FORMULATION AND CHARACTERIZATION OF PARENTERAL

DOSAGE FORM CONTAINING AN ANTINEOPLASTIC AGENT BY

LYOPHILIZATION TECHNIQUE

M. Pharm Dissertation Protocol

Submitted to

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA

BENGALURU

By

Mr. RIZWAN AHMED

M.PHARM, PART-I

Under the guidance of

Dr. Dinesh B.S. M.Pharm, Ph.D

Professor

DEPARTMENT OF PHARMACEUTICS

PRIYADARSHINI COLLEGE OF PHARMACYKORATAGERE, TUMKUR

2012-2013

ANNEXURE – II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the Candidate and Address

RIZWAN AHMED

PRESENT ADDRESS:M.PHARM 1ST YEAR, DEPT OF PHARMACEUTICSPRIYADARSHINI COLLEGE OF PHARMACY,KORATGERE, TUMKUR

PERMANENT ADDRESS: S/O M D HUSSAINNEAR MASJID, MAIN ROAD,POST: KARDAKALTQ:LINGSUGURDIST: RAICHUR

2. Name of the Institute PRIYADARSHINICOLLEGEOF PHARMACY,TUMKUR.

3. Course of the Study and Subject M.PHARMACY IN PHARMACEUTICS

4. Date of Admission to the Course 05 JUL 2012

5. Title of Topic

“FORMULATION AND CHARACTERIZATION OF PARENTERAL DOSAGE FORM CONTAINING AN ANTINEOPLASTIC AGENT BY LYOPHILIZATION TECHNIQUE”

1

6. BRIEF RESUME FOR INTENDED WORK

6.1 Need for the Study

Antineoplastic agents are a group of specialized drugs used primarily to treat cancer.

“Cancer is a disease of uncontrolled cell division, invasion and metastasis. It is generally

considered to be due to the clonal expansion of a single neoplastic cell. However there may

be additional somatic leading to heterogeneous cell population. It is one of the major

causes of death in the developed nations: one in three people will be diagnosed with cancer

during their lifetime.1

The anticancer drugs either kill cancer cells or modify their growth. However, selectivity

of majority of drugs is limited and they are one of the most toxic drugs used in therapy.

Treatment of malignant diseases with drugs is a rather recent development, started after

1940 when nitrogen mustard was used, but progress has been rapid, both in revealing

pathobiology of the diseases and discovery of new drugs. In addition, attempts have been

made to define optimal combinations, treatment strategies and patient support measures.

Cancer chemotherapy is now of established value and a highly specialized field.

In addition to their prominent role in leukemia and lymphoma, drugs are used in

conjunction with surgery, radiotherapy and immunotherapy in the combined modality

approach for many solid tumors, especially metastatic chemotherapy is the initial treatment

of choice in malignant diseases, drugs are used:

To cure or prolonged remission.

To palliation (alleviation of symptoms).

Adjuvant chemotherapy: Drugs are used to mop up any residual malignant

cells (micro metastases) after surgery or radiotherapy.

2

The drugs which are commonly used in treatment of cancer include Methotrexate,

Gemcitabine, Cisplatin, Carboplatin, Azacitidine, Pemetrexed, Bleomycin, Decitabine,

Epirubicine, Mitoxantrone, Ifosfamide, Mitomycin, Dactinomycin and plant alkaloids such

as Vincristine, Vinblastine, Topotecan, Paclitaxel, Docetaxel, Etoposide etc. The main

side effect of chemotherapy includes nausea and vomiting, hair loss, anemia etc. 2

The typical route of administration of anticancer drugs are oral (tablets and capsules) and

parenteral as intramuscular, Subcutaneous and intravenous. Often a pharmaceutical

product may be susceptible to physical and chemical degradation when stored as a ready-

to-use solution. The goal of the formulations scientist is to identify the right formulation

conditions, the right excipients in optimal quantities, and the right dosage form to

maximize stability, biological activity, safety, and marketability of a particular product. In

order to overcome the above listed draw backs, the solution dosage form meant especially

for parenteral use can be formulated as lyophilized products especially with antineoplastic

agents, formulating them into lyophilized product may improve their stability in the dry

state and thus improve shelf-life3.

Lyophilization is a process more commonly known as freeze-drying. The word is derived

from Greek and it means "made solvent-loving". The process of freeze-drying is used often

to stabilize delicate and heat-sensitive materials and compounds such as: proteins,

peptides, biologicals, vaccines, enzymes, microorganisms, monoclonal antibodies,

parenteral, pharmaceuticals, blood fractions, oglionucleotides, blood plasma and

diagnostics blood fractions etc4.

In the formulation of lyophilized products the typically used excipients are the

cryoprotectant and lyoproctant. Usually used cryoprotectant and lyoproctant are trehalose,

3

glucose, mannitol, sucrose and lactose in the concentration of 0.2-5%. These agents are

intended to prevent freezing stress and drying stress on the drug molecule5.

The primary mechanism that allows for freeze-drying is sublimation, whereby ice is

directly converted to water vapor, without passing through the intermediary stage of a

liquid. Rather than through heating, this is done by removal of solvent under pressure so

that the ice boils without melting.

The result is a sample whose structure is largely preserved, which can be stored at room

temperature and pressure. The process consists of three separate, unique, and

interdependent processes; freezing, primary drying (sublimation phase), and secondary

drying (desorption phase)6.

Thus the lyophilization of antineoplastic agents will result in products with following

advantages:

Ease of processing a liquid, which simplifies aseptic handling.

Removal of water without excessive heating of the product.

Enhanced product stability in a dry state.

Rapid and easy dissolution of reconstituted product.

Longer shelf life.

Less cumbersome storage, handling and distribution requirements.

Lower shipping costs because of reduced weight, especially for bulk applications7.

Decitabine (brand name Dacogen®) and pemetrexed (brand name Almita®) are anticancer

drugs belongs to antimetabolites classification available in the market as lyophilized

formulations. Lyophilization process protocols of individual drugs needs to be designed

4

and developed as and when changes are made in formulation ingredients (especially

cryoprotectants). These protocols are to be designed based upon physiochemical and

thermal characterization of individual drugs.

In the present study research work is aimed to develop a suitable injectable dosage form

for the drug candidate by working on designing of suitable lyophilization cycle by varying

the total cycle time, freezing, primary drying and secondary drying time, ramp time,

holding time and keeping all the quantities of all the active pharmaceutical ingredients

constant. Additional attempts will be made to develop scale up studies to reduce cycle time

to meet the industrial requirement. Hence there is a need to develop antineoplastic agents

such as Decitabine, Pemetrexed disodium, Mitomycin, Doxorubicin, Epirubicin,

Oxaliplatin, Melphalan etc. into lyophilized product by designing suitable lyophilization

cycle.

6.2 Review of Literature:

1. Jeffrey Cummings et al., have studied Pharmacokinetics and metabolism of

Mitomycin C (MMC) in NMRI mice bearing MAC 16 colon adenocarcinoma after

direct intratumoural injection of either 500µg free MMC or the same dose

incorporated in albumin microspheres. Microspheres produced a tumor

pharmacokinetic profile of steady state drug levels, avoiding the much higher early

peak (20.5 µg/tumor vs 98.9 µg/tumor) and lower trough of free MMC, and reducing

significantly the levels of drug reaching the systemic circulation (AUC 1.8 µg/ml x

hr for microspheres vs 6.8µg/ml x hr for free drug). 2, 7-Diaminomitosene (2, 7-

DM), a key intermediate in MMC quinone bioreduction, was used as an indicator of

drug metabolic activation in tumor tissue. Peak levels were lo-fold higher (11.2

µg/tumor vs 1.1 µg/tumor) and area under the curve 5-fold higher after free drug.

5

Even taking into account differences in tumor pharmacokinetic profiles of the parent

drug, microspheres actively inhibited 2, 7-DM formation 3-fold. However, the

microspheres generated a completely different pattern of drug metabolism where

four previously uncharacterized mitosane metabolites and elevated levels of cis and

trans 1-hydroxy 2, 7-diaminomitosene were detected. Despite similar parent drug

exposure in tumors, free drug was significantly more active (P < 0.05, Student’s t-

test) against MAC 16. These results suggest that formation of 2, 7-DM’ correlates

more closely with antitumor activity than sustained parent drug levels or appearance

of other key metabolites. Potentially, they provide the first direct evidence for an in

vivo mechanism of action dependent on bioreductive activation and formation of 2,

7-DM8.

2. Klaus Langer et al., have concluded on Freeze drying is a suitable technique to

improve the long-term storage stability of colloidal drug carrier systems such as

nanoparticles. Aim of this study was to systematically evaluate excipients for the

freeze drying and long-term stability of albumin-based nanoparticles. In our study,

nanoparticles made of human serum albumin (HSA) were freeze dried in the

presence of different cryoprotective agents and after reconstitution were evaluated

with regard to their physico-chemical characteristics. Empty, doxorubicin-loaded,

and PEGylated nanoparticles were prepared and were freeze dried in the presence

of different concentrations of sucrose, trehalose, and mannitol, respectively. The

samples were physicochemically characterized with regard to lyophilisate

appearance, particle size, and polydispersity using photon correlation spectroscopy.

For evaluation of long-term stability, the samples were stored at 2–8, 25, and 40ºC

over predetermined time intervals. In the absence of cryoprotectants, particle

growth was observed in all freeze-dried formulations. In the presence of sucrose,

6

mannitol, and trehalose aggregation of HSA nanoparticles during the freeze-drying

procedure was prevented. Although all of the excipients were identified to be

suitable stabilizers for freeze drying of HSA nanoparticles, sucrose and trehalose

were superior to mannitol, especially with regard to the long-term storage stability

results9.

3. Neeraj Kumar et al., have focused on lyophilization process, physicochemical

characterization and long-term storage stability studies of lyophilized doxorubicin-

loaded (PEG) 3–PLA nanopolymersomes. Nanopolymersomes were prepared by

nanoprecipitation method using (PEG) 3–PLA copolymer and lyophilized in the

presence of different lyoprotectants and evaluated for physicochemical properties.

The lyophilized product was studied for long-term stability at 2–8ºC, 25ºC/65%

RH and 40ºC/75% RH over predetermined periods and evaluated for changes in

physicochemical properties. In the absence of lyoprotectants, product was

collapsed with no cake formation. In the presence of inulin, mannitol, gelatin,

polyvinyl alcohol and glycine, nanopolymersome formulations demonstrated an

intact cake occupying same volume as original frozen mass. The residual moisture

content was below 2.5% w/w. The time of reconstitution was instantaneous

following addition of water without any manual shaking. However, inulin found to

be superior to the rest of lyoprotectants for overall lyophilisate physicochemical

properties. Drug loaded nanopolymersome were physically stable and no effect was

observed in terms of physicochemical properties following one year at 2–8ºC. In

conclusion, the results suggest that doxorubicin loaded nanopolymersomes could

be lyophilized using inulin 5% w/v without losing its physicochemical properties

and can be stored at 2–8ºC with a provisional shelf life of more than a year10.

7

4. Valentino J. Stella et al., have evaluated the potential of using (SBE)7m-β-CD and

HP- β -CD as enabling excipients to improve on the current melphalan injectable

formulation. Melphalan is an anti-neoplastic agent formulated for parenteral use as

a sterile, non-pyrogenic, freeze-dried powder. It is marketed by Glaxo-Wellcome as

ALKERAN® for Injection (Alkeran). A major concern with melphalan therapy,

other than its intrinsic cytotoxicity and biocompatibility, arises from its marginal

aqueous solubility and chemical stability; thus, co-solvents are used in the current

two-vial formulation. Because of the two-vial system, the product is also

inconvenient to use. Two approaches to improve melphalan’s formulation utilizing

cyclodextrins, including the use of aqueous (SBE)7m- β -CD or HP- β -CD solutions

as the reconstitution diluents, and: or the use of (SBE)7m- β -CD as a freeze-drying

excipient in a melphalan formulation, are presented. Results showed that, when the

cyclodextrins were used as diluents, the use of organic co-solvents can be

eliminated and the shelf-life of the reconstituted melphalan greatly enhanced.

When the freeze-dried melphalan:(SBE)7m- β -CD formulation was prepared, the

formulation was found to be stable; and a simplified one-vial delivery system was

achieved. In conclusion, the parenterally safe b-cyclodextrins derivatives can

provide promising alternatives and improved formulations for melphalan injectable

and perhaps similar problematic drugs11.

5. Arvind K. Bansal et al., have reviewed on excipients used in various lyophilized

formulations of small molecules. The role of excipients such as bulking agents,

buffering agents, tonicity modifiers, antimicrobial agents, surfactants and co-

solvents has been discussed. Additionally, a decision making process for their

8

incorporation into the formulation matrix has been proposed. A list of ingredients

used in lyophilized formulations marketed in USA has been created based on a

survey of the Physician Desk Reference (PDR) and the Handbook on Injectable

Drugs. Information on the recommended quantities of various excipients has also

been provided, based on the details given in the Inactive Ingredient Guide (IIG)12.

6.3 OBJECTIVES OF THE STUDY :

To achieve the ultimate goal of developing a lyophilized formulation of antineoplastic

agents to improve long term stability, the present work is designed to address the following

objectives:

Preformulation studies on the anticancer drug substance

Formulation of the injectable dosage form

Lyophilization of the injectable dosage form

Development and optimization of lyo-cycle

Evaluation of the lyophilized product as per official and In-house specification

Characterization for drug excipient interaction

Accelerated stability studies

Documentation of resultant data.

9

7.

7.0 MATERIALS AND METHODS

Materials:

Drugs: Any one anticancer drug such as Decitabine, Pemetrexed disodium, Mitomycin,

Doxorubicin, Epirubicin, Oxaliplatin and Melphalan will be chosen at the time of study.

Excipients: Mannitol, lactose, povidone etc.

Method:

Lyophilization technology

7.1. Source of Data

Review of Literature from

a) Journals such as,

1) Drug Development & Industrial Pharmacy

2) Pharmaceutical Development & Technology

3) Journal of Pharmaceuticals Science Technology

4) International Journal of Pharmaceutics

5) Cancer chemotherapy & Pharmacology

6) Pharmaceutical research

7) PDA Journal of Pharmaceutical Science Technology

8) European Journal of Pharmaceutics and Biopharmaceutics

b) Web sites such as

1) drugs.com (www.drugs.com)

2) pubmed (www.ncbi.nlm.nih.gov/pubmed)

3) sciencedirect (www.sciencedirect.com)

10

7.2 Method of Data Collection :

The physiochemical properties of the drug will be collected from drug information center,

various standard books, journals, websites and other sources like research literature data

bases.

The methodology to be followed to achieve the objectives are outlined as follows:

Preformulation studies on drug like nature, color, solubility etc. will be carried out.

Selection of suitable excipients based on compatibility results.

Lyophilization of the injectable dosage form.

Development and optimization of lyo-cycle.

Evaluation of the lyophilized product by different parameters like related

substance, pH, assay, water content, reconstitutional time, reconstitutional stability

etc.

Stability studies will be carried out for developed products as per the ICH

guidelines.

7.3. Does the study require any investigations or investigations to be conducted on

patients or Other humans or animals?

“ NO ”

7.4. Has ethical clearance been obtained from your institution in case of 7.3?

“ Not Applicable”

11

8.

LIST OF REFERENCES:-

1. www.drugs.com/cancer.html (Date of Access 24/11/2012)

2. www.answers.com/topic/antineoplastic-agents (Date of Access 19/11/2012)

3. Frank Kofi Bedu-Addo. 2004. Lyophilization. Pharmaceutical Technology: 10-18.

4. Rey L, May JC. Freeze Drying/Lyophilization of Pharmaceuticals and Biological

Products.2nd Ed. New York. Basel: Marcel Dekker, Inc:2004:1-4

5. Abdelwahed W, Degobert G, Stainmesse S, Fessi H. Freeze Drying of

Nanoparticles Formulation, Process and Storage Considerations. Adv Drug Deliv

Rev.2006; 58:1688-713.

6. Xiaolin (Charlie) Tang and Michael J. Pikal Design of Freeze-Drying Processes

for Pharmaceuticals: Practical Advice. Pharm res. 2004;21(2):191-200.

7. http://www.fda.gov/ICECI/Inspections/InspectionGuides/ucm074909.htm (Date of

Access 10/11/2012)

8. Jeffrey Cummings, Lucy Allan, John F. Smith Encapsulation Of Mitomycin C In

Albumin Microspheres Markedly Alters Pharmacokinetics, Drug Quinone

Reduction In Tumor Tissue And Antitumor Activity Implications For The Drug’s

In Vivo Mechanism Of Action. Biochemical Pharmacology. 1994;47(8):1345-

1956.

9. Marion G. Anhorn, Hanns-Christian Mahler, Klaus Langer. Freeze Drying Of

Human Serum Albumin (HSA) Nanoparticles with Different Excipients. Int. J

Pharm. 2008;363:162-9.

10. Wubeante Yenet Ayen, Neeraj Kumar. A Systematic Study on Lyophilization

Process of Polymersomes for Long-Term Storage Using Doxorubicin-Loaded

(PEG) 3–Pla Nanopolymersomes. European J Pharm Sci. 2012;46:405-14.

11. David Q. Ma, Roger A. Rajewski, Valentino J. Stella. New Injectable Melphalan

12

Formulations Utilizing (SBE)7m-Β-Cd Or Hp-Β-Cd. Int. J Pharm. 1999;189:227-

34.

12. Ankit Baheti, Lokesh Kumar, Arvind K. Bansal. Excipients Used In

Lyophilization Of Small Molecules J. Excipients And Food Chem.2010;1(1):41-

54.

13

9. Signature of the Candidate

(RIZWAN AHMED)

10. Remarks of the Guide: The topic selected for dissertation is satisfactory and feasible

11. 11.1 GuideDr.Dinesh B.S. M.Pharm, Ph.D

Professor & H.O.D,DEPARTMENT OF PHARMACEUTICSPRIYADARSHINI COLLEGE OF PHARMACYKORATAGERE, TUMKUR.

11.2 Signature of Guide:

(Dr.Dinesh B.S.)

11.3 Head of the Department:

Dr. Dinesh B.S. M.Pharm, Ph.D

Professor & H.O.D,DEPARTMENT OF PHARMACEUTICSPRIYADARSHINI COLLEGE OF PHARMACYKORATAGERE, TUMKUR.

11.4 Signature of HOD:

(Dr. Dinesh B.S.)

12. 12.1 Remark of the Principal:

The above mentioned information is found correct and I recommend the same for approval.

12.2 Principal

P.S.MINHAS PRINCIPALPRIYADARSHINI COLLEGE OF PHARMACYTUMKUR

12.Signature of the Principal

(P.S.MINHAS)

14