Induction of protective and specific antibodies against cocaine by intranasal immunisation using a glyceride adjuvant

Article abstract of DOI:10.1248/bpb.28.1038

The goal of this study was to investigate an intranasal cocaine vaccine containing the mucosal adjuvant macrogol-6-glycerol capylocaprate (RhinoVax). Cocaine-KLH conjugate was prepared and administered in two formulations. Ten mice were immunised intranasally using RhinoVax as adjuvant and ten subcutaneously using aluminium hydroxide as an adjuvant. A negative control group (n=10) received unconjugated KLH with RhinoVax intranasally. Specific cocaine antibodies in serum were measured following primary and booster immunisation. Relative antibody responses in serum indicated that the immunisation was successful. Animals were then challenged with cocaine either intranasally or intraperitoneally with subsequent measurement of drug distribution into the serum, brain and olfactory bulb. The cocaine-immunised groups revealed significantly lower cocaine levels in the brain compared to the negative control group. The inhibition of cocaine distribution to the brain in the intranasal immunised group was comparable to that of the subcutaneous immunised group. This was unexpected because the cocaine specific antibody levels in serum were fivefold lower in the intranasal immunised group. However, the presence of mucosal cocaine specific antibodies after intranasal immunisation could play an important role in hindering direct access of cocaine into the brain via the olfactory bulb.

Full text of DOI:10.1248/bpb.28.1038

1038
Biol. Pharm. Bull. 28(6) 1038—1042 (2005)
Vol. 28, No. 6
Induction of Protective and Specific Antibodies against Cocaine by
Intranasal Immunisation Using a Glyceride Adjuvant
Kolbrun HRAFNKELSDOTTIR,a Jon VALGEIRSSON,b and Sveinbjorn GIZURARSON*,a,c
a Lyfjathroun, Biopharmaceuticals; Vatnagardar 16–18, 104 Reykjavik, Iceland: b Actavis; Reykjavikurvegi 76–78, 220
Hafnarfjordur, Iceland: and c University of Iceland, Faculty of Pharmacy; Hofsvallagata 53, 107 Reykjavik, Iceland.
Received July 28, 2004; accepted March 10, 2005
The goal of this study was to investigate an intranasal cocaine vaccine containing the mucosal adjuvant
macrogol-6-glycerol capylocaprate (RhinoVax). Cocaine–KLH conjugate was prepared and administered in two
formulations. Ten mice were immunised intranasally using RhinoVax as adjuvant and ten subcutaneously using
aluminium hydroxide as an adjuvant. A negative control group (nꢀ10) received unconjugated KLH with Rhino-
Vax intranasally. Specific cocaine antibodies in serum were measured following primary and booster immunisa-
tion. Relative antibody responses in serum indicated that the immunisation was successful. Animals were then
challenged with cocaine either intranasally or intraperitoneally with subsequent measurement of drug distribu-
tion into the serum, brain and olfactory bulb. The cocaine-immunised groups revealed significantly lower cocaine
levels in the brain compared to the negative control group. The inhibition of cocaine distribution to the brain in
the intranasal immunised group was comparable to that of the subcutaneous immunised group. This was unex-
pected because the cocaine specific antibody levels in serum were fivefold lower in the intranasal immunised
group. However, the presence of mucosal cocaine specific antibodies after intranasal immunisation could play an
important role in hindering direct access of cocaine into the brain via the olfactory bulb.
Key words cocaine; addiction; intranasal; vaccine; RhinoVax; immunisation
Cocaine is a powerfully addictive psychostimulant drug the result of direct uptake into the brain.
whose abuse and withdrawal patterns differ from those of
The mucosal surfaces of the respiratory and gastrointesti-
other major illicit drugs. There is a widespread cocaine epi- nal systems are continuously exposed to exogenous agents16)
demic1) largely associated with introduction of “crack” and require effective defense systems to provide necessary
cocaine. While the need to treat such drug dependency is protection. Secretory antibodies, together with mucin, play
unequivocal, there is no proven pharmacologic therapy to an important role in preventing mucosal invasion by binding
treat cocaine addiction.2,3) A number of approaches have exogenous substances.16—18) The nasal passage is easily
been investigated;2) one involves active immunisation with an accessible and highly vascularized and therefore constitutes
immunogenic cocaine–protein conjugate to promote the pro- an attractive route for immunisation. The presence of numer-
duction of cocaine specific antibodies.4—8) Under normal ous microvilli covering the nasal epithelium generates a large
conditions antibodies cannot cross the blood-brain-barrier. absorption surface. Immune responses can be induced at mu-
However it is believed that complexation of antibodies with cosal sites distant to the site of immunisation, owing to the
free cocaine may effectively block access of cocaine to the dissemination of effectors immune cells in the common
brain.6,9)
mucosal immune system.19) Intranasal immunisation induces
Cocaine is known to block the dopamine transporters in both mucosal and systemic immune responses, which is a
the central nervous system,1,10) with resultant heightened potential advantage for cocaine immunisation.19) If cocaine
dopaminergic stimulation at critical brain sites. The rapid specific secretory antibodies are present on the nasal mucosa,
and extreme rise of dopamine levels in the brain is thought to they could potentially bind cocaine at its site of administra-
account for the addictive nature of cocaine.11) The euphoric tion thereby hindering both systemic absorption and olfac-
effects of cocaine seem to critically depend on both the total tory absorption. Recent animal studies [unpublished] have
amount of cocaine in the central nervous system and on the shown that cocaine intranasal immunisation with RhinoVax
rate of entry into the brain.12,13) Recent studies indicate that as an adjuvant is effective in stimulating local mucosal
the latter has greater impact on the cocaine dependence.14)
Nasal insufflation of cocaine is a popular administration
cocaine specific antibody responses in the buccal mucosa.
Cocaine is a small molecule and must be covalently linked
route among drug addicts because the drug is rapidly ab- with an immunogenic macromolecule in order to be im-
sorbed from the nasal mucosa.15) Cocaine, however, induces munogenic. To ensure Th2 immune responses towards the
intense vasoconstriction at the site of absorption, which may molecule, the vaccine must be formulated with a suitable ad-
affect the absorption into the systemic circulation.14) Follow- juvant. The balance between toxicity and immunogenicity of
ing insufflation a substantial amount of the drug is swallowed adjuvants is very delicate. Most adjuvants have the inherent
into the gastrointestinal tract with subsequent absorption and problem of being irritating or even toxic (e.g. Freund’s com-
first-pass metabolism.14) Cocaine enters the brain from the plete adjuvant and cholera toxin) which limits their use in hu-
circulation through the blood-brain-barrier. A paper by Chow mans. In 1998 Gizurarson et al. published their work on the
et al.15) also indicates that a small fraction of intranasally ad- RhinoVax, mucosal adjuvant system. This adjuvant is non-
ministered cocaine can be transported directly from the nasal toxic and has been studied with a number of antigens in vari-
cavity to the brain via the olfactory system. Therefore, the ous animals as well as in humans.20,21) When administered in-
rapid onset of action observed for insufflated cocaine may be tranasally, the adjuvant was found to stimulate both local and
∗ To whom correspondence should be addressed. e-mail: sveinbj@hi.is
© 2005 Pharmaceutical Society of Japan

June 2005
1039
systemic immune responses, providing an interesting alterna-
Immunisation Animals were randomly divided into
tive to parenteral immunisation. The adjuvant consists of a three groups with ten animals in each group. (1) Intranasal
mixture of mono- and diglycerides of caprylic- (C-8) and cocaine immunisation; 5 ml of preparation (A) was adminis-
capricacid (C-10) where all free alcohol groups are pegy- tered into both nostrils. (2) Subcutaneous cocaine immunisa-
lated.
tion (positive control group); 60 ml of preparation (B) was in-
The aim of this work was to investigate whether an in- jected into the anterior dorsal section of the back. (3) Nega-
tranasal cocaine vaccine using RhinoVax as a mucosal adju- tive control (intranasal KLH immunisation); 5 ml of prepara-
vant, could induce specific cocaine antibodies and, if so, tion (C) was administered into both nostrils. The primary im-
whether these antibodies were able to prevent cocaine from munisation was given at Tꢂ0 and booster doses at Tꢂ4
entering into the brain.
weeks and Tꢂ7 weeks.
Sampling Blood was drawn from the tail veins of the
animals following the primary immunisation (Tꢂ3 1/2 week)
and again following the booster immunisation (Tꢂ7 1/2
week). Approximately 0.1 ml of blood samples were col-
lected (Minicollect®, Greiner labortechnic, Frickenhausen,
Germany) and stored at room temperature for 1 h. Then the
samples were centrifuged at 3000 rpm for 15 min and serum
collected and stored at ꢃ20 °C until analysis.
Antibody Measurements ELISA (enzyme-linked im-
munosorbent assay) was used to measure cocaine specific an-
tibodies in the mouse sera. Succinyl cocaine (0.1 mg/ml;
100 ml in water) was covalently attached to amine activated
polystyrene 96 well microtiter plates (Covalink-NH2, Nunk
A/S, Roskilde, Denmark). Freshly prepared solutions of
sulfo-NHS (N-Hydroxyxulfosuccinimide, Pierce Co.) (3.48
mg/ml in water) and EDC (30.7 mg/ml in water) were subse-
quently added to each well. The plates were covered and
incubated overnight at room temperature with gentle agita-
tion and then washed with water to remove unbound sub-
stance.
To avoid unspecific binding, the plates were incubated for
1 h at room temperature with ELISA buffer (0.5% bovine
serum albumin and 0.05% Tween 20 in PBS) and then
washed with PBS to remove the blocking agent. The samples
and standard (benzoyl ecgonine monoclonal antibodies,
Biostride Inc., Redwood City, CA, U.S.A.), in several dilu-
tions, were incubated for 1.5 h at room temperature with gen-
tle agitation and then washed with 0.05% Tween 20 in PBS.
Horse-radish-peroxidase conjugated rabbit anti-mouse im-
munoglobulin (Dako P-260, DAKO A/S, Glostrup, Den-
mark) was added, incubated for 1 h and then washed with
0.05% Tween 20 in PBS. The peroxidase activity was deter-
mined with 1,2-o-phenylenediamine hydrochloride (DAKO
A/S) and the reaction was stopped after 15 min with 0.5 M
sulphuric acid. The absorption was measured at 492 nm in a
spectrophotometer (Perkin Elmer HTS 7000, U.S.A.).
The concentration of the cocaine specific antibodies was
calculated on the basis of a standard curve.
Cocaine Administration and Distribution Measure-
ments The animals were given 1.5 mg/kg cocaine in PBS
together with 3H-cocaine (0.02 mCi) (American Radiola-
belled Chemicals, St. Louis, MO, U.S.A.) following the
booster immunisation (Tꢂ8 weeks). Five animals in each
group received 100 ml volume intraperitoneally and the other
five received 10 ml intranasally. After 3 min the mice were
sacrificed and blood, brain, and the olfactory bulb were re-
moved. Ultima GoldTM scintillation fluid (10 ml) (Packard,
Meriden, CT, U.S.A.) was added to a known amount of
serum (0.1—0.3 ml) and stored at room temperature until
analysis. Soluene-350TM (0.75 ml) (Packard) was added to
each 100 mg of brain tissue and allowed to dissolve at room
MATERIALS AND METHODS
Preparation of Cocaine–KLH Conjugate KLH (Imject
mariculture keyhole limpet hemocyanin, Pierce Co., Rock-
ford, IL, U.S.A.) was conjugated to cocaine via succinic acid
spacer arm as follows: Norcocaine was synthesised from co-
caine by N-demethylation with 1-chloroethylchloroformate
as described by Boja et al.22) Cocaine (455 mg; 1.47 mmol)
was dissolved in 1,2-dichloroethane (25 ml) under nitrogen
atmosphere at room temperature. 1-chloroethyl chlorofor-
mate (0.5 ml) was added drop wise and the mixture then re-
fluxed for 24 h. The reaction mixture was evaporated under
reduced pressure and the residue dissolved in MeOH and re-
fluxed for 4 h. The solvent was evaporated under reduced
pressure and the residue dissolved in CH2Cl2 and washed
with dilute K2CO3 solution. The organic phase was dried
with MgSO4, evaporated under reduced pressure and norco-
caine was isolated by column chromatography (10% Et3N in
Et2O). Norcocaine was succinylated by the method described
by Swain et al.23) Norcocaine (320 mg; 1.11 mmol), succinic
anhydride (220 mg; 2.20 mmol) and triethylamine (0.3 ml)
were dissolved in CH2Cl2 (8 ml) and refluxed for 18 h. The
reaction mixture was washed with dilute aqueous HCl, dried
with MgSO4 and the solvents were removed under reduced
pressure. The residue was purified with column chromatogra-
phy (2 : 1 CHCl3–MeOH).
Succinyl cocaine (30 mg) was dispersed in distilled water
at 0 °C and dilute NaOH added until all the succinyl cocaine
had dissolved. EDC (1-ethyl-3-(3-dimethylaminopropyl) car-
bodiimide hydrochloride, Pierce Co.) (21.3 mg) was added
and five minutes later 20 mg of KLH in 2 ml PBS (pH 7.4)
was added. The reaction was left stirring at room temperature
overnight. The reaction was dialysed against 3ꢀ3 l of PBS
(12 h each). Level of haptenation was determined by analysis
of the difference in UV absorbance between conjugated and
unconjugated KLH.
Animals Thirty 7-week-old female BALB/c mice (M&B
A/S, Ry, Denmark) were used. The animals were maintained
in a temperature-controlled room (22ꢁ2 °C) and kept on a
12-h light–dark cycle. Food and water were available ad libi-
tum.
Vaccine Formulation Three preparations were used: (A)
Intranasal: 6.4 mg/ml of the Cocaine-KLH conjugate in PBS
with 5% v/v RhinoVax (Softigen 767, Sasol GmbH, Witten,
Germany); (B) Subcutaneous: 2.2 mg/ml of the Cocaine-
KLH conjugate adsorbed to 1.3% aluminum hydroxide (Al-
hydrogel, Superphos Biosector, Frederiksund, Denmark) in
water; this was allowed to reach equilibrium overnight before
use; and (C) Negative control: 6.4 mg/ml of unconjugated
KLH in water with 5% v/v RhinoVax.

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Vol. 28, No. 6
Table 1. Total Cocaine Specific Antibody Responses in Serum Following
Immunisation of Mice
temperature for two days. Finally, 1.5 ml of that solution was
diluted with Hionic FluorTM (15 ml) (Packard) and measured
for 5 min on a liquid scintillation counter (LKB-Wallac 1214
Rachbeta).
Primary immunisation
Booster immunisation
Immunisation
Tꢂ3 1/2 week (U/ml)
Tꢂ7 1/2 week (U/ml)
Statistical Analysis The difference between the groups
were analyzed by Student’s t-test. A 95% confidence interval
was used and pꢄ0.05 was considered significant.
Intranasal
Subcutaneous
Negative control
10.3ꢁ8.2
182.5ꢁ116.2
4.5ꢁ0.2
179.3ꢁ113.1
889.1ꢁ557.4
3.2ꢁ2.6
RESULTS
Preparation of Cocaine–KLH Conjugate The conjuga-
tion of cocaine to KLH was successful. The level of haptena-
tion was determined by UV-absorbance, which was a very
rudimentary assessment. UV-absorption suggested that ap-
proximately 200 molecules of cocaine were conjugated to
each KLH molecule. This reflects the minimum number
since high conjugation of KLH is known to alter the structure
of the KLH protein and expose hydrophobic surfaces that can
lead to precipitation.24) The product of the conjugation con-
tained large amounts of precipitate, which is indicative of
high conjugation. The precipitate was included in the mix-
ture used for eliciting immune response in the mice.
Antibody Measurements The relative cocaine specific
antibody response in serum was measured following both the
primary and the booster immunisation, as seen in Table 1.
Animals in both cocaine-immunised groups responded fully
to the immunisation. Following primary immunization there
was a significant increase (pꢄ0.005) in cocaine specific anti-
body level in the subcutaneously immunised group compared
to the negative control group (182.5 U/ml relative to 4.5
U/ml). The intranasally immunised group, however, did not
show a significant, increase (10.3 U/ml). The booster immu-
nisation resulted in a strong and significant (pꢄ0.005) in-
crease in cocaine specific antibodies for both the intranasal
and subcutaneous groups. These rose to 179.3 U/ml and
889.1 U/ml respectively.
Fig. 1. Cocaine Concentration in the Cerebral Tissue, 3 min after Admin-
istration of Cocaine
(A) Intraperitoneal cocaine. (B) Intranasal cocaine.
Distribution Measurements The animals in both co-
caine-immunised groups displayed lower levels of drug in the
brain following subsequent cocaine administration than the
negative control group (Fig. 1). The subcutaneous immu-
nised group showed significant (pꢄ0.05) reduction in co-
caine distribution to the brain both when cocaine was admin-
istered intraperitoneally (Fig. 1A) and intranasally (Fig. 1B),
compared to the negative control group. The intranasal im-
munised group showed significant (pꢄ0.05) reduction in co-
caine transport into the brain when it was administered in-
traperitoneally and substantial, but not significant, reduction
when administered intranasally. In Fig. 2, the cocaine con-
centration in the olfactory bulb was compared to the concen-
tration in the remaining brain tissue. In all cases, cocaine lev-
els in the olfactory bulb were higher following intranasal
than after intraperitoneal cocaine administration. This differ-
Fig. 2. The Concentration of Cocaine in the Olfactory Bulb, 3 min after
the Administration of Cocaine
ence was insignificant for both cocaine-immunised groups, Rhinovax, has been studied. Animals treated with conju-
but the negative control group had significantly (pꢄ0.005) gated-cocaine vaccines responded fully to the cocaine immu-
higher cocaine concentration in the olfactory bulb after in- nisation and demonstrated elevated specific cocaine antibod-
tranasal cocaine administration than after intraperitoneal ad- ies in the serum. Upon subsequent administration of cocaine,
ministration.
the previously immunized animals had significantly lower
levels of cocaine in the brain compared to the negative con-
trol group (i.e. unconjugated-cocaine vaccine).
DISCUSSION
Antibody Response Analysis of specific antibodies
An intranasal cocaine vaccine using the mucosal adjuvant against cocaine indicated that the immunisation was success-

June 2005
1041
ful in both conjugated-cocaine immunised groups. There cant. Concentration of cocaine in the olfactory bulb was
were, however, clear differences in the serum antibody levels found to be 2.21 mg/ml following intranasal administration,
depending on the route of immunisation. The intranasal im- compared with 0.88 mg/ml following intraperitoneal adminis-
munisation using RhinoVax as an adjuvant yielded 179 U/ml tration. However, in the cocaine-immunised groups the dif-
where the subcutaneous immunisation using aluminium hy- ference was not significant. This supports the theory that the
droxide yielded 889 U/ml.
antibodies works to block the absorption of cocaine to the
The aluminium hydroxide is a typical inducer of pure Th2 brain, both in the circulation and at site of administration.
immune response,25) and there are numerous reports in hu- Additional intranasal booster doses, should promote higher
mans and in animals showing its excellence as a primer for concentration of local antibodies on the mucosal surface,
vaccinations.26—28) This is also the case in current study, thereby effecting stronger blockage at the site of absorption.
as the subcutaneous aluminium hydroxide immunisation
The current study showed that intranasally applied
elicited a strong cocaine specific antibody response in serum cocaine-KLH antigen, together with RhinoVax, was able to
following the primary immunisation. The intranasal immuni- produce cocaine specific antibodies, which seemingly
sation was not as efficient in eliciting serum antibodies fol- blocked the absorption and reduced the level of cocaine
lowing the primary immunisation (10 U/ml). One of the entering into the brain. It will be important to study and eval-
functions of the nasal cavity is to tolerate a variety of air- uate the nature of these antibodies, their binding capacity and
borne compounds without inducing immune response to their function on the mucosal surface as well as inside the
every exogenous substance. Therefore intranasal immunisa- body.
tions are typically not as efficient in eliciting serum antibod-
ies as subcutaneously administered aluminium hydroxide
Acknowledgements The authors thank Dr. Jona Freys-
vaccines are.19,29) By contrast, the intranasal booster adminis- dottir and Dr. Orla McCallion for her helpful comments to
tration elevated the antibody responses 18 folds to 179 U/ml, this manuscript.
which is equivalent to the levels seen following primary im-
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