Camptothecin Analog Drug Profile Biology Essay

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Camptothecins belong to a class of compounds called topoisomerase inhibitors and have shown potential in the treatment of solid tumors. Camptothecin analogs are synthesized from Camptothecin; a plant alkaloid isolated from the Chinese tree Camptotheca acuminate. Camptothecins exert their mechanism of action by inhibiting topoisomerase which assist in the unwinding of DNA that occurs prior to replication. Inhibitors of the enzyme topoisomerase-I are cytotoxic for tumor cells because of their ability to cause DNA damage by forming an irreversible complex with DNA resulting in cell death. Camptothecin analog and its derivatives have shown a wide spectrum of anticancer activity against human malignancies including human lung, prostate, breast, colon, stomach, ovarian carcinomas, melanoma, lymphomas and sarcomas61-63. The commercially available compounds in this therapeutic class are usually administered either by continuous infusion or using multiple time-spaced injections, both resulting in a low patient comfort and compliance. Following parenteral administration, there has been incidence of grade 3 or higher diarrhea thus necessitating the need for development of safer, orally effective CAs to enable administration for a prolonged duration, conveniently and cost effectively on an outpatient basis64.

To overcome limitations of the currently marketed commercial product in this therapeutic class (e.g., Irinotecan Hydrochloride), a novel Camptothecin analog (CA) was synthesized by Dr. Reddy's. The novel Camptothecin analog exhibits poor wettability and low aqueous solubility. In solution phase the impurities increase as a function of time and temperature with pronounced conversion of S to R isomer under alkaline conditions65. The novel compound is currently in Phase-I clinical studies.

IUPAC Name: 5 (S)-(2'-hydroxy ethoxy)-20(S)- Camptothecin

Chemical Formula: C22H20N2O6

Molecular Weight: 408.41 g/mol

Log P: 5.282

Melting point: 238 °C

Solubility

Camptothecin analog is a pale yellow powder which is insoluble in water and ethanol, sparingly soluble in N-methyl-2-pyrrolidone (NMP) and dimethyl sulfoxide (DMSO), and slightly soluble in acetone and dichloromethane (DCM).

3.21.2 Mechanism of Action

CA's exerts their mechanism of action by inhibiting topoisomerase which assist in the unwinding of DNA that occurs prior to replication. Inhibitors of the enzyme topoisomerase 1 are cytotoxic for tumor cells because of their ability to cause DNA damage. They bind to topoisomerase 1 and form a freely reversible ternary complex along with DNA. The collision of a DNA replication fork with the ternary complex leads to a series of biochemical events - irreversible arrest of the replication fork, generation of a double-strand break, and conversion of the reversible cleavable complex into an irreversible complex - resulting in cell death66.

3.22 MATERIALS

&

METHODS

3.22.1 Materials

Table 3.22. List of Materials

S. No

Ingredients

Manufacturers

1

Camptothecin Analog

Dr. Reddy's Laboratories Ltd., Hyderabad

2

Hydroxy Propyl Methyl Cellulose (HPMC) 6 cps

Pioma Chemicals

3

Sodium Lauryl Sulphate

Qualigen Chemicals, Delhi

4

Poly Vinyl Pyrrolidone (PVP) K30

BASF, Germany

5

Poloxamer 188

Signet Chemicals BASF Germany

6

Lactose Anhydrous

DMV international, Netherlands

7

Corn Starch

Grain Processing Corporation, USA

8

Crosspovidone (Polyplasdone-XL)

ISP, USA

9

Sodium Starch Glycolate (SSG)

Amishi Drugs &Chemicals, Delhi

10

Colloidal Sillicon Dioxide

(Aerosol-A 200)

Degussa, Germany

11

Magnesium Stearate

Ferro Corporation, USA

12

Tetra Butyl Ammonium Hydrogen Sulphate

Merck, USA

13

Methanol

Ranchem, Delhi

14

Acetonitrile

Ranchem, Delhi

3.22.2 Instruments and Apparatus

Table 3.23. List of Equipments

S.No

Instruments

Manufacturer

1

Overhead Stirrer

Heidolph RZR 2051 control

2

Agitator Bead Mill

Netzsch-Feinmahltechnik Gmbh

3

Fluid bed coater

GCP 1.1

4

Mini Press II Compression Machine

Rimek, Ahmedabad

5

Tablet Hardness Tester 8M

Dr.Schleuniger Pharmatron

6

Disintegration Tester (USP)

Electrolab, India

7

Friabilator (USP)

Electrolab, India

8

Tap Density Tester (USP)

Electrolab, India

9

High Performance Liquid Chromatography (HPLC)

Waters, USA

10

Dissolution Apparatus

DISSOLAB, India

11

Aeroplex Spiral Jet Mill 50AS

Hosokova Alpine G, Augsburg, Germany

12

Ultra Turrax T25

Jahnke&Kunkel, Staufen, Germany

13

Master Sizer

Malvern, U.K

3.23 Solubility Studies

Approximately 50 mg of Camptothecin analog was added to 100 mL of purified water (n = 3) in a 250 mL stoppered conical flask, sample was vortexed to wet the drug substance, and then shaken at 200 rpm in rotary shaker water bath set at 37±0.5°C. After 24 hours of shaking the samples were centrifuged and the supernant was filtered using 0.45 µm filter. The filtered Samples were then analyzed for Camptothecin analog content by HPLC method.

3.23.1 pH Solubility Study

Different buffer media were prepared according to United States Pharmacopoeia. Approximately 50 mg of Camptothecin analog was added to 100 mL of various media (n = 3) in a stoppered conical flask. Samples were vortexed to wet the particles. The samples were shaken at 200 rpm in rotary shaker water bath set at 25±0.5°C. After 24 hours of shaking, samples were centrifuged and the supernant was filtered through 0.45µm filter. The filtered samples were analyzed for Camptothecin analog content by HPLC method.

3.24 Preparation of Nanosuspension on In-house Fabricated Equipment

The glass apparatus mimicking the media milling machine described in section 3.5 was used for preparation of nanosuspension. A predetermined quantity of a surfactant and polymeric stabilizer were dissolved in appropriate quantity of purified water. The stabilizer solution was then poured in a stainless steel vessel containing the drug substance. A Heidolf mixer (Model: RZR2051 Control, Rose Scientific Ltd., Alberta, Canada) operating at 500 rpm was used to wet and achieve a uniform, relatively coarse suspension. The solid content of the suspension was 5 % w/w. The milling media comprising 80 mL of 0.2 mm yttrium stabilized zirconium beads and additional water were added into the milling chamber. The total volume of slurry (drug substance + stabilizer + water) was 100 mL. The batch size for these development trials was 100 mL and the temperature of the suspension was maintained at 20 - 25°C during milling by circulating cold water. The milling beads were agitated using a Heidolph mixer operating at a speed of 1600 rpm. Following milling, the beads were filtered, and the nanosuspension was collected and stored at a temperature below 25°C until further processing. The concentration of surfactant and polymeric stabilizers used for production of Camptothecin analog nanosuspension is optimized. The stability of nanosuspension was evaluated upon storage at controlled room temperature conditions for a period up to two week.

3.25 Formulation Design

Formulation trials were designed to identify surfactant and polymer concentration to produce nanoparticle dispersion of the desired particle size. The concentration of surfactant and polymeric stabilizers used for production of Camptothecin analog nanosuspension is optimized. The stability of nanosuspension was evaluated upon storage at controlled room temperature conditions for a period up to two weeks.

3.26 Selection of Primary Stabilizer

For preparation of Camptothecin analog suspension, a suitable surfactant was chosen based on its ability to improve wetting property of the compound. For this purpose Sodium Lauryl Sulphate (SLS) and Tween 80 were selected as surfactants for stabilizing nanosuspension. To study the effect of surfactant concentration on particle size and suspension stability, concentration of 0.25% w/v, 0.50%w/v, 0.75%w/v and 1.0%w/v of SLS and Tween 80 were evaluated. The formula compositions with different surfactant concentrations evaluated for formula optimization are detailed in Tables 3.24 to 3.26.

3.27 Selection of Secondary Stabilizer

The primary stabilizer was chosen based on its ability to stabilize the drug particles in nanosuspension. Water soluble polymers such as Povidone (PVP K-30), Poloxamer 188 and Hypromellose 6 cps were evaluated for the selection of primary stabilizer. To study the effect of polymer concentrations on suspension stability concentration of 0.50% w/v, 1.00%w/v, 1.5%w/v and 2.0%w/v were evaluated. The formula compositions with different surfactant concentrations evaluated for formula optimization are detailed in Tables 3.27 to 3.29.

Table 3.24. SLS and HPMC Concentrations Evaluated for Formula Optimization

Formulation

Code

Formulation Composition (% w/v)

Batch size

(in mL)

CA

SLS

HPMC 6cps

NS-1

5.00

0.25

0.50

100

NS-2

5.00

0.50

0.50

100

NS-3

5.00

0.75

0.50

100

NS-4

5.00

1.00

0.50

100

NS-5

5.00

0.25

1.00

100

NS-6

5.00

0.50

1.00

100

NS-7

5.00

0.75

1.00

100

NS-8

5.00

1.00

1.00

100

NS-9

5.00

0.25

1.50

100

NS-10

5.00

0.50

1.50

100

NS-11

5.00

0.75

1.50

100

NS-12

5.00

1.00

1.50

100

NS-13

5.00

0.25

2.00

100

NS-14

5.00

0.50

2.00

100

NS-15

5.00

0.75

2.00

100

NS-16

5.00

1.00

2.00

100

Table 3.25. SLS and Poloxamer 188 Concentrations Evaluated for Formula Optimization

Formulation

Code

Formulation Composition (% w/v)

Batch size

(in mL)

CA

SLS

Poloxamer 188

NS-1

5.00

0.25

0.50

100

NS-2

5.00

0.50

0.50

100

NS-3

5.00

0.75

0.50

100

NS-4

5.00

1.00

0.50

100

NS-5

5.00

0.25

1.00

100

NS-6

5.00

0.50

1.00

100

NS-7

5.00

0.75

1.00

100

NS-8

5.00

1.00

1.00

100

NS-9

5.00

0.25

1.50

100

NS-10

5.00

0.50

1.50

100

NS-11

5.00

0.75

1.50

100

NS-12

5.00

1.00

1.50

100

NS-13

5.00

0.25

2.00

100

NS-14

5.00

0.50

2.00

100

NS-15

5.00

0.75

2.00

100

NS-16

5.00

1.00

2.00

100

Table 3.26. SLS and PVP (K-30) Concentrations Evaluated for Formula Optimization

Formulation

Code

Formulation Composition (% w/v)

Batch size

(in mL)

CA

SLS

PVP (K-30)

NS-1

5.00

0.25

0.50

100

NS-2

5.00

0.50

0.50

100

NS-3

5.00

0.75

0.50

100

NS-4

5.00

1.00

0.50

100

NS-5

5.00

0.25

1.00

100

NS-6

5.00

0.50

1.00

100

NS-7

5.00

0.75

1.00

100

NS-8

5.00

1.00

1.00

100

NS-9

5.00

0.25

1.50

100

NS-10

5.00

0.50

1.50

100

NS-11

5.00

0.75

1.50

100

NS-12

5.00

1.00

1.50

100

NS-13

5.00

0.25

2.00

100

NS-14

5.00

0.50

2.00

100

NS-15

5.00

0.75

2.00

100

NS-16

5.00

1.00

2.00

100

Table 3.27. Tween and HPMC Concentrations Evaluated for Formula Optimization

Formulation

Code

Formulation Composition (% w/v)

Batch size

(in mL)

CA

Tween-80

HPMC 6cps

NS-1

5.00

0.25

0.50

100

NS-2

5.00

0.50

0.50

100

NS-3

5.00

0.75

0.50

100

NS-4

5.00

1.00

0.50

100

NS-5

5.00

0.25

1.00

100

NS-6

5.00

0.50

1.00

100

NS-7

5.00

0.75

1.00

100

NS-8

5.00

1.00

1.00

100

NS-9

5.00

0.25

1.50

100

NS-10

5.00

0.50

1.50

100

NS-11

5.00

0.75

1.50

100

NS-12

5.00

1.00

1.50

100

NS-13

5.00

0.25

2.00

100

NS-14

5.00

0.50

2.00

100

NS-15

5.00

0.75

2.00

100

NS-16

5.00

1.00

2.00

100

Table 3.28. Tween-80 and Poloxamer 188 Concentrations Evaluated for Formula Optimization

Formulation

Code

Formulation Composition (% w/v)

Batch size

(in mL)

CA

Tween-80

Poloxamer 188

NS-1

5.00

0.25

0.50

100

NS-2

5.00

0.50

0.50

100

NS-3

5.00

0.75

0.50

100

NS-4

5.00

1.00

0.50

100

NS-5

5.00

0.25

1.00

100

NS-6

5.00

0.50

1.00

100

NS-7

5.00

0.75

1.00

100

NS-8

5.00

1.00

1.00

100

NS-9

5.00

0.25

1.50

100

NS-10

5.00

0.50

1.50

100

NS-11

5.00

0.75

1.50

100

NS-12

5.00

1.00

1.50

100

NS-13

5.00

0.25

2.00

100

NS-14

5.00

0.50

2.00

100

NS-15

5.00

0.75

2.00

100

NS-16

5.00

1.00

2.00

100

Table 3.29. Tween-80 and PVP (K-30) Concentrations Evaluated for Formula Optimization

Formulation

Code

Formulation Composition (% w/v)

Batch size

(in mL)

CA

Tween-80

PVP (K-30)

NS-1

5.00

0.25

0.50

100

NS-2

5.00

0.50

0.50

100

NS-3

5.00

0.75

0.50

100

NS-4

5.00

1.00

0.50

100

NS-5

5.00

0.25

1.00

100

NS-6

5.00

0.50

1.00

100

NS-7

5.00

0.75

1.00

100

NS-8

5.00

1.00

1.00

100

NS-9

5.00

0.25

1.50

100

NS-10

5.00

0.50

1.50

100

NS-11

5.00

0.75

1.50

100

NS-12

5.00

1.00

1.50

100

NS-13

5.00

0.25

2.00

100

NS-14

5.00

0.50

2.00

100

NS-15

5.00

0.75

2.00

100

NS-16

5.00

1.00

2.00

100

3.28 Evaluation of Nanosuspension

The coarse pre-dispersion was milled for 8 hours in the in-house fabricated bead mill. To study the effect of milling, samples were withdrawn at initial and end of the 8 hours milling cycle. Particle size analysis was performed using Laser Diffraction method (Master Sizer, Malvern instruments, U.K). The diameter of particles was calculated using volume distribution.

3.28.1 Method Development for Particle Size Analysis by Laser Diffraction Technique

3.28.2 Analysis Procedure

Following initial wetting of the sample in the dispersion unit the particle size may slowly decrease, due to the dispersion of loosely bound agglomerates under the action of the pump and stirrer. If the obscuration reduces rapidly at this point it may suggest that the material under test is soluble in the chosen dispersant. If this is case the obscuration drop will often be associated with an increase in the measured particle size, as the fines present in the sample will be dissolved most rapidly. If this is observed a different dispersant should be used. Following the initial stage of dispersion, ultrasound should be applied and the particle size followed in real-time. As the time of sonication is increased the particle size should reach a plateau where the particle size becomes stable - this represents the fully dispersed state. During sonication, if the particle size continues to reduce over time then it indicated particle breakup. Sonication may also cause agglomeration to occur - this would suggest that the dispersion is unstable, requiring the analyst to adjust the dispersion conditions. Finally, the particle size should be monitored with the ultrasound probe switched off once full dispersion is achieved. If the particle size remains stable when this occurs then the dispersion conditions are optimized. If the particle size increases the dispersion conditions will need to be adjusted, for example by moving to higher stabilizer concentrations or by using deionised water rather than tap water.

3.28.3 Specifications for Particle Size Measurement in Malvern Mastersizer

Particle size analysis for Camptothecin analog nanosuspension was done in Malvern Mastersizer, as per following specifications:

Dispersant : Water

Refractive Index of Dispersant : 1.33

Refractive Index of Drug : 1.50

Obscuration Range : 5-10

Pump speed : 2000 rpm

Sonication : No Sonication

Particle size and size distribution of the suspensions before and following milling were determined using a laser diffraction method, with a wet sampling system (Mastersizer S, Malvern Instruments, UK). The reported diameters of drug particle were calculated using volume distribution. The particle size obtained with different stabilizer compositions and their physical stability upon storage is evaluated. Based on particle size distributions obtained following milling and suspensions stability a suitable formula composition comprising primary stabilizer and secondary stabilizer were chosen for scale-up trials using the media-milling machine.

3.29 Scale-up of Manufacturing Process using Agitator Bead Mill

3.29.1 Preparation of Nanosuspension

Based on the results of milling using in-house glass milling apparatus the best formulation was selected and the process was scaled up using Netzsch bead mill (Model: Lab Star 1, Netzsch Mill, and Netzsch, Germany). The milling chamber was charged with milling media (yttrium stabilized zirconium beads) and coarse suspension. The drug concentration in the suspension was from 50 mg/ml. The process was performed in a re-circulation mode. After milling, suspension was filtered and stored at 25°C until further processing. In agitator bead mill, grinding media was accelerated in the grinding tank by an agitator shaft. The energy given to the media was passed onto the solid particles by virtue of collision and deceleration. Due to the tank being in a horizontal position, the special agitator gives an activation of grinding media over the whole tank content. Depending upon the temperature behaviour of the product the grinding chamber can be cooled. In order to achieve a good grinding result it is important to adjust size and material of construction for the grinding media. The parameters selected for preparing Camptothecin analog nanosuspension are summarized in table 3.30.

Table 3.30. Bead Mill Operating Parameters

Volume of grinding vessel

:

570ml

Media Volume

:

80 % of the grinding vessel

Media Type

:

0.2 mm Yttrium stabilized zirconium beads

Pump Speed

:

70 rpm

Mill Speed

:

2000 rpm

Coolant temperature

:

18°C

Grinding vessel coolant

:

Water

3.30 Conversion of Nanosuspensions into Solid Intermediates

3.30.1 Process Selection

Nanosuspension can be converted into solid intermediate for tabletting by following one of the processes listed below;

Spray drying

Lyophilization

Layering on to a suitable inert carrier using fluid bed coater.

Considering the process cost, duration and sensitivity of drug to high temperatures, spray granulation process was chosen for production of solid intermediates required for tabletting

3.30.2 Spray Granulation

This process involves layering of nanosuspension incorporating an inert carrier at low temperatures (40-60°C) using fluid bed processor. The spray granulation process is amenable to scale up for manufacturing larger batch sizes. The process is relatively simple and cost effective.

Selection of Suitable Carrier

Camptothecin analog exhibits poor aqueous solubility. Hydrophilic microenvironment in the tablet core would help in improving dissolution. So the criteria set forth for the selection of the suitable carrier were as follows;

Should be inert (non-reactive with the drug)

Hydrophilic and non-swellable

Water soluble to aid in recovery of drug nanoparticles

Available in granular form to aid flow and compressibility

Based on these criteria granular grade anhydrous Lactose (DCL-21) was selected for Spray granulation. The spray granulation process was used to obtain a solid intermediate granules containing re dispersible nanocrystals subsequent to the wet milling. The equipment used for this process is a Fluidized Bed Processor (Model: GPCG 1.1, Fluidized Bed Coater, Glatt GmpH, Germany), as presented in photograph 3.4.

Camptothecin analog micronized and nanosuspensions were coated over lactose anhydrous. The suspensions were sprayed into the fluid bed against the airflow by means of top spray. Drying takes place as the particles continue to move upwards in the airflow. Small droplets and low viscosity of the spray medium ensure that the distribution is uniform. The granules were dried as they moved upward, small droplets and low viscosity of the spray medium ensured that distribution was uniform resulting in granules with a narrow size distribution. The granules were then characterized for particle size distribution.

The process parameters for drug layering in Fluid bed processor are summarized in Table 3.31.

Table 3.31. Spray Granulation Conditions

Parameters Specifications

Inlet temperature

55 ± 5 °C

Bed Temperature

45 ± 5 °C

Exhaust Temperature

40 ± 5 °C

Pump Speed

4 ± 2 rpm

Atomization Air Pressure

1.2 bars

Spray Rate

15 g/min

Photograph 3.4. Fluid Bed Processor (Make: Pam Glatt)

3.31 Solid-State Characterization

3.31.1 Differential Scanning Calorimetry (DSC)

Thermal properties of the as-is drug and spray granulated nanoparticles were investigated using a Perkin-Elmer DSC-7 differential scanning calorimeter fitted with a TAC-7 thermal analysis controller and intracooler-2 cooling system (Perkin- Elmer Instruments, USA). For this analysis, about 5 mg of sample was placed in perforated aluminum sealed 50 µL pans and the heat runs for each sample was set from 40 to 300°C at 5°C/minute, an inert environment was maintained using nitrogen. The apparatus was calibrated using pure metals like Indium with known melting points and heat of fusion (rHfusion).

3.31.2 Powder X-ray Diffraction (PXRD)

PXRD diffractograms of as-is drug and spray granulated nanoparticles were recorded using a Panalytical Xpert Pro Diffractometer (PANalytical, The Netherlands) with a Cu line as the source of radiation. Standard runs using a 40 kV voltage, a 40mA current and a scanning rate of 0.02° min−1 over a 2θ range of 3 - 40° were used.

3.32 Optical Microscopic Examination

The optical microscopic examination of the suspension before and after milling was carried out by using a polarizing microscope (Make: Nikon-ECLIPSE-LV100POL) at 50X magnification to evaluate the stability of nanosuspension.

3.33 Scanning Electron Microscopy (SEM)

The surface morphology of the as-is drug and spray dried nanoparticles was examined by means of Hitachi S-520 SEM (Tokyo, Japan). The samples were fixed on an aluminum stub using adhesive tape and then the samples were made electrically conductive by coating with a thin layer of gold (~250 A°), for 35 seconds and at 35 W. The pictures were taken at an excitation voltage of 10 KV with required magnification (between 250x to 5000x).

3.34. ANALYTICAL METHODOLOGY

3.34.1 Analytical Method Development for Estimation of Camptothecin analog in Formulations

In-house analytical method was developed for estimation of Camptothecin analog content. The same method was adapted for the estimation of drug content in suspensions and tablet formulations.

3.34.2 Drug Content Estimation by HPLC

Apparatus

High Performance Liquid Chromatograph with UV Detector

Column Heater (Temperature range 25°C - 60 °C)

Chromatography Data Acquisition System

Analytical Balance: With a readability of 0.1 mg

Graduated Cylinder: 100 mL, 500 mL and 1000 mL, Class B or better

Volumetric Flasks: 5 mL, 20mL,25 mL, 50 mL and 100 mL, Class A

Glass Pipettes: 1 mL and 2 mL, Class A

Ultrasonic bath

Membrane Filters: 0.45 mm 47 mm Nylon for mobile phase filtration (UltiporÒ N66 Nylon, Part No. NX047100, Pall Life Sciences, India)

Chemicals

Camptothecin analog Working Standard: Of known purity

Potassium Dihydrogen Orthophosphate Monohydrate: AR grade (Make RANKEM)

Orthophosphoric Acid: AR grade (Make RANKEM)

Acetonitrile: HPLC grade (Make RANKEM)

Methanol: HPLC grade (Make RANKEM)

Water: HPLC grade or equivalent

3.34.2.1 Chromatographic Conditions

Buffer Preparation: Weigh 3.40g of potassium dihydrogen orthophosphate monohydrate in 1000 mL of water and mix well to dissolve completely. Adjust the pH to 6.8 ± 0.1 with dilute potassium hydroxide solution. Filter through 0.45 mm membrane filter.

Mobile Phase: Mix 680 mL of buffer and 320 mL of acetonitrile. Filter and degas by sonication.

Diluent : Mix 300 mL of methanol, 600 mL of 50 % orthophosphoric and 100 mL of acetonitrile

HPLC Column: Waters Symmetry C18, 250 x 4.6 mm, 5 µ (Part No. WAT054275)

Column Temperature: 25°C

Flow Rate: 1.0 mL per minute

Wavelength: 257 nm

Injection Volume: 10 mL

Run Time: 15 minutes

3.34.2.2 Standard Preparation

Weigh accurately 30 mg of Camptothecin analog working standard into a 50 mL volumetric flask. Dissolve and dilute to volume with diluent and mix. Pipet 2 mL of above solution into a 20 mL volumetric flask and dilute to volume with diluent and mix. Filter through a 0.45µ filter discarding the first 2 mL.

3.34.2.3 Sample Preparation

Weigh accurately amount equivalent 30 mg of Camptothecin analog in to a 50 mL volumetric flasks. Add about 40 mL of diluent. Sonicate in an ultrasonic bath for 20 minutes with intermittent shaking. Dilute to volume with diluent and mix well. Filter the solution through a 0.45m disk filter (Part No: ZWGSFN 13045). Pipet 2 mL of filtrate into a 20 mL volumetric flask. Dilute to volume with diluent and mix well. Filter through a 0.45µ filter discarding the first 2 mL.

3.34.2.4 Preparation of System Suitability Solution

Weigh accurately 30 mg of Camptothecin analog working standard into a 50 mL volumetric flask. Dissolve and dilute to volume with diluent. Dilute 2 mL of the resulting solution to 20 mL with diluents and mix. Use this solution as the resolution test preparation.

3.34.2.5 Calculations

Calculate the amount of Camptothecin analog present in mg/capsule using following formula.

mg/unit dose =

Where,

A is the peak area of Camptothecin analog peak in sample preparation

B is peak area of Camptothecin analog in standard preparation

Sw is the weight of Camptothecin analog working standard in mg

Tw is the weight of sample taken in mg

P is the purity of Camptothecin analog working standard in percent

AW is the Average weight in mg

3.34.3 Saturation Solubility of Drug Nanoparticles

Saturation solubility evaluations were carried out in buffer media at different pH conditions using a shake flask method. In this method excess amount (100 mg/mL) of drug substance ("as-is" and dried suspension containing microparticles or nanoparticles) was added to 25 mL of each buffer maintained at 37°C and shaken for a period up to 24 hours. The samples were filtered using 0.10µm pore size Millex-VV PDVF filters (Millipore Corporation, USA) prior to analysis. Samples were diluted and concentrations were determined using an HPLC method.

3.35 Tablet Preparation

3.35.1 Selection of Tabletting Excipients

Super disintegrants like Sodium starch glycollate, Corn starch and crospovidone were selected for the evaluation based on tablet disintegration time.

Magnesium stearate was chosen as lubricating aid.

Anhydrous colloidal silicon dioxide was selected as glidant to improve the blend flow.

Spray granules containing drug nanoparticles was mixed with extra granular excipients in a double cone blender, for 10 minutes. The blend was then processed into tablets using a mini compression machine (Model: Mini-II B, Rimek, India) fitted with 6.5 mm round, standard concave type D tooling. The physical properties of tablets, hardness, friability and disintegration time, were measured. The tablet hardness was measured using a hardness tester (Model: 8M, Dr Schleuniger Pharmatron, USA). Each hardness value reported is an average of ten measurements. The disintegration time was measured in purified water at 37 ± 0.5°C, using a disintegration tester (Model: ED2L, Electrolab, India), using sintered disks. The disintegration time reported is an average of six measurements. Tablet friability was calculated as the percentage weight loss of 20 tablets after 100 rotations using a friabilator (Model: EF2, Electrolab, India).

3.35.2 Preparation of Tablets using Processed Camptothecin analog Granules

The granules containing drug nanoparticles of Camptothecin analog were blended with extra-granular excipients using a double cone blender. The blend was subsequently compressed into tablets at the desired strength. Before compressing the blend, the bulk properties of blend was measured.

3.36 Evaluation of Blend Physical Properties

3.36.1 Angle of repose

The angle of repose for the each formulation was determined by the funnel method. The fixed amount of blend was allowed to flow out of the funnel orifice fixed at a height of 2 cm from the surface on a plane paper kept on the horizontal platform. The gradual addition of the blend from the funnel mouth forms a pile at the surface this is continued until the pile touches the stem tip of the funnel. A rough circle is drawn around the pile base and the radius of the powder cone was measured. Angle of repose was then calculated with the use of the following formula:

tan θ = h / r

Where, θ = angle of repose

h = height of the pile

r = average radius of the powder cone

3.36.2 Density

The bulk density of spray granulated powder was determined by placing a known amount of powder (approximately 5 g) under gravity into a calibrated measuring cylinder and recording the volume occupied by the powder.

The tapped density of the spray granulated powder was determined by measuring the volume occupied by the powder following tapping using a tamping volumeter (Tapped Density Assessor: Copley Scientific Ltd., Nottingham, UK) until no further change in the powder volume was observed.

3.36.3 Carr's Index:

The Carr's Index value gives an indication of powder flow; a value less than 25% indicates a fluid powder (good flow) whereas, a value greater than 25% indicates a cohesive powder with poor flow. Carr's Index values for spray granulated powder were derived from bulk density and tapped density data, according to the Equation 2.

3.36.4 Hausner ratio

A Hausner ratio value less than 1.20 is indicative of good flow whereas, a value > 1.5 indicates poor flow. Hausner ratio was calculated from tapped and bulk density using the equation 3.

3.37 Evaluation of Tablet Physical Properties

The following physical properties of tablets incorporating nanoparticle Camptothecin analog were evaluated

3.37.1 Average Weight: Twenty (20) tablets were weighed on an analytical balance and average weight was calculated.

3.37.2 Tablet Thickness: The thickness in millimeters (mm) was measured individually for 10 pre-weighed tablets by using a digimatic digital outside micrometer (Mitutoyo, Japan).

3.37.3 Tablet Hardness: The tablet hardness was measured using a hardness tester

(Model: 8M, Dr Schleuniger Pharmatron, USA). Each hardness value reported is an average of ten measurements. Crushing strength was recorded in kP.

Friability: Tablet friability was calculated as the percentage weight loss of about 6.5 g of tablets after 100 rotations in a friabilator (Model: EF2, Electrolab, India).

Disintegration time: The disintegration time was measured in purified water at 37 ± 0.5°C, using a disintegration tester (Model: ED2L, Electrolab, India), using sintered disks. The disintegration time reported is an average of six measurements.

Moisture Content: The moisture content of tablets is determined by using Karl Fisher auto titrator ( Metrohm 794 basic titrino, Switzerland).

3.37.7 Dissolution Studies

From the solubility study it was found that the solubility of Camptothecin was low in 0.1 N HCl (pH 1.2) and it also showed clear discrimination between micronized and nanonized drug solubility. Therefore 0.1N HCl was chosen as the discriminating dissolution medium based on its ability to differentiate the impact of changes in composition and particle size on dissolution rate. A USP dissolution apparatus (Model: DISSO 2000, Labindia, India) type II (paddle method) with a paddle operating at 50 rpm was used for dissolution studies. All dissolution tests were carried out on an equivalent of 25 mg of CA (in suspension and powder state) and tablets. The volume and temperature of the dissolution medium were 500 mL and 37°C, respectively. Samples were withdrawn at predetermined time intervals and assayed using an HPLC method.

3.37.7.1 Apparatus

Dissolution Apparatus: USP Apparatus II (paddle)

High Performance Liquid Chromatograph with UV Detector

Column Heater (Temperature range 25°C - 60 °C)

Chromatography Data Acquisition System

Analytical Balance: With a readability of 0.1 mg

Graduated Cylinder: 20 mL, 100 mL, 500 mL and 1000 mL, Class B or better

Beaker: 10 L

Volumetric Flasks: 20 mL, 50 mL and 100 mL, Class A

Pipettes: 1 mL 2 mL, 4 mL and 5 mL, Class A

Membrane Filters: 143 mm, 0.45 mm Nylon (MDI, Part No. 4B192945) for dissolution media filtration.

Ultrasonic bath

3.37.7.2 Dissolution Conditions

RPM: 50

Medium: 0.1 N HCl, 500 mL

Temperature: 37 ± 0.5°C

Sampling Time Point: 45 minutes

3.37.7.3 Calculations

Calculate the percent Camptothecin analog released as a percent of labeled amount using the following equation:

% Released =

Where,

A is the peak area of Camptothecin analog sample preparation

B is the peak area of Camptothecin analog standard preparation

Sw is the weight of Camptothecin analog working standard in mg

LC is label claim

P is the percent purity of Camptothecin analog working standard

3.38 In-Vivo Pharmacokinetic Study

Male Wistar rats weighing 230 ± 20 g were fasted for 12 - 14 hours prior to dosing but allowed free access to water. Eight rats were assigned to two groups and they received suspensions comprising either spray granulated drug microparticles or drug nanoparticles, respectively at a dose level of 10 mg/kg. Rodent feed was returned 3 hours post dosing. Blood samples were collected from the orbital sinus at pre-determined intervals (0, 0.5, 1, 3, 5, 8, 10 and 24 hours) post-dosing. Plasma was separated immediately by centrifugation (13,000 RPM for 2 minutes at 4°C) and stored in polypropylene vials below -10°C until analysis. The animal experiments were carried out in accordance with the guidelines provided by the Institutional Animal Care and Ethics Committee.

3.38.1 Bioanalysis by HPLC

Apparatus

High Performance Liquid chromatography Analyst® software

Analytical Balance: With a readability of 0.1 mg

Pipettes: 100 µL, 200 µL and 1 mL

Ultrasonic bath

Tarsons vortex mixer

Membrane Filters: 0.45 mm 47 mm Nylon for mobile phase filtration (UltiporÒ N66 Nylon, Part No. NX047100, Pall Life Sciences, India)

Chemicals

DRF-1042 and DRF-1152 Working Standard: Of known purity

Triethyl amine: AR grade

Methanol: HPLC grade (Make RANKEM)

Acetonitrile: HPLC grade (Make RANKEM)

Water: HPLC grade or equivalent

3.38.2 Chromatographic Conditions

Buffer Preparation: 1 mL of Triethyl amine was pipetted in 100 mL of water and mixed well. Filter through 0.45 mm membrane filter.

Mobile Phase: Mix Triethyl amine (1 % v/v) and acetonitrile in the ratio of 80:20. Filter and degas by sonication.

HPLC Column: Supelcosil®-LC318, 250 X 4.6mm 5 µ, Stainless steel column

Flow Rate: 1.0 mL per minute

Detection: Fluoresence

Injection Volume: 20 mL

Run Time: 12.5 minutes

3.38.3 Sample Preparation

A simple and sensitive high-performance liquid chromatography (HPLC) method has been developed for the determination of Camptothecin analog, in human plasma. The sample preparation was a simple deproteinization with acidified cold methanol yielding almost 100% recovery of Camptothecin analog. An isocratic reverse-phase HPLC separation was developed on a Supelcosil-LC318 column (250 Ã- 4.6 mm, 5 µm) with mobile phase consisting of 1% v/v triethylamine acetate, pH 5.5 and acetonitrile (80:20, v/v) at a flow rate of 1.0 mL/min. The eluate was monitored with a fluorescence detector set at excitation and emission wavelengths of 370 and 430 nm, respectively. An aliquot of 100µL plasma (stored at 8° C) was precipitated with 4000µL of acidified methanol for the estimation Camptothecin analog. Following mixing on a cyclo mixer for 2 minutes and centrigated for 4 minutes at 12,800 rpm in a centrifuge, clear supernant was separated into a 300 µL auto-sampler vial and 20µL of this was injected on to analytical column for High Pressure Liquid Chromatography analysis. Standard curves were obtained from linear square regression analysis of drug/internal standard peak area ratio as a function of plasma concentration versus time data was analyzed, and the oral pharmacokinetic data were developed.

3.39 Stability Studies

The physical stability and dissolution characteristics of tablets formulation incorporating drug nanoparticles evaluated upon storage at accelerated (40°C/75% RH) and room temperature conditions (25°C/60% RH) for a period up to 3-months. The physical properties such as tablet description, hardness and disintegration on storage were evaluated along with dissolution characteristics.

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