Download formulation development and stability studies of vitamin D3

“FORMULATION DEVELOPMENT AND STABILITY STUDIES OF VITAMIN D3:
COMPARISON AMONG DIFFERENT PREPARATION METHODS”
DISSERTATION PROTOCOL
SUBMITTED TO
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
KARNATAKA, BANGALORE.
BY
VENKATRAO MANNAM
M.PHARM, PART-I
DEPARTMENT OF PHARMACEUTICS
NARGUND COLLEGE OF PHARMACY
BANGALORE-85
(2010-2012)
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES,
KARNATAKA, BANGALORE
ANNEXURE-II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR P.G DISSERTATION
1.
NAME OF THE CANDIDATE
AND ADDRESS
VENKATRAO MANNAM
NARGUND COLLEGE OF PHARMACY,
DATTATREYA NAGAR, II MAIN,
100 FEET RING ROAD,
BSK III STAGE,
BANGALORE-85,
KARNATAKA.
2.
NAME OF THE INSTITUTION
NARGUND COLLEGE OF PHARMACY,
DATTATREYA NAGAR, II MAIN,
100 FEET RING ROAD,
BSK III STAGE,
BANGLORE-85,
KARNATAKA.
3.
COURSE OF STUDY AND
SUBJECT
MASTER OF PHARMACY IN
PHARMACEUTICS
4.
DATE OF ADMISSION OF
COURSE
3rd APRIL 2010.
5.
TITLE OF TOPIC
“ FORMULATION DEVELOPMENT AND
STABILITY STUDIES OF VITAMIN D3:
COMPARISON AMONG DIFFERENT
PREPARATION METHODS ”
6.
BRIEF RESUME OF THE INTENDED WORK:
6.1 NEED FOR THE STUDY:
Vitamin D is also called the Antirachitic vitamin, as it counteracts rickets, breakingdown of the mineral substance in fully grown bones (Osteodistrophy), bone and joint
deformations, growth disorders, spontaneous brittleness of bones and deficient eggshell
strength.
The term vitamin D actually refers to a pair of biologically inactive precursors of a
critical micronutrient. They are vitamin D3, also known as Choleacalciferol, and vitamin
D2 also known as Ergocalciferol. Choleacalciferol (D3) is produced in the skin by a
photoreaction on exposure to UV light from the sun (wavelength 290 to 320nm).
Ergocalciferol (D2) is produced in plants and enters human diet through consumption of
plant sources. D2 and D3 forms are inactive as such but once present in the circulation,
enter the liver and kidneys where they are hydroxylated to form active forms of D 2 and
D3. D2 has shorter half life than D3.
Vitamin D3 plays an important part in human and animal nutrition. It regulates the
absorption, metabolism and excretion of calcium and phosphorus from the intestine and
kidneys. Further it controls the incorporation of calcium and phosphorus in the skeleton.
It is common to this fat-soluble agent that, in its pure form, it can be handled only with
difficulty or not at all, because it is oxidation-sensitive substance. Furthermore, a fine
dispersion of the agent is advantageous for optimal absorbability and thus
bioavailability. These substances are therefore often supplied in the form of emulsions
or in the dry powders or as solution in physiologically tolerated oil or embedded in a
fine dispersion in a protective colloid. All of these methods have common drawbacks
such as rapid deformation and disintegration due to heat or physical force and also due
to aqueous solution employed. Decreased content uniformity increased running cost as
decreased productive are the other disadvantages. Recently soft capsules containing a
vegetable oil solution of active vitamins D3 have also developed. However, soft capsules
have their particular drawbacks such as that are limited in their form and that the process
of their preparation is complicated.
So an attempt will be made to produce an efficient formulation of granule or powder
forms which will be stable throughout its shelf life, dispersible in aqueous solutions and
overcome the above mentioned draw backs.
Powdering or granulating of vitamin D3 can be accomplished by any or all of the
following methods:

By Micro Encapsulation:
 In this procedure the desired polymer is dissolved in a suitable organic solvent
followed by the addition of the desired substances to be encapsulated. The
substance is either dissolved or dispersed in the organic solvent. The resulting
organic solution is dispersed in an aqueous phase to obtain oil in water emulsion
where oily micro particles are dispersed in the aqueous phase. Upon complete
removal of the solvent from the micro particles the microcapsules are formed.
This method will be modified if required or any suitable method will be
employed.

By dry emulsion:
 A process for preparing a dry powder composition containing vitamins D3 by
homogenizing vitamin D3 in an aqueous solution containing one or more
protective colloids for a time and under conditions effective to produce an
emulsion containing vitamin D3, wherein said aqueous solution optionally
contains one or more sugars or other additives and drying the mixture optionally
in the presence of a coating material, to yield a dry powder. Modification of this
method or any other suitable method will be employed.

By spray drying:
 Emulsifying the drug and oil at ordinary temperature together with soluble casein
or a cellulose in an aqueous solution and spray-drying the emulsified liquid, so
as to obtain powder or
 After addition of the vitamins to a solvent and agitation, the solvent is removed
and the residue is subjected to powdering. The powdering may be effected by
spray drying or
 Any other methods through which stable granules may be obtained will be
employed.
6.2 REVIEW OF LITERATURE:
 Vitamin D3 including its physical, chemical, biological properties and its
pathological importance are discussed extensively in Martindale 34th edition (1).
 Marc S. Micozzi and Bethesda Maryland in their review article “Confronting the
Worldwide Epidemic of Vitamin D Deficiency” reported the physiological
Importance of Vitamin D in various disease state and normal daily intake (2).
 Stephen Allen Christensen in his article “Is Vitamin D3 better than D2?” has
explained why vitamin D3 is more stable than D2 and clearly pointed the need of
Vitamin D3 (3).
 Hahnlein, Wolfgang, Hansen, Morten Mohr, Olesen, Jes Elenius et al. have clearly
reported the need for the stable formulation. They also demonstrated dry powder
formulation with much emphasis on particle size, homogeneous fine dispersion, and
stability concluding its importance for optimal absorbability and bioavailability (4).
 Masaaki Nakahara, Akihiko Kurosaki, Hiroshi Otake, Hitoshi Kanai, Hiroshi
Yokokawa, Kiyoshi Kumabe et al. have clearly demonstrated the conventional
methods for powdering oil-soluble substance mentioning their common drawbacks.
They discovered that elution of the same to heat, pressure, water, or the like, can be
prevented by allowing the same to adsorb on a calcium component in an aqueous
solution in the presence of surfactant (5).
 Gregory Paul Dittmar Andrew Irvine Sokolik in their patent application title
“Vitamin D content uniformity in pharmaceutical dosage forms” have come up with
new dosage forms of vitamin D3 and calcium carbonate having improved content
uniformity. The improvements are realized through modifications to the formulation,
the raw material specifications, and the process of manufacture (6).
 Vladimir Babtsov, Kiryat Shmona, Yury Shapiro, Givat shmuel, Emma Kvitnitsky,
Kiryat Shmona in their patent article “Methods of Microencapsulation” have used
excipients like polymethylacrylate, cellulose acetate, polyvinyl alcohol, sodium
Lauryl acetate etc in their preparation involving Microencapsulations of water
insoluble drugs (7).
 Hahnlein, Wolfgang, Hansen, Morten Mohr, Olesen Jes Elenius et al. have prepared
a dry emulsion preparation of vitamin D3 by homogenizing vitamin D3 in an
aqueous solution containing one of more protective colloids for a time and under
conditions effective to produce an emulsion containing vitamin D3. They said
aqueous solution optionally contains one or more sugars or other additives and
drying the mixture optionally in the presence of a coating material, to yield a dry
powder (8).
 Yajima and Mizuo have demonstrated the powdering of vitamin D3 by spray drying
a method that involves addition of the vitamins to a solvent and agitation. The
solvent is removed and the residue is subjected to powdering. The powdering may be
effected by spray drying, drying in vacuum, freeze-drying in drum of other know
drying methods. They concluded that when a diluted acid is used as a solvent, the pH
Should preferably be adjusted to 3 to 4. On the contrary, when diluted ammonia is
used, the pH should preferably adjust to 10-11. Subsequently, vitamins are added (9).
 Makino, Yuji Suzuki, Yoshiki, have introduced a pharmaceutical solid preparation
of active form of vitamin D3 of improved stability which comprises of active form
of vitamin D3 dispersed in an excipient readily soluble in organic solvent and a basic
substance. They also examined the stability and ‘residual percentage of active
vitamin D3’ of the prepared specimen comparing with the control (standard vitamin
D3) (10).
 Ishii, Kuniaki, Toriumi, Yumiko Itai, Shigeru Hayashi, Hidefumi, Nemoto, Manami
have prepared solid pharmaceutical preparations containing a vitamin D3 derivative.
The composition of this preparation consisted of excipients like Mannitol, sugar,
Hydroxypropyl cellulose, and a binder polyvinyl pyrrolidone (11).
 Moroi, Masami, Yokoyama, Toshio, Iwasa and Akira have demonstrated the methid,
for the preparation of a stable dosage-form of active vitamin D3, which comprises an
active vitamin D3 and a stabilizer selected from polyvinylacetal diethylaminoacetate
and Hydroxypropylcellulose adding a pharmaceutically-acceptable carrier to the
resultant mixture (12).
 Nemoto, Kaoru, Igusa, Kazuo, Ogasawara, Toshichika in their patent article
“Stabilized active forms of vitamin D3” demonstrated the preparation containing an
active form of vitamin D3 which is stabilized by incorporation of an amino acid.
They concluded amino acids that do not contain a sulphur atom or an acid amino
group in its structure stabilized the vitamin D3 (13).
 John M. Ballard, LImin Zhu, Eric D.nelson, Randal A. seburg conducted LC-UV
profile of a thermally stressed vitamin D3 tablet and concluded that four major
degradants formed are identified as the Octanoate and Decanoate esters of D3 and
pre-vitamin D3. This observation reinforces the need to be aware of potential
interactions when designing formulations of apparently inert excipients and active
pharmaceutical ingredients. They also concluded that even minor drug-excipent
reactivity can be significant in the long-term stability of pharmaceutical products due
to the stringent quality standards to which pharmaceutical formulations are held (14).
 Semih Otles, Yasar Hisil have clearly demonstrated the determination of vitamin D3
by high pressure liquid chromatography (HPLC). They estimated the quantity of
vitamin D3 in hen egg and the recovery study of vitamin D3. They finally concluded
that HPLC method is rapid simple, sensitive, reproducible and very efficient
technique for the determination of vitamin D3 in hen eggs (15).
6.3 Objectives of the study :
The aim of the present work is to formulate and evaluate the stable vitamin D3
formulation and comparison of different methods.

It is therefore an objective of this study is to provide a composition for solid
pharmaceutical preparations of active vitamins D3 where in the stability of said
active vitamins D3 is remarkably improved.

It is another objective of the invention to provide a process for the preparation of a
composition for solid pharmaceutical preparations of active vitamins D3. It should be
simple and efficient method where by a composition uniform in size distribution and
homogeneous in active vitamins D3 content can be obtained.

Further, this invention is to provide solid pharmaceutical preparations of active
vitamins D3, wherein the stability of active vitamins D3 to light, heat, oxidation, etc.
is remarkably improved.

It is yet another objective of this invention is to provide a composition of active
vitamins D3 which is extremely suited as a composition for making solid
preparations such as tablets, powders, granules, etc. of active vitamins D3 given as
such or added to milk powder, rice flour, soft drinks etc.

The other objective is to prepare in compatible with taste masking, to retain drug
properties and to improve flow property of powders.

Finally in-vitro analysis of different formulations produced using standard analytical
tools like HPLC, UV, etc.
6.4 MATERIALS AND METHODS:
Materials:
1. Active forms of vitamin D3: wherein the active form of vitamin D3 is at least one is
selected
from
the
group consisting of
dihydroxycholecalciferol,
1α-hydroxycholecalciferol,
1α,24-dihydroxycholecalciferol,
1α,25-
1α,24,25-
trihydroxycholecalciferol, 1α-hydroxy-24-oxocholecalciferol, 1α,25-dihydroxy-24oxocholecalciferol,
1α,25-dihydroxy-cholecalciferol-26,23-lactone,
dihydroxy-cholecalciferol-26,23-peroxylactone,
1α,25-dihydroxy-cholecalciferol,
1α,25-
26,26,26,27,27,27,-hexafluoro-
25-hydroxycholecalciferol,
24-
hydroxycholecalciferol, 24-oxocholecalciferol, 24,25-dihydroxycholecalciferol, 25hydroxy-24-oxo-cholecalciferol, 25-hydroxycholecalciferol-26,23-lactone and 25hydroxycholecalciferol-26,23-peroxylactone,
2. Excipient readily soluble in an organic solvent is at least one selected from the group
consisting
of
polyvinyl
Hydroxypropylmethylcellulose,
pyrrolidone,
methylcellulose,
Hydroxypropylcellulose,
cholesterol,
β-Sitosterol,
Campesterol and Deoxycholic acid etc.
3. Excipient which is slightly soluble in an organic solvent crystalline cellulose, starch,
casein, cyclodexrtin, lactose, Hydroxypropyl starch, dextrin, gelatin, etc.
4. Organic solvents: methanol, ethanol and propanol; solvents of halogenated
hydrocarbons such as dichloromethane and chloroform; and such ethereal solvents a
s diethyl ether, etc.
5. Other excipients: D-mannitol or white sugar, starch, dextrin, tragacanth, gelatin,
PVP, HPC, PVA, calcium carbonate etc,
6. Coloring agents, such as dyestuffs of tar source, sunset yellow.
7. Antioxidants: butyl hydroxytoulene (BHT), butyl hydroxyanisol (BHA), lecithine, αtocopherol, hydroquinone, ascorbic acid, octyl gallate and dodecyl gallate as
corrigents, citric acid, fumaric acid, menthol and citrus perfume.
8. Surfactants: sodium dodecylsulfate and sodiumlauryl sulfate, etc.
9. Calcium components like calcium carbonate, calcium citrate etc.
10. Thickening and stabilizing agents like xanthum gum, guar gum, locust bean gum,
etc.
11. Reducing sugars like glucose, fructose, lactose, maltose etc.
12. Amino acids such as vlanine, valine, proline, phenylalanine, tryptophan, leucine,
isoleucine, glycine, serine and basic amino acids such as lysine, arginine and
histidine.
Method:
Some of the commonly used methods which are to be employed for the
formulation of the stable vitamin D3 preparations are:
1. Spray drying method.
2. A method using hardening of oil.
3. Encapsulation method.
4. Hydrogenation method.
5. Dry emulsion method.
6. Clathration with bile salts, cyclodextrin.
7. Complexation with sterols.
8. Dispersion with polyvinyl pyrolidine, etc.
7.
7.1. Source of Data
Data will be obtained from Pubmed, Science Direct, Medline, and other Internet
facilities, literature search and related articles from library of Nargund College of
Pharmacy.
A. Journals & articles :
 Advanced drug delivery reviews.
 International Journal of Pharmaceutical Sciences.
 Journal of Controlled Release.
 International Journal of Pharmaceutics.
 Journal of Pharmaceutical Sciences.
 European Journal of Pharmaceutics and Biopharmaceutics.
B. Internet Browsing.
 www.sciencedirect.com
 www.google.com
 www.pubmed.com
7.2. Method Of Collection Of Data:
1. Literature survey using internet and scientific journals.
2. Experimental studies which includes.

Formulation development and evaluation.

Powder or granular properties or tablet properties and in-vitro dissolution
studies for the developed formulation.

Spectrophotometric, HPLC, UV, FTIR methods etc for the estimation of drug
and analysis of in-vitro dissolution samples.

Stastical analysis of all the results.
7.3. Does the study require any investigation or intervention to be conducted on
patients or other humans or animals? If so, please mention briefly.
-NOT APPLICABLE-
7.4. Has ethical clearance been obtained from your institution in case of 7.3?
-NOT APPLICABLE-
8.
LIST OF REFERENCES:
1. Martindale-The complete Drug Reference. Thirty-fourth edition. Edited by Sean
Sweetman C. page no 1461-3.
2. Marc Micozzi S, Bethesda Maryland. Confronting the worldwide Epidemic of
Vitamin D Deficiency. Nutri News. October 2008.
3. Stephen Allen Christensen, Is vitamin D3 is Better than D2? Available from:
http://vitaminsminerals.suite101.com/article.cfm/is_vitamin_d3_better_than_d2.2008
jun 22. Accessed on: Nov 25th 2009.
4. Hahnlein, Wolfgang, Hansen, Morten Mohr, Olesen, Jes Elenius et al, inventors.
BASF Aktiengesellschaft (Ludwigshafen, DE), assignee. Stable emulsions and dry
powders of mixtures of fat-soluble vitamins, their preparations and use. United
States Patent 6531157. 2003 Nov 03.
5. Masaaki Nakahara, Akihiko, Kurosaki, Hiroshi, Otake, Hitoshi et al, inventors. Kitti
Corporation, assignees. Method for producing calcium component powder
containing oil-soluble substance. Patent application no; 20080305170. Available
from; http://faqs.org/patents/app/20080305170. accesed on Nov 28th 2009.
6. Gregory Paul Dittmar and Andrew Irvine Sokolik, inventors. Taisho pharmaceuticals
Co. Ltd. (Madison, JP), Sumitomo pharmaceuticals Co. Ltd. (Madison. JP),
Wisconsin Alumni Research Foundation (Madison, WI), assignee. Composition for
solid pharmaceutical preparations containing vitamin D3 derivative, United States
Patent 5328903. 1994 Dec 07.
7. Vladimir Babtsov, Kiryat Shmona, Yury Shapiro, Givat Shmuel, Emma Kvitnitsky,
Kiryat Shmona, inventors. Tagra Biotechnologies Ltd, assignee. Methods of
microencapsulation. US patent 6932984. Aug 23rd 2005.
8. Hahnlein, Wolfgang, Hansen, Morten Mohr, Olesen, Jes Elenius et al, inventors.
BASF Aktiengesellschaft (Ludwigshafen, DE), assignee. Stable emulsions and dry
powders of mixtures of fat-soluble vitamins, their preparations and use. United
States Patent 6531157. Mar 11th 2003.
9. Yajima and Mizuo, inventors. Asama Chemical Co. Ltd. (Tokyo, JP), assignee.
Method of preparing a stabilized vitamin powder. US patent 4935257. June 19th
1990.
10. Makino, Yuji, Suzuki, Yoshiki, inventors. Teijin Ltd. (Osaka, JP), assignee. Solid
preparation of active vitamin D3 having improved stability. European patent
0413828. Apr 17th 1996.
11. Ishii, Kuniaki, Toriumi, Yumiko, Itai, Shigeru, et al. inventors. Taisho
pharmaceuticals Co. Ltd. (Madison, JP), Sumitomo pharmaceuticals Co. Ltd.
(Madison. JP), Wisconsin Alumni Research Foundation (Madison, WI), assignee.
Composition for solid pharmaceutical preparations containing vitamin D3 derivative,
United States Patent 5328903. 1994 Dec 07.
12. Moroi, Masami, Yokoyama, Toshio, Iwasa, Akira Moroi, inventors. SS
pharmaceutical Co. Ltd. (2-12-4 Nihonbashihama-cho, Chuo-ku, Tokyo, 103, JP),
assignee. Compositions for the preparations of dosage-form active vitamins D3 and
process for preparing stable dosage-form active vitamins D3 by using the same. Dec
05th 1993.
13. Nemoto, Kaoru, Igusa, Kazuo, Ogasawara, Toshichika, inventors. Chugai Seiyaku
Kabushiki Kaisha (Tokyo, JP), assignee. Stabilized active forms of vitamin D3.
United States patent 4836957. June 6th 1989.
14. John Ballard M, Limin Zhu, Eric Nelson D, Randal Seburg A. degradation of
vitamin D3 in a stressed formulation: The identification of esters of vitamin D3
formed by a trans esterification with triglycerides. Journal of Pharmaceutical and
Biomedical Analysis. 2007 (43): 142-0.
15. Semih Otles, Yasar Hisil. The determination of vitamin D3 by high pressure liquid
chromatography (HPLC). GIDA; 1994.19(16): 377-9.
9.
SIGNATURE OF THE
CANDIDATE
( VENKATRAO MANNAM )
10. REMARKS OF THE GUIDE
RECOMMENDED FOR DISSERTATION WORK.
11. NAME & DESIGNATION OF
11.1 GUIDE
Dr. GOPAL MURALIDHARAN
PROFESSOR
DEPT.OF PHARMACEUTICS
NARGUND COLLEGE OF PHARMACY
11.2 SIGNATURE
11.3 HEAD OF THE
DEPARTMENT
11.4
Dr. C.S.R. LAKSHMI
PROFESSOR
HEAD OF THE DEPARTMENT OF
PHARMACEUTICS
NARGUND COLLEGE OF PHARMACY
SIGNATURE
12. 12.1 REMARKS OF THE
PRINCIPAL
FORWARDED AND
RECOMMENDED FOR FAVORABLE
CONSIDERATION.
Prof. DAYANAND. S. PURANIK
PRINCIPAL
12.2 SIGNATURE.