University of Nigeria Research Publications

Aut

hor

NZEKWE, Ifeanyi Thaddeus PG/M.Pharm/02/32730

Title

Evaluation of Sodium Benzoate Solution as Alternative Vehicle for Parenteral Diazepam

Facu

lty

Pharmaceutical Sciences

Dep

artm

ent

Pharmacy

Dat

e

June, 2007

Sign

atur

e

EVALUATION OF SODIUM BENZOATE SOLUTION AS ALTERNATIVE VEHICLE F0.R PARENTERAL

DIA2XPAM

EVALUATiON OF SODIUM BENZOATE SOLUTION AS ALTERNGTI[VE VEHICLE EY3R PARENTERAL

DIAZEPAM

VEHICLE Fm PARENTERAL DIAZEPAM

DR V.C. OKORE SUPERVISOR

Attamrr, Dr. C.O. kimono, Dr. K.C. Qkkansi, Mrs. P.0, N m m i Mr. Daw Uu&u !l@sBaH

and Mr. Pred Otuu, for their Ftieodliness, and to all other rndm ofthe Department of

Demmnent oPPRsrmmmwu. and the M n f Desire Cornmrtcfs. for b a r immense help-

June 2007.

ABSTRACT

administrahn In this study, the effects of sodium bmaoate on h e mtubtliry,

I 1 A q w u s solubility of drugs as a formulalion factor

1 1 1 M~~ solubilfeation

1,1.4 Sdtfbmation

1.1.5 Prodrugging

1,1.6 Cn-8olvency

1.1.7 0th dubilizina; methods

1.2.1 Routes of injection

1.2.1.1 Intrmuscular route

1.2 1.2 Iimwmous route

i

ii

iii

iv

v.

vi

1.2.2.5 Other paremteral vehicles

1.2.3.1 Red lime stabilily testing

1-2.3.2 A d m t e d stability MhU

1 3 QHalig. mM ofpatateras

1.3.1 SteriIity test

1.3.2 Pyrogenicily test

1.3.3 Clarity tst

1.3.4 Leak test

1.4 In vim hmolysis by drugs and solvents

1.5.4 A formulation dilemma

1.6 Sodium bemate: a non-toxic hydrotrope

CHAPTER TWO

Appaldix VI E H a m ~ i r & far diazepam in dim k n r s ~ sokuion

Appendix VI b: Haerna&sis d h fbr dimpain in propflena glycol

Apmdix W c: Haemolysis data f ir diazepam in normal saline

Appendix Vtl €x l-htmol+ data far progryiene glycol in water 9 3 a

CHAPTER ONE

MTRODUCTtON

whih for the British Ph-peia, it i s v e ~ y sli&@ soluble in vvmber. Accodmg tO

dnm. which is ~racticallv imuluble in water. however. the Connulation of this rndecule as

the Fh4C of a sutmaam i s low, the initial d b t l of z&ro solutilizaiion may not be

iod lb mlubn due to i d solubility bycorn-h bdmim idide ha and in&e

espwidy for parentend we. %ere ia also IC drnger of drug precipitation on dilution in

k4y fhfdr, which dtw drug ~olwxntr ipn at the Bite of e n m 1 . DilutionB2 hi a

0 6 p m matMa h r t o r c h the gracipitatioon ppensity dfonndllboq nd lydtotrope

prodwe additive eohbilimtion e f f a due to reduction d CMC of the ampkiph'ie'lbq

Combinttiorrs af cettlin Wrottopes have been.lcnown ro giw mod tmuhm. It it

particle size reduction, as well as solid-state modifications (polymorphs). Advanced

techniques like combinatorial chemistry and molecular modeling have yielded drug

candidates more closely resembling natural mediators in the body, so that

pharmacokinetic-related problems are minimized. Hydrophilic solubilization technology

(HST) and lipophilic solubilization technology (LST) are examples of new solubilization

technologies83.

1.1.8 Combined solubilization

Efforts have been made to optimize drug solubility by the combined use of several

solubilizers. Cyclodex~ns have been combined with polysorbates in the solubilization of b

n i ~ i n ~ e n i n ~ ~ . Salicylic acid is ordinarily soluble in 550 parts of water, but the solubility

increased several fold when it was solubilized in hydroalcoholic systems containing

polysorbate8'. Studies on combinations of hydrotropes and co-solvents on the solubility

and stability of diazepam showed that increase in co-solvent concentration increased the

synergistic solubilizjng effectg6. The use of self-ernulsifiing systems containing

surfaclants, co-solvenls and oils that together form a spontaneous emulsion in contact with

aqueous medium has been described8'. Mixtures of hydrotropes and surfactants may

produce additive solubilkation effect due to reduction of CMC of the amphiphile88-g9.

Combinations of certain hydrotropes have been h o r n to give good resultsg0. It is

noteworthy that not all combinations of solubilizing agents give synergistic results or

additive results. Combinations of 2HP-p-CD with sodium deoxycholate or 1, 2-propylene

glycol or sorbitol reduced the solubilizing capacity of ~H~-P-CD", while additive

hydrotropic efrects were observed behveen 2HP-0-CD and urea or nicotinamide. Negaiive

effects were also observed in combination of acetone or dimethylsulfoxide with urea or

nicotimamide in the solubilization of riboflavin", In a simulated study to examine the

effect of combinations of solubilizers, usug betacyclodextrins and surfactants, the

particle size reduction, as well as solid-state modifications (polymorphs). Advanced

techniques like combinatorid chemistv and molecular modeling have yielded drug

candidates more closely resembling natural mediators in the body, so that

pharmacohetic-related problems are minimized. Hydrophilic solubilization technology

(HST) and lipophilic solubilization technology (LST) are examples of new solubilization

technologie~~~.

1.1.8 Combined solubilization

Efbrts have been d e to optimize drug solubility by the combined use of several

solubilizers. Cyclodextrins have been combined with polysorbates in the solubilization of b

naringeninN4. Salicylic acid is ordinarily soluble in 550 parts of water, but the solubility

increased several fold when it was solubilized in hydroalcoholic systems containing

polysorbate85. Studies on combinations of hydrotropes and co-solvents on the solubility

and stability of diazepam showed that increase in co-solvent concentration increased the

synergistic solubilizing effectg6. The use of self-emulsifying systems containing

surfactants, co-solvents and oils tha~ together form a spontaneous emulsion in contact with

aqueoils medium has been described8'. Mixtures of hydrotropes and surfactants may

produce additive solubilization effect due to reduction of CMC of the amphiphile88-89.

Combinations of certain hydrotropes have been known to give good results". It is

noteworthy that not all combinations of solubilizing agents give qnergistic results or

additive results. Combinations of 2HP-P-CD with sodium deoxycholate or 1, 2-propylene

glycol or sorbitol reduced the solubilizing capacity of ~H~-P-cD'', while additive

hydrotropic effects were observed between 2HP-6-CD and urea or nicotinamide. Negative

effects were also observed in combination of acetone or dimethylsulfoxide with urea or

nicotimamide in the solubilization of riboflavin". In a simulated study to examine the

effect of combinations of solubilizers, using betacyclodestrins and surfactants, the

combined value for solubility was less than the sum of the individual solubility values in

cyclodextrins and surfactants".

1.2 Parenteral dosage forms

A parenteral product is defined as a pharmaceutical dosage form intended for

administration through one or more layers of skin or mucous membrane".

Parenteral products are required to comply with very high standards of purity and

safety because they are readily bioavailable and are injected into sterile compartments.

The formulation of a parenteral product requires a careful choice of excipients. Privary

considerations include solubility of drug in water, stability, intended site of administration

or clinical use. and compatibility between excipients, as well as sterility of formulation.

The most important consideration for a non-vascular injection is bioavailability,

and this is influenced by the physico-chemistry of the drug, the vascularization of the

injection site. exercise, etc. The physico-chemistry of the drug determines the choice of

vehicle or dosage form (solution, suspension or emulsion). Once in circulation in the

blood vessel, therapeutic effect is influenced by concentration of drug at the site of action,

extent of distribution and binding to plasma proteins, as well as elimination processes.

A primary consideration in the formulation of a drug is the choice of vehicle,

which is dependent on solubility behaviour. A good solvent must achieve a good

solubility level of the drug, and ensure stability, while not adversely altering the

pharmacodynamics of the drug. Again, the solvent should produce little or no tissue

irritation, which is observed with many organic solvents94.

The pH of an injectable formulation should be as close to physiologic pH as

possible to avoid pain or tissue necrosis. Adjustment of pH is done according to the

Hendersen-Hasselbalch equation for buffer species (Equation 1).

Equation 1

where Csalt and Cacid represent the molar concentrations of salt form and acid form

in the buffer solution.

An injection may also need an antioxidant. The choice of antioxidant may depend

on the redox potential of the active ingredient since the antioxidant must have a lower

redox value to permit preferential oxidation, thereby protecting drugs and or excipients.

The Nernst equation is the mathematical representation of the relationship between the

factors affecting a redox system.

where Eo represents the standard redox

concentrations, apro,jucts and a,,,,,,,,, being

b

Equation 2

potential and E is the redox potential at

the number of moles of oxidized form and

reduced forms, respectively. Sodium bisulfite is commonly used as an antioxidant in

parenteral formulations. Other parenteral antioxidants include thiourea and sodium

formaldehyde sulfoxylate.

Multiple dose injections contain preservatives to combat accidental contamination,

but this is not necessary for large volume parenterals or single dose ampoules. The

necessity for a preservative may also depend on the injection site, as certain regions, e.g.

spinal cord, must not be administered with injections containing preservatives. Paraben

esters are commonly used.

Isotonicity enhancers may be necessary in hypotonic formulations. Sodium

chloride is frequently used for this purpose. Hypertonic solutions do not need tonicity

enhancers, and may frequently cause swelling at injection site.

Containers for parenterals are commonly made of glass or, to a lesser extent,

plastics. Ampoules and vials are commonly made of borosilicate glass9'. Plastic materials

that are comrnonly produced by blow fill and seal (BFS) lechnology are increasingly used,

but interaction with ingredients will remain an issuew.

The problems associaled with injectable preparations are tissue irritation, pain and

sometimes tissue necrosis. Poorly water-soluble drugs may also show aggregation-related

problems, such as embolism, and this limits the use of coarse particles in intravenous

injection, particularly un-emulsified oils. Prodrugging may help to reduce the pain

associated with injection of drugs formulated with organic co-solvents or surfactants. A

prodrug that shows enqme-catalysed reversibilily inslead of chemical catalysis in vivo is

preferable, but some systems show the reverse9'.

1.2.1 Routes of injection

Depending on composition, volume and intended site of action, an injection may

be administered through one or more of several routes.

1.2.1 .l lntwmuscular route

This is the most popular route of injection. The drug is injected into a mass of

muscle in the vascularized and less innervated areas of the body. Comtnonly employed

muscles are the deltoid, vastus lateralis, ventrogluteal and dorsogluteal musles.

Intramuscular in.iections are not easily amenable to self-medication.

The contraindications for the use of the intramuscular route are hrombocytopenia

and coagulopathy, since hematoma may develop.

1.2.1.2 Intravenous route

This is commonly employed in emergency and intensive care medicine. I1

provides a fast reliable route of drug administration followed by rapid distribution. It is

the most reliable route because for all other routes, blood flow may be inadequate in

acutely ill patients, making absorption from even the intramuscular or subcutaneous routes

unreliabIe. Caution is, however, needed with bolus intravenous injections because of the

possibilrty of shock. Also, the danger of occlusion of fine capillaries in the brain

precludes the use of dosage forms with large particle sizesg8.

1.2.1.3 Subcutaneous route

In this mode of delivery, drug is introduced into the subcutis, being the area

direcli!. beneath the dermis and epidermis. Insulin is a conunon esarnple of drug

frequently administered through this route. Absorption foHotving subcutaneous

administration could be enhanced by the use of hyaluronidase enzyme, w-hlch increases

permeabilily of the subcutaneous matrix. Subcutaneous injections are amenable to self-

medication as in insulin management. They can also be employed for depot medication. b

1.2.1.4 lntraspinal route

This is frequently employed in chemotherapy: anesthesia or laboraton! diagnosis.

Special syringes are used for introduction of drugs into a particular area of the spinal

column, classified further as intrathecal, intracisternal, epidural, etc. Volume of injections

into these regions should not exceed 25 rnl and the formulation should not contain a

bactericidew.

1.2.1 -5 Other injectioil routes

There are many other injection routes, such as intracardiac, intradermal.

intraosseous, intramterial, etc. The intracardiac route is a direct injection of drug into the

heart tissue commonly used in cardiopulmonary resuscitation md also for the

administralion of streptokinase in myocardial infarction, Intraosseous delivery indirectly

mc&es use of the intravenous route because the bone marrow drains into a vein, and this is

commonly employed in paediatric and emergency medicine in cases of inaccessible vein.

Intradermal injection of drug (into the dermis) is mostly employed in allergen testing.

Intra-arterial injection is a specialized procedure, which employs an artery instead of a

vein. Commonly employed drugs for intraarterial administration are vasodilators for

countering spasm and also thrombolytic drugs intended to dissolve emboli. InIraarterial

and intracardiac injections should not contain a bactericide*.

1.2.2 Non-aqueous vehicles for injections

The use of non-aqueous vehlcles may be inevitable in cases of drugs with poor

aqueous solubility, hydrolytic instability or unfavourable partitioning in plastic containers.

If the drug is also inactive by oral route, formulation in the form of a non-aqueous

injection may be employed.

Formulations in oily vehicles can be modified to achieve cellain effects, e.g. depot

medication, as described previous. In these cases, there is improved patient compliance b

due to reduced dosing frequency. It is worthy of note that a distribution coefficient unduly

100-101 favouring the lipid vehicle may lead to drug concentration in fatty tissues .

1.2.2.1 Fixed oils

These include cottonseed oil, olive oil, arbs oil, castor oil, sesame oil, etc. They

are relatively non-toxic and bland, but some allergic reactions have been reported in

patients injected with such oil-based preparztionslO1. They are not miscible with water and

cannot therefore be employed in unemulsified intravenous products. Arachis oil is used in

dimercaprol injection. Oils may be stabilized against oxidation by including oil-soluble

antioxidants (e.g. tkio~ycollate) or by vacuum sealing.

1.2.2.2 Propylene glycol

Propane-1, 2-diol is a viscous liquid that is miscible with water and chloroform,

but not fixed oils. It is relatively non-toxic and is the safest glycol known. It is widely

employed as a co-solvent with water or alcohol. It has been used in the formulation of

phenobarbitone, diazepam and melarsoprol injections. The major draw back with

102-103 propylene glycol is the high incidence of pain and thrombopMebitis .

1.2.2.3 Ethyl oleate

Ethyl oleate is a vehicle for progesterone injection. It is yellowish, immiscible

with water, but miscible with ether, alcohols and fixed oils. It is rapidly absorbed from the

tissues, but may undergo discoloration on standing.

1.2.2.4 Propane-1,2,3-trio1 (Glycerin)

This is a clear viscous high boiling point liquid that is miscible with water. It has

very low toxicity and is well tolerated on injection Glycerin is used alone or in

104-105 combination in several pain remedies .

1.2.2.5 Other parenteral vehicles +

Other parenteral vehicles include bemy1 benzoate, ethyl carbonate and the

proprietary whicle, Cremophore ~ 1 ' ' ~ .

1.2.2.6 Emulsions as parenteral vehicles

Microemuisions are inicellular dispersions of nanometer-sized droplets of oil-

dissolved drugs in water. A new method of microemuision formulation consists of a lipid

drug reservoir with a proprietary combination of surfactants and co-surfactant, which

together form a microemulsion on contact with an aqueous environment. The emulsion-

solubilized drug is then hrther solubilized in the body tissues and, therefore, absorbeds3.

Emulsion formulations have been applied to d i a ~ e ~ a m ' ~ ~ - ~ ' ~ , tetra~epam"~,

antimycotics'14, pr~xicam"~, naproxenH%d 10razeparn~~~. The choice of oil component

for such emulsions parbcularly for intravenous delivery is important118..

1.2.3 Stability of parenteral products

Stability is the capability of a particular formulation in a specific container/closure

system to remain within the physical, chemical, microbiological, therapeutic as well as

toxicological specifications11g, Stability is important to the formulation pharmacist

because it affects the quality, purity and identity of the product, as well as its safety and

potency or strength. Legal requirements and consideralions make the early determination 14

of stability veq7 crucial. Stability is commonly expressed in terms of the expiry date or

she!r life 01 a product. But this determination is only true for a particular container-

closure system under specified conditions of storage. Shelf life is predicted from stability

120-121 tests under generaVnorma1 conditions of storage , or by accelerated testing under

extreme conditions. Red time stability testing is normally applied to reference materials

and clinical chemistry reagents'22. Manufacturers' quality assurance criteria typically

require that a product must retain at least 90 % of the initial value throughout its life'".

Stabilitjc tests, besides predicting the shelf life of products, also define the best storage

conditions and packaging materials and choice of escipients. Different testing criteria are +

used for new drug moieties and for established formulated substances. For formulations,

stability iests are based on changes in formulation properties. Non-quantitative methods

include colour changes, taste, odour, palatability, calung, cracking, etc. Quantitative

methods depend on the nature of the formulation and may determine the content of active

ingredient (or excipients), viscosity or yield value, sediment volume, redispersibility

number, solubility, dissolution rate, etc.

1.2.3.1 Real time stability testing

Experiments are done to determine the physical; biological, biopharmaceutical and

microbiological characteristics of a formulation, during the expected shelf life and under

the expected storage conditions. The testing protocol must permit distinction between

percent degradation and inter-assay variation. This calls for use of analytical reagents that

are suficieritly stable so that a single batch can provide a constant reaction or change.

Instrumental validation is also very important. High performance liquid chromatography

(HPLC) is commonly used for assay of drugs and impurities.

1.2.3.2 Accelerated stability testing C

This increases the rate of physical and chemical changes by using exaggerated

conditions as part of formal testing protocol. The data generated is used to corroborate 15

those of real time stability studies and to further predict longer-term effects. These tests

are also called stress tests. Commonly employed stress conditions include high

temperature, high relative humidity, high intensity of light, centrifugation and freeze-thaw

cycles. However, we will be most concerned with accelerated chemical degradation tests

as a basis for shelf life determination.

Chemical stability is the ability to maintain the molecular identity of the drug.

Changes in molecular identity (and therefore strength or quality) are caused by several

mechanisms, notably hydrolysis, oxidation and photolysis. @

The chemical stability of a drug is challenged by major stresses such as

temperature. light, pH and high relative humidity. Hydrolysis is the most important means

of drug degradation, since it can be catalyzed by both temperature and pH.

Most accelerated testing protocols employ the Arrhenius equation123

Equation 3

where k~ and k l are reaction rate constants at temperatures, Tz and T I respectively (the

temperatures are expressed in Kelvin). E, is the activation energy and R is the molar gas

constant. If the activation energy is known or calculated, the Arrhenius equation permits a

projection of degradation rate and, therefore, shelf life at ambient temperatures from those

obtained at higher temperatures'24.

For a first order degradation reaction,

ln [D] = ln [D,,] - K! Equation 4

where [Do] and [Dl represent the initial drug concentration and concentration remaining

after time, t; and K is the degradation rate constant. By substituting InrD] with ln[0.9D0],

the time to have 90 percent of initial drug concentration (T~o , also regarded as the shelf

life) is given as:

Equation 5

Since active ingredients may degrade by more than one me~hanis rn '~~ , it is necessary to

validate activation energies and shelf lives calculated from accelerated studies with real

time stability tests.

1.3 Quality control of parenterals

There are three general areas of quality control, namely incoming stock,

manufacturing (or processing) and the finished product. *

Incoming stock may be tested in such areas as microbial load, pyrogens on raw

materials and equipment, glass tests on containers as well as identity tests on rubber

closures. Process control involves all the tests, observations, readings and measurements

in the production process, such as cycling time, sterilization temperature, f i l l volume, and

label identity. Finally, a finished parented product may be subjected to sterility tests,

pyrogen tests, clarity tests and leak tests in addition to the chemical analysis applicable to

all dosage forms.

1.3.1 Sterility test

Sterility is an absolute term, which means freedom from the presence of viable

microorganism. It is a strict uncompromising requirement of an injectable product.

Sterility tests involve incubating representative samples of the finished product for

microbial growth. A sterility test is based on the assumption that provided the growth

requirements are optimal - proper nutrients, pH, temperature, oxygen (or lack of it),

sufficient incubation time - a single microbial cell will grow by geometric progression

until the number of cells and their metabolic products exceed the solubility capability of

the culture medium, causing turbidity.

The main components of a sterility testing protocol are the application of a

statistically sound sampling procedure to get true representative batch samples, and the use

17

of aseptic slcllls lo prevent accidental contamination of test products. Because of these

limitations. the oficial sterility test provides an estimate of the probable, not actual,

sterility of a batch of products because the actual product administered to a patient has not

been tested, and the sampling technique may fail to give a true representation of the

sterility of the whole batch. However, it does provide an endpoint check that

"representative" samples of the batch did not disclose contamination. Sterility is the end

result of conformity to good manufacturing practice, even in the selection of raw

materials

Some products like terminally-sterilized large volume parenterals may not undergo t

sterility testing, as long as the sterilization procedure has been experimentally validated to

have a high Sterility Assurance Level (SAL). Release of products based on validated

sterilization procedure without endpoint sterility testing is called parametric release.

Exhaustive sterility tests must be performed on all products sterilized by margnal

sterilization procedures like aseptic filtration. There may be sterilization failure if any

aspect of the sterilization protocol, e.g. improper loadmg of material, is omitted. A

detailed description of the sterility test procedure, as well as sterilization methods for

thermo-labile and thermo-stable products, can be found in the USP (2001)'~'.

1.3.2 Pymgenicity test

Pyrogens are derived mainly from the lipopolysaccharides (LPS) found on the

outer membrane of Gram-ve bacteria. However, all bacterial cells produce pyrogens.

Pyrogens cause a number of changes on injection, the most noticeable being ppresia

Pyrexia, though rarely fatal, may be of serious concern in severely il l patients receiving

l x ~ e volume parenterals.

Lipopolysaccharide is composed of lipid A and polysaccharides. The lipid A alone

lacks biological activity, but toxicity results in combination with the polysaccharide and

this may be altribulable to increases in solubility of lipid A and to antigenicity effects. 18

Pyrogem can be eliminated by the use of depyrogenated materials and equipment,

followed by adherence to strict procedures. Pyrogens on packagng material can be

controlled by heating alone or in combination with alkali or strong oxidizing solutions or

by washing with detergent'25. Some proprietary liquids are used to depyrogenate heat

sensitive surhces, or even surfices amenable to standard heat Some

pharmaceutical compaqies render plasiic containers pyrogen-free by waslung with alkaline

agent (pH 9-1 0) on a machine integrated nith the filling line. When this choice is made,

any residual clearing material must be adequately removed by rinsing with pyrogen-free

water for injection. b

The procedural detail and specifications for pyrogen testing with rabbits are

contained in the USP (2001)'~'

The gelling property of the lysate of the amoebocytes of Limulus polyphemus has

been developed for pyrogenicity testingI2'. This method has gained wide acceptance

following favourable results'28.

1.3.3 Clarity test

It has been shown that formation of granulomas in the vital organs of the body

could be traced to fibres, rubber Fragments and other solids present in intravenous

infusions129.

Clarity testing represents a very difficult quality control parameter Tor injections.

Visual inspection is the oldest method, and i t is still encouraged by the IJSP, although

instrumental methods of particulate matter testing using light blockage and video imaging

give more objective results. New procedures combining light obscuration followed by

filtration and microscopic examination have been describedI3'

1.3.4 Leak test

Another commonly performed test on injections is leak test (for ampoules), which

is a package integrity test. Leak tests are designed to ensure a hermetic seal. Invisible 19

cracks on ampoules may permit exchange of materials with the environment and lead to

leahage or contamination. Leak test employs negative pressure to detect leakage in

ampoule wall to enable dye penetration.

1.4 In vitro haemolysis by drugs and solvents

Many pharmaceutical agents are formulated with excipients for parenteral

administration. Many of these excipients induce haemolysis on intravascular

administration.

The goal of in v i m haemolysis studies is to determine the potential of a

formulation to cause intravascular haemolysis. The methods employed may reveal extent b

of inlra\~ascular hacmolysis or amount oC cell damage alter intramuscular administration.

A number of agents have been shown to cause haemolysis in v i m - drugs, chemicals,

solvents and even siliceous materials. Diazepam solubilized in sodium salicylate was

reported to cause a high level of haemolysisl". A comparative study of the haemolytic

activities of diazepam in sodium salicylate and saline led to the conclusion that different

solvent systems altered the haemolytic fragility of erythrocytes to chemical substances.

A similar study with lorazepam in emulsion or organic solvent vehicles revealed

remarliable haemolysis with the organic solvent-based formulation, prompting the

researchers to discourage the use of the organic solvent (propylene glycol) as a vehicle for

Iorazepam""

Solvents like dimethylsulfoxide (DMSO)'"-'~" propylene glycol and glycerin131

have been shown to cause substantial haemolysis. Drugs that have been linked with hgh

haemolytic activity include chlorpromazine and clema~tine'~~, procaine hydrochloride,

l ~ r a z e ~ a m " ~ , and elomidate'".

Studies have been conducted or. the use of protective agents and formulations for

inhibiti~g the haemolytic activities of drugs. Wurster el reported that urea caused

substantial haemolysis even in low concentration, which could be counteracted by the 20

addition of sodium chloride. They dso showed that the presence of a non-penetrating

electrolyte lihe dextrose stabili~ed [he cells by increasing the osmotic resistance, and that

those electrolytes with polyvalent anions caused better inhibition of haemolysis than the

monovalent ions. Destrins have been shown to inhibit the haemolytic activities of

chlorpromazine and clemastine'"". Cyclodexlrin polysulfate offers protection against a

wide range o r haemolyhcally active substances by reducing the inter'action of haemolytic

agent with membrane surface'". Formulation of etomidatc as an emulsion in a study was

round to reduce the haemolytic activity, compared to its formulation in propylene

glycol I". b

A paradox esists with the haemolytic role of qclodextrins. Whde they have been

137-138 shown to inhibil haemolysis in a variely of situations , they are also reported to cause

massive haernolysis and shape changes in red blood cells in other situations. In a study,

qclodextrins protected erythrocytes from haemolysis and shape changes caused by

chlorpromazine and flufenamic acid1". This was explained in terms of a decrease in

effective drug concentration by inclusion complexation. In another study, Jrie et a/. 13',

found out lhat different grades of destrins caused haemolysis of human erythrocytes in the

order of bel:lcyclodestrin r alphacyclodestrin > gammacyclodestrin. They concluded that

the double effect of cyclodextrin was due to removal of membrane components at hlgh

concentration. In yet another it was found that alphacyclodexhin had high

potency for mduction of shape change of erythrocyte from discocyte to spherocyte, while

betaqclodestrin caused Iysis before shape change was completed. The order of

haemolytic potencies agreed with their binding affinities for membrane cholesterol,

confirming hat haemolysis was secondary to membrane des t r~c t ion '~ . It has been

reported that dilution with sodium citrate solution or normal sdine significantly lowered

the haemolytic potency of diazepam injection, while mannitol and dextrose infusions

offered no protection14'. 21

The choice of a suitable solubilizing agent should therefore include a consideration

of the haemolytic potential of the excipient since many excipients and solubilizers, apart

142- 145 from cyclodextrins, have been demonstrated to cause considerable haemolysis . The

discovery of highly effective surfactants with low haemolytic activity has partly solved the

major drawback in the use of surfactants as parenteral so~ub i l i ze r s '~~ .

1.5 Diazepam

1 S.1 Physico-chemistry

Diazepam is 7-chloro- I , 3-dihydro- 1 -methyl-5-phenyl- I, 4-benzodiazepine-2-one.

It has a molecular formula of C16H13N20C1 and a molecular weight of 284.7 g/mole. The

structural formula of diazepam is shown below.

Molecular structure of diazepam

Physically, it is a white to yellowish, powder with a melting point range of 13 1°C

to 135OC1, a dissociation constant, pK,, of 3.3 at 20 OC, and maximum stability at pH 5.5

Diazepam degrades in aqueous solution by hydrolytic cleavage of the 4: 5-

azomethine bond to give an intermediate, which undergoes further hydrolysis to produce

2-methylamino-5-chlorobenzophenone and a glycine derivative. The reactions are

reversible and pH dependentlj7. All catalyses in the pH range of 1-10 give one

intermediate''' while further catalysis of the product in suitable media give additional

prodt~ci\ 1 1 ' ) I \ I . Anionic surlirctant\ can inhibit the acid hydrolysis of dia/cpani, and this

cl'lcct Increase\ us the h~drophohic nature ol'thc surlhctant incrcascs'".

I)ia/cpalii ix a long-acting bcn/odiazcpinc ~ ~ i t l i anticonvulsant, anxiolylic, sedative,

mu~c l c rcla\ant and atnncsic propcrtics. and ir is thc ~ i los l wiilcly ilscd drug liw treatment

I .: of' ir~sonini:i. li.brilc convulsion, statils cpilcplicus and :~lcohol witlitlr.awal symplonis .

I all bcrl/odia/cpincs. i t has a rapid onset ol'action oncc delivered into tlic C'NS and is

- 1 - i very \ale . It is used in lhc managcmcnt ofansicty. tension. ilcprcssion. inson~nia and

t agltatioii. It i s also i ~ s c l i ~ l in the managcmcnt ol' ~ C L I ~ C shcIct;~I musclc spacni ol'ccntral,

pcriplicra l or tctanic origin. Il has bccn ilscd to provide rcl icl' in alcohol w itlidra\val

situatioris lo combat agitations, rrcmors and impending delirium.

13cn/otIia/cpincs control corivulsion and sei/i~rc states by inhibiting thc

conhidcrctl to be tlic clt11~ ol'clioicc i n tonic-clonic, abscncc. ar~d hcmiclonic and mjoclonic

plia\c I nictabolicm in the livcr lo givc N-dcsmclhy lcliazcp:i~ii and tcma/.cpam. Secondary

1.5.3 Side-effects

Common side effects include loss of psychornotor skill, lassiiude, lightheadedness

and anterograde amnesia. Habituation is common, and cognitive impairment and

158-159 confusion have been associated with use of benzodimepines . From results of studies

on the effects of acute administration of diazepam on learning, il was found to produce

dose-dependenl increases in percent error in the acquisition component, while generally

not affecting percent errors in the performance component, but tolerance developed to the

errors with chronic administration'".

Other common side effects are headache, nausea, weakness, vertigo, joint and b

chest pains Dependence may develop, and withdrawal symptoms may include insomnia,

anxiety. irritability, anorexia, unpleasanl dreams, sweating and faintness.

Concomitant administration with alcohol may be fatal.

1 S.4 A formulation dilemma

Various attempts have been made in the past to present a dosage form applicable to

the \vide range of therapeutic uses of diazepam. The most important problems stem from

its poor water solubility, haemolytic activity of solvents, and the need to formulate an

acceptable dosee form for emergency situalions like convulsive states. In seizure

conditions or convulsive states, the oral route is inapplicable. The rectal route has been

studied as a means of delivery of diazepam in such conditi~ns'~'"~~. Diazepam

suppositories may show slow. erratic absorption, which limits lheir use in the management

of acute seixure'". Formulation of diazepam as a hydrogel for rectal application has been

extensively studied, and has been reported to be as effective as the intravenous route165.

However, such rectally-administered drugs may leak from the rectum, causing inadequate

dosing 311d treatment failure, and therefore requiring a nlodulalion of their rheological

properties1".

Attempts at formulating parenteral diazepam applicable to emergency conditions

have utilized such approaches as use of co-solvents, cyclodextrins, pH control, surfactants,

as well as a combination of mechanisms. Holvoet et 01.~' used cyclodextrins in an attempt

to find alternative formulation with no co-solvent-related effects. With diazepam,

however, the viscosity of cyciodextrin solutions at the solubilizing concentration and more

importantly, the haemolytic activity of ~ ~ c l o d e x t r i n s ' ~ ~ make their use worrisome.

Alvarez and co-workers'68 posit that co-solvency is the best approach to the formulation of

parenteral diazepam based on studies done with co-solvents, cyclodextrins, pH cq t ro l ,

surfactants and combinations of these agents. Their report, however, ignores the clinical

problems associated with the use of co-solvents, and evaluates only stability and solubility

parameters.

The different emulsion-based formulations that have been studied must be

viewed in the light of the potential hazards associated with parenteral

administration of emulsions and the need to determine and regulate oil droplet

size16'

1.6 Sodium benzoate: a non-toxic hydrotrope

molecular structure of sodium benzoate

Sodium benzoate is a white crystalline powder with a density of 1.44 gem" and a

melting point of 300" C. It is generally stable but may be moisture sensitive. It is

incompatible with oxidizing agents, mineral acids and alkalis. Sodium benzoate finds use

1 70- 1 72 as a preservative in a number of consumable products . Studies have indicated a lack

of toxicity on intake17'.

Following oral administration, it is metabolized in the liver by conjugation with

glycine to form hippuric acid17'. This conjugation process has been described as saturable

in h ~ r n a n s ' ~ ~ , d fo rm the basis of a liver function test.

Sodium benzoate has a strong hydrotropic property that has been utilized in the

analyses of d r ~ ~ s ' ~ ~ - ~ ~ ~ and in the formulation of injectable products'78, where it replaces

the more tosic arid costiy organic solvents.

CHAPTER TWO

EXPERIMENTAL

2.1 Materials

Propylene glycol (E-Merck, Germany), sodium benzoate (BDH, England): normal

saline (DANA, Nigeria), phenobarbitone sodium (Renaudin, France) were used as

obtained from the manufacturers. Diazepam 1irz1s donated by Fasil Rakwon Ltd (Nigeria).

Fresh human blood was obtained from McChuks Laboratories Ltd (Nigeria). Distilled

water was obtained from Jokem Ltd (Nigeria). All other reagents were of analytical grade

and were used as such. White albino mice weighmg between 22 - 43 g were used. , 2.2 Methods

2.2.1 Determination of equilibrium solubility of diazepam

An excess mount of diazepam powder was added to each of six conical flasks

containing, respeclively, 50 ml of different concentrations (0.05 M to 1.0 M) o r sodium

benmate. The flasks were shaken in a thermostated water bath (01 T 643, Helo,

Denmark) at 30 "C for 24 hours. The dispersions were allowed to equilibrale for a further

1-hour period md then filtered through a filter paper (Whatman number 1). An aliquot of

the filtrate was analyzed spectrophotometrical$r (UV-2102, Unico. U.S.A) at 31 1 m A

similar procedure was followed for solubility determinations at 40 "C and 60 "C

respeclively.

2.2.2 Determination of mechanisms of solubilization

(i) Speh-al llieasure~nf The spectra of two concentrabons (5 rng % and 10 mg %) of

diazepam in 0.12 M sodium benzoate solution were recorded in a spectrophotometer

(UV-2102, Unico, U.S.A.), between 200 nrn and 400 nrn.

(u) Conductivity measurement The conductivrty of sodium benzoate solutions (0.05 M -

1.0 M) was determined at 31.050.4 OC using a conductivity meter (CO 150, Hach,

England)

(iii)l/iscosity and density measurements The relative viscosity o r sodium benzoate

solutions (0.05 M - 1 0 M) was determined (?t 30*2 OC using Ostwald U-tube

viscometer and a stopwatch (Stopstar 2, Hantart, Germany). The density of each solution

was determmed at 30*2 "C using a 10 ml pycnometer.

2.2.3 Stability testing

pH measurements were performed on each of different sodium benzoate solutiqn al

30*2 O C usmg a digital pH meter (Labtech, India). Three concentrations (0.12 M, 0.35 M,

and 0.80 M) of sodium benzoate solutions were then used to achieve three solubility levels

of diazepam. Each solution of diazepam was distributed into three amber-coloured glass

bottles and placed respectiilely at 40 "C, 50 "C and 60 OC. Samples were withdrawn at

four-day intervals for 4 weeks and assayed for content of diazepam.

2.2.4 Haemolysis studies

This was camed out according to the method described by Okore er 0 1 . ' ~ ' . The

defibrinated red blood cells were obtained by centrifuging 10 mI of whole blood at 3000

rpm for !he minutes. The supernatant was decanted, and the packed cells were re-

suspended in normal saline, stirred gently with a glass rod and centrifuged again. This

cycle of washing and centrifuging was repeated until the supernatant became colourless.

The cells were now made up to original volume with normal saline and refrigerated until

required for use. A stock solution of 1 % diazepam in propylene glycol was prepared, and

from ttus, dilutions were made either with normal saline, 0.12 M sodium benzoate solution

or propylene glycol to produce solutions having concent~ations of 5 mg % to 80 mg %

diazepam.

A five-millilitre volume of each concentration of diazepam was incubated at 30 "C

with 0.5 ml of red cell suspension for 30 minutes. The resulting dispersion WE centrifuged

at 3000 rpm for 5 minutes, in order to separate the cellular materials. Haemoglobin

released from lyzed cells formed part of the supernatant solution. The haemoglobin

solutior~ was diluted appropriately with normal saline and its optical density was measured

with a photoelectric calorimeter (AE-11 D, B. Bran, England) at 531 nrn. Readings were

expressed as percentages of those obtained after total haemolysis achieved by laking red

cells in distilled water at 30 O C for 2 hours. The determination was conducted in triplicate.

Similar determinations were carried out for the blank vehicles (without diazepam) to b

dete~mine their intrinsic haemolytic activities.

2.2.5 Animal studies

These were necessary to ascertain the effects of sodium benzoate on the

pharmacodynamics of diazepam, and to compare such eflects with the values for the

propylene glycol vehicle, which is commonly used for delivery of diazepam.

2.2.5.1 Barbiturate hypnosis test17'

Mice of allelerage weight of 35.4 g were divided into three groups of three each.

N o d saline was administered at a dose of 10 mlkg i.p. lo the first group. Diazepam (in

0.8 M sodium benzoate solution) at a dose of 1 mg/kg i.p. was administered to the second

group, while the third group received 1 mgkg i.p. of diazepam in propyfene glycol.

Afier 30 minutes of initial drug administration, sodium phenobarbitone (40 mg/kg

i.p.) was administered to the animals in each group. The onset and duration of sleep were

determined by the time to loss and duration of loss of righting refledgo respectively, after

administration of the second drug.

2.2.5.2 Pentytenetetrazole seizure test

The test mice were divided into three groups of three each Normal saline (10

mlkg i.p.) was administered to each animal in group one, diazepam (1 rnglkg i.p.) in 0.8 29

M sodium benzoate was administered i.p. to each animal in the second group and

diazepam (1 mg/kg i.p.) in propylene glycol was administered lo each animal in the third

After 30 minutes of initial administration, pentylenetetrwole (40 mgkg i.p.) was

administered to each of the animals. The time taken for onset of clonic convulsions, the

duration of clonic convulsions and the percentage of seizure and mortality protection were

recorded'". Ar, animal was deemed as protected if no seizure occurred &er one hour.

2.3 Statistical analysis

Where applicable, values were presented as mean st s.e.rn. Statistical analyses #

were done using Student's t-1-est. Values of P< 0.1 imply statislical significance.

CHAPTER THREE

RESULTS AND DISCUSSION

3.1 Solubility of diazepam

The solubiliQ, curves for diazepam at the three test temperatures presented in Fig 1.

show that the solubility of diazepam increased substantially with increases in

concentration of sodium benzoate. The solubility of diazepam increased from about 7 mg

% in the 0.05 M solution of sodium benzoate to 29 mg % in the 1 M solution. The higher

temperalures gave higher solubilily values, confirming that dissolution is endothermic. A

non-linear relationship was observed with gradual changes in solubility in the first 1

portions of the curve, with subsequent increase in the rate of change of solubility. Changes

in solubility were gradual in the first portions of the curves, and then rapidly changed,

showing marked increases in solubility. This positive deviation, which occurs over a

narrow range of concentrations, is characteristic of hydrotropic solubilizationS5. The

hiphasic phenomenon is indicative of the multifactoral influences involved in the

mechanisms of solubilization. Hi&er hydrolrope concentrations would, therefore, give

much hgher solubility values than in an othenvise linear relationship. Improvements in

the solubilities of organic molecules undergoing interactions have been attributed to the

formation of complexes having better solubilities than one or bolh of the interacting

molecules'R2. Whatever the mechanism(s), the hydrophobic nature of the solute was

counteracted in a manner that permitted a better interaction of solute and solvent to give a

soh tion.

0.00 0.20 0.40 0.60 0.80

Concentration of soduim benzoate (M)

Fig.1: Effect of sodium benzoate on solubility of diazepam at different temperatures

- m - at 30 C ---~t- at 40C -+- at 6 0 C

3.2 Spectral analyses

Fig. 2 shows the uv spectrum of diazepam in the absence of sodium benzoate.

This reveals two definite peaks, a major peak at 242 nm and a minor peak at 3 1 1 nrn. On

the other hand, the spectrum produced by sodium benzoate solution alone shows no

distinctive peaks but rather, a cluster, giving rise to an irregularly broad band of peaks

(Fig. 3). Figs. 4 and 5 show the spectra due to varying concentrations of diazepam in 0.20

M sodium benzoate. This concentration of sodium benzoate was chosen because

molecular compleses have been reported to occur at low hydrotrope con~entrations'~,

while salting in'" or other influences predominate at higher concentrations. The smaller t

peak due to diazepam is evident at 3 1 lnrn (Fig. 4) or 3 12 nrn (Fig. 5), and is distinct from

the broad bands due to sodium benzoate, which probably mask the higher peak at 242 nm.

The peaks vary in magnitude in direct proportion with the concentration of diazepam in

the system. The absence of new peaks due to the combination indicates that molecular

complexation is absent. However, new peaks may not appear even if complexation is

presenl, particularly if the extenl of complexation is small. Such a small reaction may not

give rise to appreciable difference in absorption around the peak of maximum

ab~orpt ion~~. Also, if very weak complexes are formed, the bonds may be too weak to

manifest any pronounced changes in spectral characteristics of the associating

molecules'xs Furthermore, the UV study, apart from revealing no new peaks, shows no

shifts in the A,, of diazepam. Th~s eliminates the possibility of permanent interaction.

Test Report Test Date: 613012007 User Name: Nzekwe lfeanyi Test Mode: SCANNING Graph's Name: Sample scan of Diazepam in Distilled water Start Wavelength: 200.0 nm End Wavelength: 401.0 nm Scan Interval : 3nm nm

peak: WL01=242,0 Abs=2,425 WLO2=3ll.O Abs=0,412

Fig. 2: Spectra of diazepam in distilled water

3 4

Test Report Test Daten 02-23-2007 User Name: Nzekwe Test Mode: SCANNING Graph's Name: Scan of 0.2 M sot~um benzoate in distilled water. Starl Wavelength: 200,O nm End Wavelength: 400.0 nm Scan Interval : Inm nm

Fig. 3: Spectra of sodium benzoate solution

35

Test Report Test Date 02.23-2%; User Name Yzekwe Test Mode SCANNING Graph's Nape Scan of 5 mgCh d ?zepanln 0 2 FA sodlum kenzoate Start Wavelength 220 O rn Erd Wavelength 4130 0 nm Scan lnlerial I i m nm

Fig. 4: Spectra of diazepam (5 mg %) in the presence of sodium benzoate.

3 6

Test Report Test Date: 02-23-2007 User Name: Nzekwe Test Mode SCANNINGtItni Graph's Name: Scan of Qmg% ~ i a z e p a ~ i n 0.2 M sodium benzoate . Start Wavelength 200.0 nm End Wavelength: 400.0 nm Scan Interval : Inm nm

Abs

peak: WL01=312.0 i\bs=0.939

Fig. 5: Spectra of diazepam (10 mg %) in the presence of sodium benzoate

3.3 Conductivity, density and viscosity measu~wnents

To further understand the exact nature of the mechanism(s) of solubilization, the

conductisities, viscosities and densities of sodium benzoate solutions were plotted as a

function of the molar concentrations. These relationships are depicted in Figs. 6 to 8.

In the conductivity curve (Fig. 6), there is an initial linear relationship between the

conductance of the solution and its concentration. This linearity, however, was interrupted

by a break at around the concentration value of 0.5 M, after which the slope falls slightly,

showing a decrease in conductance per unit concent~ation change. This is very instructive

because in an ionized system, the number of ions carrying electric charges increases b

direclly with concentration. Since the number of sodium benzoate molecules remains

constant, the break in conductivity is highly suggestive of molecular aggregations6.

Density and viscosity measurements follow essentially similar patterns. The density curve

(Fig. 7) showed a decrease in slope about the same point, and this is indicative of increase

in partial mold volume, suggesting aggrewe formation50. The relative viscosity cunre

(Fig. 8) shows a change in slope at the same concentration, indicating that higher shear

rates are needed to cause moleculru displacement. 731s is co&~rmative of the involvement

of moleculm aggregation'86, similar to micellization phenomenon in surfactant systems.

At a certain critical concentration, the molecules undergo a change in intermolecular

attraction, causing adhesion of molecules and forming a characteristic matrix in which

solute particles are trapped. Such an interaction with greater intermolecular bonding will

no doubt raise the viscosity of the system, necessitating an increase in shear force to cause

displacement. Th~s concentration is sometimes described as the minimum hydrotropy

concentration. It is expected that the physical properhes of the solution notably surface

tension, density and viscosity will change over this narrow range of concentrations.

0 0.2 0.4 0.6 0.8 1 1.2

Sodium bemate concentration (M)

Fig. 6: Plot ofconductivrty vs molar concentration ofsodiurn bemate

Concentratjon (M)

Fig. 7: Plot of density against molar concentration of sodium benzoate

0.2 0.4 0.6 0.8

concentration of sodium benzoate (M) Fig. 8: Plot of relative viscosity against concentration of sodium benzoate

3.4 Stability. The effects or concentraion 01 sodium benzoate on the degradation kinetics

of diazepam are presented in Fig. 9.

Also, the pH values of different solutions of sodium benzoate are presented in Fig.

10, while data on the kinetics of stability are presented in Table I. Diazepam showed

stability in the three concentrations of sodium benzoate. Stabilily frequently results from

interactions between drug and ligand, even though there are reports on the fncilitation of

degradation or participating molecules upon molecular intera~tion'~~. The 0.12 M solution

of sodium benzoate gave an extrapolated degradation rate constant, K, of 2.42~1 ~ ~ ~ b h r - ' at

25 O C , while the 0.35 M and the 0.80 M solutions gave extrapolated rate constants of

2 .63~10" hr-' and 2.8 1x1 o - ~ hr-' respectively. These values were calculated from the

Arrhenius equation (Eq. 4) at 298K The corresponding shelf lives of 5.0 years, 4.6 yrs and

4.3 years respectively were computed by means of Eq. 6. The activation energy of

hydrolysis was calculated as 22.0 kcal mol -I, 22.20 kcal mol-' and 22.35 kcalmol-',

respectiveiy, for the three cot~entcations of sodium benzoate.

0.12 M 0 0.35 M A 0.80 M

Ihea r (0.80 M) - - - - - Linear (0.35 M) - - - - Linear (0. 12 M) ~ n s 01 s o d i ~ m bcnrnalc

Fig. 9: Arrhenius plots for diazepam at the three concentrations of sodium benzoate

0 0.2 0.4 0.6 0.8 1 1.2

Conc. (M)

Fig. 10: Plot of pH against concentration (M)

Table 1

Kinetics of stability data for the test concentrations.

Conc. of

sodium

Shelf life (yrs>

benzoaie

(MI

0.12

0. 12

0.12

Temp (K)

313

32 3

333

Rate Of

degradation of

3.195

3.096

3.003

Ln k Activation Energy (Kcal/xnol)

diazepam. k

(hr- I )

1.54 s

4.35 x lo4

1 .28 s I 0"

-1 1.08

-10.04

-8.96

22.00

The formation of micelles or inclusion aggregates lead to a two-phase system in

which it is expected that the rate of degradation in the micellar phase will be lower than in

188-189 the continuous phase due to protection of drugs from attacking ions . The aggregation

of sodium benzoate molecules at about the concentration of 0.5 M has been illustrated.

The 0.8 M formulalion was, therefore, expected to display the highest slabilily. The results

obtained (Fig. 9 / Table 1) point to the greater influence of pH on stability of diazepam in

sodium ben~oate solution, suggesting that degradation was by hydrolytic mechanisms. A

better understanding of this high stability is permitted by the earlier discussion on the

chemistry of diazepam. Diazepam degrades in a reversible hydrolytic pathway to give an b

intermediate product, which undergoes further hydrolysis to give other end products. This

hydrolysis is catalyxed by both hydrogen (H') and hydroxyl (OFF) ions. i.e. both acidic

and alkaline conditions with maximum stability of diazepam occurring at around pH 5.5.

The pH values of the solutions of sodium benzoate used for stability studies were 6.81,

6.94 and 7.13, for concentrations of 0.12 M, 0.35 M and 0.80 M respectively (Appendix

IIId). The 0.12 M system has the nearest pH value to the pH of maximum hydrolytic

stability of diazepam, and hence the lowest degradation rate and the longest shelf life. The

pH value of 7.13 due to the 0.80 M solution is farthest from this maximum stability value,

and this condition is expected to produce marked instability of diazepam. However, the

shelf life 01'4.3 years due to lhis system couid be attributed to the formation of protective

188-189 aggregates

The high stability of hydrotropically-solubilizd systems is well known1g0. But it

is necessary to corroborate stability values from stress tests with shelf stability because

123-124 multiple mechanisms may be involved in the degradation process .

The temperature effects are predictable for all the formulations. The rate of

degradation In the 0.35 M system increased by a factor of 9 between 40 "C and 60 "C, and

ths furher confirms the hydrolytic nature of the degradation, which is accelerated by 46

Ic~i ipcralulc. I'his can be a(tr ibu~cd to the highcr hinctic energy 0 1 ' the molcculcs a1

\,aluc ;IS c ! o x as possible to the pl 1 ol 'masimum stubilitj, ol'tlrc dvi~g,. 'l'his choice can be

made 1.1-0111 s consideration 01' Ihc pK, ol' the agcnt. 'Thc hul'lkring propc~ ly oi' sodiunr

bcwoatc i 4 ;in advantage in this regard.

3.5 I laemnlyis

I hc rc\uIl\ ol ' l l ic h a c l n o l j s i ~ tests arc pvcscntctl in I:ig\. I I to I ? .

Incrca4ing conccntra~icr~rs oi'di;~/cpam in pwpylcnc glycol a n d sodiurn bcn/o;-ltc systems

rc4~1Itcd 111 i~lcrc:~sed Ic\!eI.\ 01' hacmolysis (I:ig. I I ). I'his conccntr:rtion-dc~>cndc~it cfl'cct

whcvc a rapid Increase in hacmol~sis occurred. 13ct\vccr, S mg %, and 40 mg 'Yo. the lcvcl

[lie hacmcrl>\l~ \aluc4 arc 0.6 '%) (sot l iun~ bcnzoatc) or 45 '35 (prop) Icric glycol). while at

40 Ing '%,. Ihc hacmc1ly\i4 \/aluc\ arc 36 'XI and 70 '$0 rc \pccl i \ t l ) (Appcndi\ VI).

0 10 20 30 40 50 60 70 80 90

Concentration of diazepam (mg O h )

Fig. I I : Haemolysis curves for diazepam in different vehicles

+ 0.12 M Sodium Benzote + Propylene Glycol +Normal Saline

0 2 0 40 6 0 8 0 100 120

Concentration of propylene glycol (%)

Fig. 13: Haemolysis curve for propylene glycol in water

In the sodium benzoale-based formulalions, haemolysis values remain relalively

constant over the range of 10 mg % to 40 mg % of diazepam, while the lowest haemolysis

\ d u e of 43 % is obtained at 25 mg % concentration of diazepam.

A 0.72 % (0.05 M) solution of sodium benzoate (without diazepam) gave a high

haeniolytic value of 50 % (Fig. 12); which may be attributed lo osmotic effects of dilute

solutions. The 1.73 O/o (0.12 M) and 5.04 % (0.35 M) solutions gave 5 % haemolysis and

10 % haemolysis respectively. An 11 solulion (0.76 M) solution gave 17 % haemolysis

(Appendix VII). b

In comparison, all the concentrations oC propylene glycol gave haemolysis values

131, 135 greater than 80 %. This is in agreement with the findings of earlier workers . This

massive haemolysis elicited by propylene glycol in all concenimtions, is oE lnajor concern

and seriously questions the continued use of propylene glycol as a vehicle, particularly for

drugs with proven haemolflic activitI\,, such as diazepam.

3.6 Hypnosis test result

The result of hypnosis test is presented in Table 2.

The average lag time prior to onset of sleep for the negative control, sodium

benzoate-based md pr-opylene glycol-bmed diazepam formulations were 31.66 * 8.33,

16.00 * 4.51 and 0.00 f 6.51 minutes respectively. On administering the diazeparn-in-

sodium bemoate formulation sleep latency was reduced from 3 1.66f 8.33 to 16.00k4.5 1

minutes, while total sleep time was prolonged from I10.0W 30.41 lo 287.00k38.69.

Table 2: Potentiation of phenobarbitone-induced sleep

Formulation Dosei'

NS (without

No of animals per group is 3

1 0 mlkg

diazepam) .. - -

DSB

(15 ~ng %)

Onset of sleep (m%)

. ~ - - -

1.0 mg/kg

* Significant at p < 0.1

NS means normal saline

DSB means diazepm in 0.8 M sodium benzoate

Duration of sleep (hn)

DPG means diazepam in propylene glycol.

a: This dose was followed after 30 minutes with phenobarbitane sodium, 40 mgkg i.p.

This pattern of response is common to modulators of gamma-amino bulyric acid

(GABA) receptors such as barbiturates, ben/.odiazepines cytolil~les~ zopiclone and

neuroactive steroids and also the selective GABAR agonist (musimol) that have been

found to increase sleep continuity and promote non-REM sleep at higher dose.

The propylene glycol formulation produced a shorter time to onset of sleep of

9.00*0 5 1 minutes. w}iich was statistically significant when compared with data obtained

for the control g~oup. Also, the total sleep time of 259.00 k 22.23 minutes obtained for the

propylene glycol group was also statistically different from (he control.

Propylene glycol is highly lipophilic, and this may explain the slightly shorter time I

to sleep onset and the shorter total sleep time. There may have been faster absorption and

eliniination rates of propylene glycol-based formulations, compared to the sodium

ben~ode-based formulations. There is, however, no statistical significance in the time

values either Ibr onset of sleep (P > 0.1) or the total sleep times (P > 0.1) between the two

fot~nulations.

3.7 Seizure test result

The pentylenetetrazole ,mticonvulsant test gave similar results for the two

formul,?lions; as presented in Table 3.

Both formulations provided 1 00 percent protection from penqlenetetrazole-

induced seimre as against the negative control group where zero protection was recorded

(100 96 mortality). In experimental animals administration of PTZ causes instant

~onvulsion''~'. The effects of the two formulations in idubiting this reaction are consistent

with GABA-mediated inhibition because PTZ acts at GABA receptors to bring about

hyper cxcitabilityl".

Table 3: PI-otection by diazepam against pentylenetetrazole-induced seizure.

DSB (15 mg%)

DPG (20.6 mg %)

No ol' animals per group is 3

* S~gnificanlatp<O.I

NS means nnrmal saline

DSB means diazepam in 0.8 M sodium benzoate

DPG means diazepam in propylene glycol

n . Thls dose was foilowed after 30 minutes with pentylenetetrazole, 40 mgkg i.p.

~ o s e ~

10 mllkg

I.Omg/kg

1 .O mdkg

Perccnt protection (%)

0

100

#

100

Quanta1 protection (out ofthree)

013

CIonic seizure Onset Duration (mi n) (min)

9.67*3.08 1.33+0.33

-

-

1

313

-

a) Sodium benzoate improves the solubilih. of diazepam in water by hydrotropic

mechanisms. The \ d u e of 6.9 mg % obtained in the most dilute solution of sodium

ben~oate tested (0.05 M) at 30 'C in this study is in fair agreement with the experimental

solubility vdue of 5.7 mg % obtained by LoTtsson and ~ re insdo t t i r ' ~~ in pure water at

room temperature using a similar method. Higher values could be obtained by using

higher concentrations of hydrotropic salt since the solubility relationship shows positive

deviation from linearity-.

b) The mechanism of hydrotropy of sodium benzoaie is by self-association at a 8

threshold concentration, determined here as about 0.5 M, at which a marked improvement

in solubility or co-solute and change in solution properties occur, comparable to the

behaviour of ampiphiles at their CMC.

c) Diazepam is very stable in sodium benzoate solution. This is due to favourable pH

profile of sodium benzoate solutions and to the f o d o n of protective molecular

aggregates.

d) Diruepam has an appreciable haemolytic activity in vitro. Since it is used in small

therapeutic doses, massive haemolysis may not occur in vivo.

e) Propylene glycol causes massive haelnolysis of red blood cells in dl proportions

with water. The continued use of propylene glycol as vehicle in diazepam injections

should be reviewed on the basis of our findings and evidence highlighted in the literature.

f) Fortnulation of diazepam in sodium benzoate gives lower levels of haemolysis than

in propylene glycol in vitro

g) There is no statistical difference between the pharmacodynamic activities of

diazepam in propylene glycol and sodium benzoate solution, adh respect to potentiation

of' phenobarbitone-induced sleep, or control of pmtylenetetrazole-induced seizures.

3.9 Conclusion

This evaluation of sodium benzoate solution as a vehicle for parenteral diazepam

was carried out by testing the effect of sodium benzoate on the solubility, stability,

haemolytic activity and pharn~acological actions of diazepam. An attempt at esplaining

the outcome on the basis of interaction between sodium benzoate and diazepam in the

aqueous medium has been made. Preliminary data generated in this work indicate that

sodium beruoate solution can be successCully applied as vehicle in the formulation of

diazepam injection, and that such a formulation will not exhibit the high toxicity of

proprietary diazepam-in-propylene glycol injection. Besides achieving fair solubility b

levels, the stability of diazepam is also enhanced under suitable pH conditions without the

necessity for other buffering agents.

Hydrotropes. however, are frequently used in equirnolar amounts with the drug,

and sometimes at very high concentrations, lhereby making the formulation to exhibit lhe

sensual properties (odour, colour, etc.) associated with the hydrotrope. Therefore, a final

choice of a suitable concentration of the salt for enhancing the solubility of diazepam must

be based on s compromise between drug solubility and maGmum acceptable interferences

in sensual properties or formulations.

The bioavailability and precipitation propensity on dilution are other assessments

that should be done before the final choice of a suitable concentration of sodium benzoate

is made. Tlus is because the solubilized drug is like a reservoir and the pharmacological

activi:). is due to free drugl9> whch should be yielded on administration or dilution. Also,

it may be necessary to investigate the influence of sodium benzoate on diazepam with

respect to other pharmacologicd actions such as its anxiolytic and musle relaxant effects,

to ensure that such a formulation will retain the capaci@ for application in all the

conditions where the propylene glycol-based formulation has proved effective.

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Boern plot of disznparn In sodlurn benzoate solullon

Appendix I b

Table for Beers Plot

A = 0.0723 C, where A is the absorbance and C is the concentration of diazepam

r Conc. (mg %) Absorbance

Conc. of sb (M)

---- 0.05

0.12

0.20

0.35

0.50

0. HO

1.00 -

Appendix 11 a

Table for solubility values at 30 OC

Absorb. I Absorb. 11 -- Mean

absorb.

0.501

0.510

0.632

0.926

1.130

2.028

2.147

Cone, of diazepam (mg %)

sb means sodium benzoate

Appendix 11 b

Table of values for solubility at 40 O C

Absorb. I Absorb. 11 Mean Conc, of diazepam (mg %)

absorb.

Appendix I1 c

Table of values for solubility at 60 O C

Conc. of sb (M)

0.05

0. 12

0.20

0.35

0.50

0.80

1 .OO

Absorb. I Absorb. II

0.690, 0.707

0.919, 0.925

1.163, 1.163

1.807, 1.836

2.681, 2.681

2.681, 2.681

2.681 2.681

Mean

absorb.

0.700

0.922

1.163

1.822

2.68 1

2.68 1

2.681

Conc. of diazepam (mg %)

9.68

12.75

16.09

25.26

37.08

37.08

37.08

Appendix LII a

Conductivity data

Conc. of sb (M) Conductivity (ms)"

a: ms is millisiemens

Appendix I l l b

Viscosity data for sodium benzoate solutions

Conc. of sb (M) Mean time Rel. visc.

--

Cone. of sb (M)

Appendix 111 c

Density data for sodium benzoate solutions

Weight of 1 Weight of bottle

bottle (g) 1 + solution (g)

Weight of

solution (g)

9.860

9.880

9.935

9.979

10.070

10.1 83

10.324

1 0.42 1

Density @m")

Appendix IIId

pH values of different concentrations of sodium benzoate solution

Conc. (M)

0.00

0.05

0.12

0.20

0.35

0.50

0.80

1.0

PH 6.35

6.71

6.8 1

6.89

6.94

7.01

7.03

7.22

APPENDIX IV a

STABILITY DATA FOR DIAZEPAM IN 0.12 M SODIUM BENZOATE AT 40 OC

Tim. (hrs.)

0

72

168

240

336

408

504

576

624

Absorb (av.)

0.32 I

0.32 1

0.320 '

0.320

0.319

0.319

0.319

0.3 18

0.318

Conc. of diazepam (av.)

4.439

4.439

4.426

4.426

4.412

4.412

4.412

4.3 98

4.398

Ln (conc. of diazepam)

I .49O4

1.4904

1.4875

1.4875

1.4843

1.4843

1.4843 b

1.481 1

1.4811

APPENDlX 1V b

STABILITY DATA FOR DIAZEPAM IN 0.35 M SODIUM BENZOATE AT 40 OC

Tim. (hrs.)

0

72

168

240

336

408

504

576

624

Absorb (av.)

0.641

0.639

0.638

0.638

0.637

0.636

0.635

0.634

0.634

Cone. of diazepam (av.)

8.866

8.838

8.824

8.824

8.81 1

8.800

8.783

8.770

8.770

Ln (conc. of diazepam)

APPENDIX IV e

STABILITY DATA FOR DIAZEPAM IN 035 M SODIUM BENZOATE AT 50 "C

Tim. (hrs.) Absorb (av.) Conc. of diazepam (av.) Ln (conc. of diazepam)

APPENDlX 1V f

STABlLITY DATA FOR DIAZEPAM IN 0.80 M SODIUM BENZOATE AT 50 "C

Tim. (hrs.) Absorb (av.) 1 Conc of diazepam (av.) Ln (conc. of diazepam)

#

APPENDlX 1V 11

STABILITY DATA FOR DIAZEPAM IN 0.35 M SODIUM BENZOATE AT 60 "C

STABILITY DATA FOR DIAZEPAM IN 0.12 M SODIUM BENZOATE AT 60 "C

Tim. (hrs.)

0

7 2

168

240

336

408

5 0.1

576

624

Tim. (hrs.)

0

72

168

240

336

408

504

576

624

Absorb (av.)

0.64 1

0.634

0.625

0.6 19

0.6 1 1

0.605

0.600

0.59 1

0.586

Cone. of diazepam (av.)

8.866

8.769

8.645

8.562

8.45 1

8.368

8.299

8.174

8.105

Absorb (av.)

0.321

0.318

0.314

0.31 1

0.307

0.304

0.30 1

0.298

0.296

Ln (conc. of diazepam)

2.1 822

2.1712

2.1570

2.1473

2.1343

2.1244

2.1161

2.1010

2.0925

Conc. of diazepam (av.)

4.439

4.398

4.343

4.302

4.246

4.205

4.1 63

4.122

4.094

7

Ln (conc. of diazepam)

1.4904

1.481 1

1.4686

1.459 1

1 .446C)

1.4363

1.4262

1.4163

1.4095 I

APPENDIX lV i

STABILITY DATA FOR DIAZEPAM IN 0.80 M SODIUM BENZOATE AT 60 OC

Tim. (hrs.) Absorb (av.)

1.787

Conc. Of diazepam (av.)

24.716

24.398

Ln (conc. of diazepam)

3.207 5

3.1945

3.1 803

Appendix V a

0 100 200 300 400 500 600 700

Time (hours)

Degradation of diazepam in 0.12 M sodium benzoate at 4 0 ' ~

Appendix V b

Time (hours)

Degradation of diazepam in 0.35 M sodium benzoate at 4 0 ' ~

Appendix V c

Time (hours)

Degradation of diazepam in 0.8 M sodium benzoate at 4 0 ' ~

Appendix V d

0 100 200 300 400 500 600 700

Time (hours)

Degradation of diazepam in 0.12 M sodium benzoate at 50 OC

Appendix V e

100 200 300 400 500

Time (hours)

Degradation of diazepam in 0.35 M sodium benzoate at 50 OC

Appendix V f

0 100 200 300 400 500 600 700

Time (hours)

Degradation of diazepam in 0.80 M sodium benzoate at 50' C