Pharmaceutical preparation for topical administration of antigens and/or vaccines to mammals via a mucosal membrane

A novel type of formulation for the topical administration of antigens and/or vaccines to mammals via mucosal membranes comprising one or more adjuvants/vehicles selected from (a) polyoxyethylene sorbitan monoesters, (b) polyoxyethylene castor oil, (c) caprylic/capric acid glycerides and (d) gangliosides in an amount of 0.01 to 15% (v/v) calculated on the total volume of the preparation. This formulation enhances the immunological response n a mammal following mucosal administration, e.g. nasal, oral, rectal or vaginal application.

The present invention relates to novel pharmaceutical preparations for 
topical administration of antigens and/or vaccines to mammals, including 
humans, via a mucosal membrane. The invention also relates to the use of 
certain compounds (to be defined in more detail below) as adjuvants or 
vehicles in such preparations. 
The parenteral (intramuscular and subcutaneous) administration of antigens 
and/or vaccines is normally regarded as the most convenient way of 
administration. However, the administration by injection presents a range 
of disadvantages. Thus it requires the use of sterile syringes and may 
cause pains and irritations, particularly in the case of repeated 
injections, including the risk of infection. More significantly, in the 
case of intramuscular injections there is also a risk of the infection 
being poorly tolerated. There is likely to be an induration (hardening of 
tissue), haemorrhage (bleeding) and/or necrosis (local death of tissue) at 
the injection site. Besides, injections cannot be administered 
satisfactorily by untrained persons. 
Administration of attenuated virus, bacteria or parasites has been 
attempted intranasally as well as through other mucosal surfaces. The 
elicitation of an immune response by such antigens through mucosal 
surfaces cannot be considered unexpected in such cases, because the 
modified live pathogens of the vaccine is following the natural route of 
infection of the wild-type pathogen creating immunity through a 
sub-clinical infection. The use of modified live pathogen to effect 
immunization entails a certain risk, however, because the more purified 
antigens are very poor immunogens and thus require effective formulations 
and adjuvants to produce a clinically protective immune response. 
Mucosal administration is currently receiving special interest, attempting 
to stimulate locally produced antibodies (secretory IgA antibodies) and 
also to avoid the inconveniences caused by the direct intervention into 
the organism in connection with parenteral administration. Additionally, 
this route of administration may conveniently be used as an alternative to 
parenteral injection, since it may well be performed by an untrained 
person. Furthermore, small children will avoid the psychological 
irritation during injection (vaccination). 
In order to be an attractive alternative to parenteral administration, the 
intranasal administration should be capable of stimulating humoral and 
cellular immune factors both systemically (mainly of the IgG isotype) and 
at mucosal surfaces where most pathogens enter the host by locally 
produced antibodies of the secretory IgA (IgA.sub.s) isotype. Several oral 
vaccines have been shown to induce appropriate IgA.sub.s responses in 
remote secretions including saliva, lachrymal fluid and fluids obtained 
from nasal and gastrointestinal washes. Such intranasally administered 
vaccines and/or antigens may not cause any considerable pain or irritation 
to the patient nor any irreversible damage or irritation to the mucosal 
surfaces. 
In nasal administration, the antigen and/or vaccine must be applied to the 
mucosa in such a condition that it is able to penetrate or to be absorbed 
through the mucosa. In order to penetrate the mucus the vehicle must be 
biocompatible with the mucus and hence have a certain degree of 
hydrophilicity. 
Vaccines and/or antigens are not able to be administered in pure form. It 
is necessary to blend them with other components to obtain a preparation 
which is ready for use. Dependent on the chemical properties of the 
antigen and/or vaccine it will be necessary to take various considerations 
into account before a pharmaceutical preparation for humans or animals can 
be produced. 
BRIEF DESCRIPTION OF THE DRAWINGS 
FIG. 1 illustrates a phase diagram of a composition according to the 
present invention. 
It has now surprisingly been found that the topical administration of 
antigens and/or vaccines to mammals via mucosal membranes can be performed 
in a new and significantly improved manner by using a novel type of 
formulation, said preparation being characterized by comprising one or 
more adjuvants/vehicles selected from 
(a) polyoxyethylene sorbitan monoesters of the general formula 
##STR1## 
wherein R is selected among laurate, palmitate, stearate and oleate, and 
wherein the sum of w, x, y and z is 4, 5 or 20; 
(b) polyoxyethylene castor oil produced by reacting 1 mole of castor oil or 
hydrogenerated castor oil with 10-45 moles of ethylene oxide; 
(c) caprylic/capric acid glycerides of the general formula 
##STR2## 
wherein each R.sup.1 independently is H or a C.sub.8 -C.sub.10 acyl group 
containing 1-6% free glycerol, 45-50% monoglycerides, 30-40% diglycerides 
and 5-9% triglycerides, and 
(d) gangliosides of the general formula 
##STR3## 
wherein Gal is galactose, Glc is glucose, Cer is ceramide (N-fatty acyl 
sphingosine) and NeuAc is N-acetyl neuraminic acid (sialic acid), and 
wherein R.sup.2 may be one or more substances selected among N-acetyl 
galactosamine, galactose, N-acetyl neuraminic acid or combinations 
thereof, and R.sup.3 is H or N-acetyl neuraminic acid 
in an amount of 0.01 to 15% (v/v) calculated on the total volume of the 
preparation. 
The nasal epithelial membrane consists of practically a single layer of 
epithelial cells (pseudostratified epithelium) and it is therefore even 
more suited for antigen and/or vaccine administration than other mucosal 
surfaces having squamous epithelial layers, such as the mouth, vagina, 
etc. These surfaces, however, are also well suited for the application of 
antigens and/or vaccines with the delivery system according to the 
invention. The extensive network of blood capillaries under the nasal 
mucosa is--together with the high density of T and B cells--particularly 
suited to provide a rapid recognition of the antigen and/or the vaccine, 
which may also provide a quick immunological response. 
For liquid compositions it is essential that the effective amount of the 
antigen and/or the vaccine can be administered in a volume of less than 
about 300 .mu.l for human subjects. A larger volume can be disagreeable to 
the patient and will evidently drain out anteriorly through the nostrils 
or posteriorly toward the pharynx. The result is that a part of the 
antigen and/or the vaccine is lost from the absorption site. 
The volume is preferably from about 20 .mu.l to about 125 .mu.l and 
preferably administered into both nostrils. 
A variety of vehicle systems for the delivery of antigens and/or vaccines 
have been developed. The literature to date has suggested that uptake of 
antigens and/or vaccines from the nasal mucosa is frequently made possible 
by incorporation of a special vehicle system into the formulation, adding 
certain amount of absorption enhancing agents or a certain amount of 
adjuvants. 
Much has been written regarding the potential use of various vehicles as 
drug delivery systems for intranasal administration. In such vehicle 
systems, the medicament is rapidly absorbed into the blood stream. One of 
the problems encountered in using such vehicle systems is that the antigen 
and/or the vaccine is absorbed and degraded without recognition and, 
therefore, without stimulating an immunological response. The system 
according to the invention describes a vaccine/antigen delivery system 
which provides a clear immunological response in spite of the short 
contact time inside the nasal cavity. 
A possible enhancement of the immunological response after mucosal 
administration of polyoxyethyl-35-castor oil, caprylic/capric acid 
glycerides and/or gangliosides together with an antigen or a vaccine has 
not been suggested anywhere in the prior art. 
U.S. Pat. No. 4,610,868 describes a lipid matrix carrier for parenteral 
administration of drugs. This system requires a lipid matrix carrier 
comprising a hydrophobic compound, an amphipathic compound and a bioactive 
agent with a globular structure of a diameter between 500 and 100,000 nm. 
Here the hydrophobic compound may comprise a mixture of glycerides and the 
amphipathic compound may comprise a sphingolipid. Furthermore, this 
formulation may be administered into the nasal area. However, this system 
is not acceptable as a nasal formulation, due to the rapid clearance 
inside the nose and the large globular structure. Therefore, this system 
will be transferred into the stomach by the cilia before the bioactive 
agent is released. 
U.S. Pat. No. 4,985,242 describes an intranasally applicable powdery 
pharmaceutical composition comprising a polypeptide with physiological 
activity, a quaternary ammonium compound, and a lower alkyl ether of 
cellulose. Typical surfactants in this composition are polyoxyethylene 
sorbitan fatty acid esters. This powdery pharmaceutical composition is 
stated to have an excellent preservability and chemical stability of the 
polypeptides. Further, when the composition is administered to the nasal 
cavity in the form of a spray, the polypeptides are absorbed effectively 
through the nasal mucosa. However, the surfactant concentration is 
critical since, on the one hand, high concentrations lead to sticky 
preparations without powder characteristics. On the other hand, low 
concentrations will not enable the induction of an immunological response. 
If the purpsose of U.S. Pat. No. 4,985,242 had been to induce an 
immunological response, which is not the case, this would be regarded as a 
serious drawback when protein and peptide drugs were to be administered. 
These surfactants would therefore not be usable for the purpose of the 
present invention. 
Several other references relating to the use of a polyoxyethylene 
derivative of a sorbitan ester in nasal preparations are known. However, 
no reference describes the substance according to the invention as an 
adjuvant or as an immunomodulator. This effect is indeed surprising and 
unexpected. A novel method of administering the natural female sex 
hormones 17.beta.-oestradiol and progesterone as solutions, suspensions, 
gels and ointments, containing 1% to 2% Tween 80, is described in U.S. 
Pat. No. 4,315,925. From EP Patent No. 246,625 is known an aqueous steroid 
formulation for nasal administration of an anti-inflammatoric steroid 
preparation containing propylene glycol, polyethylene glycol 400 and 1% to 
4% Tween 20. EP Patent No. 242,643 describes an intranasal administration 
of drugs, especially insulin, using e.g. 0.01% to 0.5% Tween 80 to reduce 
the nasal irritation by other absorption promoters. Finally, in 
PCT/AT87/00015 a sprayable, Tween-containing formulation for e.g. 
benzodiazepines is described. However, this formulation requires the use 
of a propeller gas. 
The present invention presents a new and significantly improved method for 
the administration of antigens/vaccines, using the above new type of 
formulation. The method provides protective immune response in recipients 
of the antigen and/or the vaccine, both systemically and locally, which 
are elicited after intranasal immunization. 
The primary object of the invention is to provide an intranasal 
composition, which is capable of producing a high systemic immune response 
(humoral and cellular, mainly of the IgG isotype) as well as locally 
produced antibodies of the secretory IgA isotype at mucosal surfaces 
without causing unacceptable damage to the nasal epithelial membrane. 
It is another object of the invention to provide a controlled delivery 
system for intranasal application, which is biocompatible with the mucus 
and which is capable of dissolving required amounts of antigens and/or 
vaccines in small volumes. 
According to an aspect of the invention the present delivery system is also 
usable for other mammalian surfaces such as the vagina, eye, mouth, lungs, 
ear, genital tract, gastrointestinal tract, rectum, skin etc. 
As mentioned previously, the pharmaceutical preparation of the present 
invention is characterized by comprising one or more substances selected 
from 
(a) polyoxyethylene sorbitan monoesters, (b) polyoxyethylene glycerol 
triesters, (c) caprylic/capric acid glycerides, and (d) gangliosides. 
The preferred polyoxyethylene sorbitan monoester (a) is Polysorbate 20, 
which is a laurate ester of sorbitol and its anhydrides copolymerized with 
approximately 20 moles of ethylene oxide for each mole of sorbitol and 
sorbitol anhydrides. 
The polyoxyethylene glycol triester (b) is preferably Polyoxyl-35-castor 
oil. This compound is mainly the triricinoleate ester of ethoxylated 
(about 35 moles) glycerol with smaller amounts of polyethylene glycol 
ricinoleate and the corresponding free glycols. Polyoxyl-35-castor oil is 
commonly known as Cremophor EL. 
The caprylic/capric acid glycerides (c) are principally a mixture of mono-, 
di- and triglycerides in which the acid groups are only caprylic and 
capric acid groups. They are known commercially under the trade name 
Imwitor. 
The gangliosides (d) of the above formula IV are principally a mixture of 
asialo-, monosialo-, disialo- and trisialogangliosides. 
The composition according to the invention may comprise one or more 
additional pharmaceutical excipients, selected among surfactants and 
absorption promoters, such as polyoxyethylene alcohol ethers, bile salts 
and derivatives thereof, fusidic acid and derivatives thereof, oleic acid, 
lecithin, lysolecitines, Tween 21 to 85, etc, water absorbing polymers, 
such as glycofurol, polyethylene glycol 200 to 7500, polyvinylpyrrolidone, 
propylene glycol or polyacrylic acid, gelatine, cellulose and derivatives, 
etc.; substances which inhibit enzymatic degradation, such as aprotinin, 
etc.; alcohols, such as ethanol, glycerol, benzyl alcohol, etc.; organic 
solvents such as ethyl acetate, benzyl alcohol, etc.; hydrophobic agents, 
such as vegetable oil, soybean oil, peanut oil, coconut oil, maize oil, 
olive oil, sunflower oil, "Miglyols" or mixtures thereof, etc.; 
pH-controlling agents, such as nitric acid, phosphoric acid, acetic acid, 
citrates, etc.; preservatives and osmotic pressure controlling agents, 
such as glycerol, sodium chloride, methyl paraoxybenzoate, benzoic acid, 
etc.; liposome and/or emulsion formulations, such as lecitines, etc.; 
microencapsulated formulations; propellants, such as butane; water etc. 
The use of propellants is not compulsory in the preparation according to 
the invention. 
The pharmaceutical preparation of the invention may comprise any antigens 
and/or vaccines. The vaccines may be selected among all the vaccines 
causing diseases in humans or animals. These include bacterial vaccines 
such as chlamydia, cholera, diphtheria, haemophilus influenzae, leprosy, 
meningococcal, pertussis, pneumococcal, shigella, tetanus, tuberculosis, 
etc.; virus vaccines such as hepatitis viruses, herpes viruses, human 
immunodeficiency viruses (HIV), influenza viruses, measles virus, mumps 
virus, parainfluenza virus, paramyxo viruses, polio virus, rabies viruses, 
respiratory syncytial viruses, rhinovirus types, rotavirus, rubella virus, 
etc., and parasite vaccines such as vaccines for leishamaniasis, 
schistosomiasis and trypanosomiasis, which may be used to produce local 
and/or systemic antibodies. 
The invention is described in further detail in the following examples.

EXAMPLE I 
A tetanus vaccine formulation consists of (a) tetanus toxoid (22.5 .mu.l), 
gangliosides (10.0 .mu.l) and Tween-20 (7.5 .mu.l); (b) tetanus toxoid 
(22.5 .mu.l) and a solution of an Imwitor/cremophor mixture (1:1) (17.5 
.mu.l); (c) tetanus toxoid (22.5 .mu.l) and isotonic saline (17.5 .mu.l). 
Formulations a, b and c are administered intranasally to mice (2.5 
.mu.l/nostril) under i.p. nembutal anaesthesia. Each mouse received 1.5 Lf 
tetanus toxoid. Three weeks later the mice are boosted with the same 
formulations and one week after, they are sacrificed and serum and nasal 
wash antibodies are measured. The excess serum samples are furthermore 
measured in living animals receiving live tetanus toxoid in the 
neutralisation test. The following results were obtained: 
______________________________________ 
Formulation 
Blood IgG Nasal IgA 
Neutralisation 
______________________________________ 
Control (s.c.).sup.a) 
1.09 105 0.5 
Formulation a 
2.45 625 0.5 
Formulation b 
1.54 1132 0.8 
Formulation C 
0.0007 30 0.000 
______________________________________ 
.sup.a) Commercially available product, single administration. 
EXAMPLE II 
A diphtheria vaccine formulation consists of (a) diphtheria toxoid (7.5 
.mu.l), gangliosides (12.5 .mu.l) and Tween-20 (20.0 .mu.l); (b) 
diphtheria toxoid (7.5 .mu.l), PBS-saline (12.5 .mu.l) and a solution of 
an Imwitor/cremophor mixture (1:1) (20.0 .mu.l); (c) diphtheria toxoid 
(7.5 .mu.l) and isotonic saline (32.5 .mu.l). Formulations a, b and c are 
administered intranasally to mice (2.5 .mu.l/nostril) under i.p. nembutal 
anaesthesia. Each mouse received 1.5 Lf diphtheria toxoid. Three weeks 
later the mice are boosted with the same formulations and one week after 
they are sacrificed and serum and nasal wash antibodies are measured. The 
excess serum samples are furthermore measured in the neutralisation test. 
The following results were obtained: 
______________________________________ 
Formulation 
Blood IgG Nasal IgA 
Neutralisation 
______________________________________ 
Control (s.c.).sup.a) 
0.354 34 0.012 
Formulation a 
0.004 36 0.025 
Formulation b 
2.22 352 0.020 
Formulation c 
0.0004 30 0.000 
______________________________________ 
.sup.a) Commercially available product, single administration. 
EXAMPLE III 
An influenza vaccine formulation consists of (a) influenza virus vaccine 
(5.0 .mu.l), gangliosides (10.0 .mu.l), a solution of an Imwitor/cremophor 
mixture (1:1) (6.0 .mu.l), distilled water (16.5 .mu.l) and a PBS solution 
(2.5 .mu.l); (b) influenza virus vaccine (5.0 .mu.l) and isotonic saline 
(35.0 .mu.l). The formulation was administered intranasally to mice (2.5 
.mu.l/nostril) under i.p. nembutal anaesthesia. Each mouse received 0.2 
.mu.g influenza HA. Four weeks later the mice were sacrificed and the 
serum HI titer measured. The following results were obtained: 
______________________________________ 
Formulation 
HI test 
______________________________________ 
Control (s.c.).sup.a) 
1/80 
Formulation a 
1/160 
Formulation b 
1/20 
______________________________________ 
.sup.a) Commercially available product. 
EXAMPLE IV 
A tetanus and diphtheria vaccine formulation consists of (a) tetanus toxoid 
(510 .mu.l), diphtheria toxoid (169 .mu.l), gangliosides (75 .mu.l) and 
Tween-20 (750 .mu.l); (b) tetanus toxoid (510 .mu.l), diphtheria toxoid 
(169 .mu.l) and a solution of an Imwitor/cremophor mixture (1:1) (220 
.mu.l). Six rabbits were divided into 3 groups of 2 rabbits each (4 
nostrils in each group). Formulations a and b were administered 
intranasally (50 .mu.l into each nostril) under unanaesthesized condition. 
Each rabbit received 18 Lf tetanus toxoid and 18 Lf diphtheria toxoid. The 
last group served as control and received only a single intranasal dose of 
isotonic saline. The rabbits were sacrificed by intravenous injection of 
pentobarbital 31/2 after dosing. Each nasal cavity was opened and 
individually evaluated macroscopically. The evaluator was blind as to the 
dosing scheme. The data show that the lesions observed were distributed 
almost evenly over the control and the test groups. Small focal nature and 
anterior location of some lesions were obtained, corresponding to the 
abrasion from the tip of the applicatior pipette. No macroscopic 
difference was observed between isotonic saline and the formulations a and 
b. 
EXAMPLE V 
Three solvents, phosphate buffered saline (PBS), caprylic/capric acid 
glycerides (CCG) and polyoxyethylene sorbitan monoesters (PS), were mixed 
together in various concentrations in order to see their interrelationship 
(phase diagram). The figure shows that within certain concentration rages 
an emulsion or a semisolid solution is achieved. CCG and PBS show a 
heteogeneous solution upon mixing when little or no PS is present in the 
system. Viscosity, bioadhesiveness, sprayability and homogenicity (in the 
case of an emulsion delivery system) may be controlled, dependent on the 
concentration of each substance. 
EXAMPLE VI 
A tetanus vaccine formulation consists of (a) tetanus toxoid (510 .mu.l), 
gangliosides (75 .mu.l), polyoxyethylene sorbitan monoesters (750 .mu.l) 
and saline (169 .mu.l); (b) commercially available tetanus/diphteria 
vaccine, adsorbed to aluminum hydroxide. Formulation a was administered 
intranasally to rabbits (50 .mu.l/nostril) using no anaesthesia nor 
sedation, and formulation b was administered subcutaneously. Each rabbit 
received 18 Lf tetanus toxoid and 18 Lf diphtheria toxoid. Three weeks 
later the rabbits received a booster of the same formulations. Weekly 
serum samples were collected from the marginal ear vein, and the samples 
were measured using the ToBi technique. The following results were 
obtained (IU/ml): 
______________________________________ 
Formulation 
2 weeks 3 weeks 4 weeks 
______________________________________ 
a 0.034 1.012 0.847 
b 0.477 1.572 1.456 
______________________________________ 
EXAMPLE VII 
The synergistic effect between caprylic/capric acid glycerides (CCG) and 
polyoxyethylene sorbitan monoesters (PS) was determined as follows: 
Six diphtheria (1.5 Lf) vaccine formulations were made: (a) in phosphate 
buffered saline (PBS); (b) commercially available Al(OH).sub.3 adsorbed 
vaccine for subcutaneous injection; (c) in PBS solution containing 40% 
polysorbate 20; (d) in PBS solution containing 40% polysorbate 20 and 25% 
polyoxyethylene castor oil; (e) in 40% polysorbate 20 and 10% 
caprylic/capric acid glyceride (mono- and di-glycerides); and (f) in 40% 
polysorbate 20, 25% polyoxyethylene castor oil and 10% caprylic/capric 
acid glyceride (mono- and di-glycerides). The formulations were 
administered intranasally to mice (2.5 .mu.l/nostril) under i.p. nembutal 
anaesthesia. Three weeks later the mice received a booster containing the 
same formulations, and a further week later they were sacrified and serum 
antibodies were measured. The following results were obtained: 
______________________________________ 
Formulations Diphth. IgG 
______________________________________ 
a 0.0004 
b 0.354 
c 0.448 
d 0.127 
e 7.3 
f 0.115 
______________________________________ 
It appears that neither PS nor CCG alone can provide a satisfactory effect. 
This is only the case with combinations of PS and CCG. 
EXAMPLE VIII 
In this example the synergistic effect between caprylic/capric acid 
glycerides (CCG) and polyoxyethylene sorbitan monoesters (PS) was 
investigated further. 
Seven influenza A vaccine formulations were made: (a) in phosphate buffered 
saline (PBS); (b) in PBS solution containing 25% polyoxyethylene castor 
oil; (c) in PBS solution containing 25% polyoxyethylene castor oil and 10% 
caprylic/capric acid glyceride (mono- and di-glycerides); (d) in PBS 
solution containing 40% polysorbate 20; (e) in PBS solution containing 40% 
polysorbate 20 and 25% polyoxyethylene castor oil; (f) in 40% polysorbate 
20 and 10% caprylic/capric acid glyceride (mono- and di-glycerides); and 
(g) in 40% polysorbate 20, 25% polyoxyethylene castor oil and 10% 
caprylic/capric glyceride (mono- and di-glycerides). The formulations were 
administered intranasally to mice (2.5 .mu.l/nostril) under i.p. nembutal 
anaesthesia. Three weeks later the mice received a booster, containing the 
same formulations, and a further week later they were sacrificed and the 
serum antibodies were measured. The following results were obtained: 
______________________________________ 
Formulation 
IgG 
______________________________________ 
a 0.072 
b 0.053 
c 0.073 
d 0.114 
e 0.038 
f 0.354 
g 0.037 
______________________________________ 
Caprylic/capric acid glycerides were not tested alone, since they are 
insoluble in water. 
EXAMPLE IX 
This example illustrates the selection of the optimal CCG and PS 
concentration. 
Seven diphtheria vaccine formulations were made: (a) in phosphate buffered 
saline (PBS); (b) in PBS solution containing 35% polysorbate 20; (c) in 
PBS solution containing 57.5% polysorbate 20; (d) in PBS solution 
containing 35% polysorbate 20 and 10% caprylic/capric acid glyceride 
(mono- and di-glycerides); (e) in PBS solution containing 57.5% 
polysorbate 20 and 10% caprylic/capric acid glyceride (mono- and 
di-glycerides); (f) in PBS solution containing 35% polysorbate 20 and 24% 
caprylic/capric acid glyceride (mono- and di-glycerides); and (g) in 57.5% 
polysorbate 20 and 24% caprylic/capric acid glyceride (mono- and 
di-glycerides). The formulations were administered intranasally to mice 
(2.5 .mu.l/nostril) under i.p. nembutal anaesthesia. Three weeks later the 
mice received a booster, containing the same formulations, and one further 
week later they were sacrificed and the serum antibodies were measured. 
The following results were obtained: 
______________________________________ 
Formulation 
IgG 
______________________________________ 
a 0.365 
b 1.22 
c 0.092 
d -9.65 
e -2.33 
f 1.31 
g -26.6 
______________________________________ 
Caprylic/capric acid glycerides were not tested alone, since they are 
insoluble in water. 
EXAMPLE X 
The selection of the optimal CCG and PS concentration is further 
illustrated in this example. 
Seven diphtheria and tetanus vaccine formulations were made by using fixed 
caprylic/capric acid glyceride (mono- and di-glycerides) concentration 
(10%) but variable polysorbate 20 (mono-ester) concentration, ranging from 
28% (a) with 2% increments up to 40% (g). The formulations were 
administered intranasally to mice (2.5 .mu.l/nostril) under i.p. nembutal 
anaesthesia. Three weeks later the mice received a booster, containing the 
same formulations, and one additional week later they were sacrificed and 
the serum antibodies were measured. The following results were obtained: 
______________________________________ 
Formulation Diphth. IgG 
Tetan. IgG 
______________________________________ 
a 0.07 0.04 
b 0.17 0.04 
c 0.10 0.02 
d 0.16 0.03 
e 1.60 0.01 
f 1.25 0.06 
g 0.27 0.004 
______________________________________ 
EXAMPLE XI 
This example concerns the selection of polyoxyethylene fatty acid esters. 
Such polyoxyethylene fatty acid esters are found as mono- and trimesters. 
Diphtheria toxoids were formulated in the following different 
compositions: (a) in isotonic phosphate buffered saline (PBS); (b) in PBS 
solution containing 47% polysorbate 80 (tri-ester); and (c) in PBS 
solution containing 47% polysorbate 20 (mono-ester). The formulations were 
administered intranasally to mice (2.5 .mu.l/nostril) under i.p. nembutal 
anaesthesia. Four weeks later the mice were sacrificed and the serum 
antibodies were measured. The following results were obtained: 
______________________________________ 
Formulation 
IgG 
______________________________________ 
a 0.001 
b 0.002 
c 0.006 
______________________________________ 
EXAMPLE XII 
The selection of glyceride esters was performed as follows: Six tetanus 
(1.5 Lf) and diphtheria (1.5 Lf) vaccine formulations were made. The 
formulations were administered intranasally to mice (2.5 .mu.l/nostril) 
under i.p. nembutal anaesthesia. Four weeks later the mice were sacrificed 
and serum and nasal wash antibodies were measured. The following results 
were obtained: 
______________________________________ 
Formulation Diphth. IgG 
Tetan. IgG 
______________________________________ 
Negative control 0.0013 0.0078 
C.sub.8 and 10 diglyceride 
0.0003 0.0030 
ester (Miglyol 829) (3.5%) 
C.sub.8 and 10 mono-diglyceride 
0.0027 0.2580 
ester (Imwitor 742) (7%) 
C.sub.16 triglyceride ester 
0.0014 0.0057 
(Dynasan 116) (2.5%) 
______________________________________