the PGIs by introducing process control.

PGIs can not be treated as other impurites as per RG1 Q3A/Q3B guideline, because the

tests performed to demonstrate the Genotoxicity of DS may not full fill the criteria to

demonstrate the stability of PGIs. The TTC approach for control of PGI is a general

appropriate approach till further unless specific risk management approach is

proposed. The current research work will states examples where the impurities are

monitored and controlled during the manufacturing process of DS and DP. It is also

highlight the current trend like staged threshould of toxicological concerned as per

latest guideline on controlling PGIs.

However, genotoxic Impurities must be detected, identified, and then reported before

clinical trial initiation to ensure patient safety and eventual drug approval, but every drug

and its process has a unique set of process impurities. To confirm drug approval, the

industry has heavily invested resources to address the GTI concerns and stay compliant

with limits of genotoxic and potential genotoxic impurities.

The genotoxic impurities that are identified, detected and reported in the thesis are well

supported through Historical and deductive structural alerts (DEREK-Deductive

Estimation of Risk from Existing Knowledge).

UPLC and HPLC offer an interesting tool for rapid and sensitive analytical method

development for the determination of GTIs/PGIs in APIs. These techniques are simpler

compared to other highly sophisticated techniques such as LCMS/GCMS.

The present study describes a sensitive, simple, rapid and accurate validated Liquid

chromatography method by using UV detection for estimation of potential GTIs/PGIs in

the drug substance. The liquid chromatographic method was found to be selective,

sensitive, precise, linear and accurate for the determination of the described GTIs.

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