Oral delivery of biologically active substances bound to vitamin B12 or analogues thereof

An orally administered complex of a drug, hormone, bio-active peptide, or immunogen with the carrier molecule, such as vitamin B12 or analogue thereof, and a method for delivering said complex to the intestine of a host vertebrate in order to deliver the complex to the circulation of the host and thereby elicit a pharmacological response to the drug, hormone, or bio-active molecule or to elicit a systemic immune response to the immunogen. The invention also provides a method for the production of the complex. Further the invention provides medicaments containing the complex.

TECHNICAL FIELD 
The present invention relates to oral delivery systems. More particularly 
the invention relates to enhancing the absorption of active substances by 
administering these substances bound to vitamin B12 (VB12) or an analogue 
thereof. 
BACKGROUND ART 
The oral route of administration is perhaps the most preferable means of 
delivering an antigen or pharmaceutically active agent to man. This route 
does however suffer from the major disadvantage that there is generally 
poor uptake of antigens or pharmaceutically active agents by the 
gastrointestinal tract and some agents may be destroyed by prolonged 
exposure to proteolytic enzymes. In this regard, attemps to orally 
immunize man or animals in the past have met with limited success. 
Effective vaccination has generally only been achieved by the 
administration of large quantities of antigen or by combining parenteral 
priming with oral boosting. Recent work by us utilizing a number of 
molecules with the ability to bind to the intestinal mucosa has 
demonstrated effective oral immunization using low doses of these binding 
proteins or by coupling various antigens or haptens to these carriers. 
Uptake and delivery to the circulation of these molecules from the 
intestine seemed to be due to receptor mediated endocytosis. 
It has been known for some time that a number of specific uptake mechanisms 
exist in the gut for uptake of dietary molecules. Thus there are specific 
uptake mechanisms for monosaccharides, disaccharides, amino acids and 
vitamins. Most of these uptake mechanisms depend upon the presence of a 
specific protein or enzyme such as monosaccharidase or disaccharidase 
situated in the mucosal lamina propria which binds to the molecule and 
transports it into the cells lining and lamina propria. 
Two notable exceptions to these uptake mechanisms are found with iron 
transport and VB12 uptake. In both these cases a specific binding protein 
is released into the intestine, which binds to its ligand in the lumen of 
the gut. 
Thus, during iron uptake in the intestine transferrin is released from the 
stomach, binds to iron and is in turn bound by a receptor on the duodenal 
mucosa. The receptor-transferrin-iron complex is then taken up by receptor 
mediated endocytosis. 
Similarly, the absorption of physiological amounts of VB12 by the gut 
requires that it be complexed with a naturally occurring transport protein 
known as intrinsic factor (IF) (1-5). This protein is released into the 
lumen of the stomach by parietal cells in the fundus. Once bound to 
intrinsic factor, the VB12.IF complex interacts with a membrane bound 
receptor for IF located on the terminal ileum of the small intestine. The 
receptor-IF-VB12 complex is then internalized by a process of receptor 
mediated endocytosis (RME). Allen and Majerus (7) demonstrated that it is 
possible to chemically modify VB12, couple it to a resin and use the 
VB12-resin to affinity purify IF. This finding suggested to us that it may 
be possible to couple large macromolecules (such as the resin used by 
Allen and Majerus) to VB12 and to still preserve it's ability to interact 
specifically with intrinsic factor. By coupling molecules to VB12 in such 
a way as to preserve the ability of VB12 to interact with intrinsic factor 
it was hoped that we could use the natural uptake mechanism for VB12, to 
deliver various proteins, drugs or other pharmaceutically active molecules 
to the circulation. 
It is thus the object of this invention to utilize the VB12 uptake 
mechanism to transport active substances such a drugs, hormones, antigenic 
material and the like, covalently coupled to VB12 or an analogue thereof, 
from the intestinal lumen into the circulation. 
DISCLOSURE OF THE INVENTION 
In a first embodiment the invention provides a complex which comprises at 
least one active substance linked to at least one carrier molecule which 
is VB12 or an analogue thereof wherein the ability of the carrier to 
undergo the binding reactions necessary for uptake and transport of VB12 
in a vertebrate host and the activity of the active substance are 
substantially maintained. 
In the context of the present invention, the term active substance includes 
all, part, an analogue, homologue, derivative or combination thereof, of a 
hormone, bio active peptide, therapeutic agent, antigen or hapten. 
Preferred active substances for delivery according to the invention 
include: hormones and bioactive peptides such as luteinizing hormone 
releasing hormone (LHRH), insulin, testosterone, interferon, pregnant mare 
serum gonadotrophin (PMSG), human chorionic gonadotrophin (HCG) and 
inhibin; therapeutic agents such as neomycin, salbutamol cloridine, 
pyrimethamine, penicillin G, methicillin, carbenicillin, pethidine, 
xylazine, ketamine hydrochloride, mephanesin and iron dextran; antigens or 
haptens including allergens, proteins, polysaccharides and secretory 
products such as grass pollens (for instance barley and couch), weed 
pollens (e.g. clover, dock) tree pollens (e.g. ash, cyprus), plant pollens 
(e.g. broom), epithelia (e.g. cat hair, dog hair, pig hair) and house dust 
mite, wheat chaff and kapok; a protein derived from or immunogens against 
influenza, measles, Rubella, smallpox, yellow fever, diphtheria, tetanus, 
cholera, plague, typhus, BCG, tuberculosis causing agents, Haemophilus 
influenzae, Neisseria catarrhalis, Klebsiella pneumoniae, pneumococci, 
streptococci; a secretory product derived from diphtheria, tetanus, 
cholera, plague, typhus, tuberculosis causing agents, Haemophilus 
influenzae, Neisseria catarrhalis, Klebsiella pneumoniae, pneumococci, 
streptococci, Streptococcus mutans, or is derived from a malarial parasite 
or the causitive agent of coccidiosis in chickens. 
Preferred analogues of VB12 include cyanocobalamin (CN-Cbl), aquocobalamin, 
adenosylcobalamin, methylcobalamin, hydroxycobalamin, cyanocobalamin 
carbanalide, and 5-o-methylbenzylcobalmin [(5-OMeBza)CN-Cbl] as well as 
the desdimethyl, monoethylamide and the methylamide analogues of all of 
the above. Also included are the various analogues and homologues of 
cobamamide such as coenzyme B12 and 5'-deoxyadenosylcobalamin. Other 
analogues include chlorocobalamin, sulfitocobalamin, nitrocobalamin, 
thiocyanatocobalamin, benzimidazole derivatives such as; 
5,6-dichlorobenzimidazole, 5-hydroxybenzimidazole, trimethylbenzimidazole, 
as well as adenosylcyanocobalamin [(Ade)CN-Cbl], cobalamin lactone, 
cobalamin lactam and the anilide, ethylamide, monocarboxylic and 
dicarboxylic acid derivatives of VB12 or its analogues. 
Preferred derivatives of VB12 include the mono-, di- and tricarboxylic acid 
derivatives or the proprionamide derivatives of VB12. Carriers may also 
include analogues of VB12 in which the cobalt is replaced by zinc or 
nickel. The corrin ring of VB12 or its analogues may also be substituted 
with any substituent which does not effect its binding to IF. 
In a preferred embodiment of the invention there is provided a complex 
comprising the lys-6 form of LHRH and VB12. 
The complexes of this invention, of coupled active substances can be used 
to deliver these substances to any uni or multicellular organism with a 
requirement for, and a specific transport mechanism for VB12. For example, 
bacteria resistant to a particular antibiotic where the resistance is 
mediated by the loss of ability to transport the antibiotic inside the 
cell, could be overcome by this procedure. A VB12-antibiotic complex could 
thus be effectively delivered inside the bacterial cell via the VB12 
transport mechanism. This could lead to an ability to reutilize a number 
of antibiotics whose current use has become limited by development of 
bacterial resistance. Delivery of active substances, of the type described 
above, could be achieved to a wide variety of organisms, particularly 
parasites of humans or animals. 
In another embodiment the invention provides a process for the production 
of a complex comprising, at least one active substance linked to at least 
one carrier molecule, said carrier molecule being VB12 or an analogue 
thereof, wherein the ability of the carrier to undergo the binding 
reactions necessary for uptake and transport of VB12 in a vertebrate host 
and the activity of the active substance are substantially maintained 
which process comprises one or more of the following steps: 
a) reacting the active substance with the carrier to form said complex: 
b) chemically modifying the active substance to provide at least one 
functional group capable of forming a chemical linkage, and reacting the 
active substance and carrier to form said complex; 
c) chemically modifying the carrier to provide at least one functional 
group capable of forming a chemical linkage and reacting the active 
substance and carrier to form said complex; 
d) chemically modifying the active substance and the carrier to provide 
functional groups capable of forming a chemical linkage, and reacting the 
active substance and carrier to form said complex; 
e) reacting the active substance with at least one cross-linking agent and 
reacting the active substance and the carrier molecule to form said 
complex; 
f) reacting the carrier with at least one cross-linking agent and reacting 
the active substance and carrier to form said complex; 
g) reacting the active substance and carrier with at least one 
cross-linking agent and reacting the active substance and carrier to form 
said complex. 
A preferred process of the invention comprises; 
(i) preparing the mono-acid derivative of VB12 by mild acid hydrolysis, and 
purifying the derivative; 
(ii) chemically modifying an active substance to provide at least one 
functional group capable of forming a chemical linkage; and 
(iii) reacting the modified active substance and mono-acid derivative of 
VB12 to form said complex. 
The cross-linking agent may contain a disulfide bond or be cleavable by 
acid, base or periodate. Examples of cross-linking agents include 
N-(4-azidophenylthio)phthalimide, 4,4'-dithiobisphenylazide, 
dithiobis-(succinimidylpropionate), 
dimethyl-3,3'-dithiobispropionimidate.2HCl, 
3,3'-dithiobis-(sulfosuccinimidylpropionate), 
ethyl-4-azidophenyl-1,4-dithiobutyrimidate,HCl, 
N-succinimidyl-(4-azidophenyl)-1,3'-dithiopropionate, 
sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-1,3'-dithiopropionate 
, sulfosuccinimidyl-2-(p-azidosalicylamido)-ethyl-1,3'dithiopropionate, 
N-succinimidyl-3-(2-pyridyldithio)propionate, 
sulfosuccinimidyl-(4-azidophenyldithio)-propionate, and 2-iminothiolane. 
Preferred cross-linking agents are disuccinimidyl tartrate and 
bis-[2-(succinimidyloxycarbonyloxy)-ethyl]sulfone. 
Suitably, cross-linking of the carrier and active substance may be achieved 
by acid hydrolysis of the amide groups of the propionamide side chains 
adjacent to rings A, B and C of VB12 and coupling to suitable groups of 
the active substance. 
In a further embodiment of the invention there is provided a medicament 
which comprises a complex according to the invention together with a 
pharmaceutically acceptable carrier or diluent. 
Examples of pharmaceutically acceptable carriers and diluents include 
typical carriers and diluents such as sodium bicarbonate solutions and 
similar diluents which neutralize stomach acid or have similar buffering 
capacity, glycols, oils, oil-in-water or water-in-oil emulsions, and 
include medicaments in the form of emulsions, gels, pastes and viscous 
colloidal dispersions. The medicament may be presented in capsule, tablet, 
slow release or elixir form or as a gel or paste. Furthermore, the 
medicament may be provided as a live stock feed or as food suitable for 
human consumption. 
The invention also provides an antibacterial formulation comprising a 
complex according to the invention, in which the active substance is an 
antibacterial active substance together with a carrier or diluent 
therefor. 
In another embodiment the invention provides a method of enhancing a host 
vertebrate's response to an orally administered active substance which 
method comprises the oral administration of an effective amount of said 
active substance as a complex according to the invention, or of a 
medicament according to the invention. 
The invention also provides a method of selectively modulating the 
magnitude and/or type of immune response to an antigen or hapten, which 
method comprises orally administering an effective amount of said antigen 
or hapten as a complex according to the invention, or of a medicament 
according to the invention. 
The invention also provides a method of delivering an active substance to 
any unicellular or multicellular organism, including bacteria, protozoa, 
or parasites, which has a requirement for VB12 as well as a specific 
uptake mechanism for the same, which method comprises administering a 
complex of the invention to the organism. In this manner bacteria which 
are resistant to an antibiotic due to the loss of their ability to 
transport the antibiotic into the cell could be once again made sensitive 
to the antibiotic by coupling the antibiotic to VB12 and using the natural 
VB12 uptake system of the bacteria to deliver the antibiotic into the 
cell. In this fashion a number of antibiotics whose use has been 
discontinued due to the occurrence of bacterial resistance could regain 
pharmacological significance. 
In a further embodiment of the invention there is provided a method of 
delivering an active substance across the blood/brain barrier or across 
the placenta into a developing foetus by administering a complex of the 
invention. Delivery of such substances would occur through the natural 
VB12 uptake mechanisms at these barriers. 
BEST MODE FOR CARRYING OUT THE INVENTION 
Materials 
Bovine serun albumen (BSA), VB12, p-nitrophenol, LHRH acetate salt, and 
neomycin sulfate were all purchased from Sigma Chemical Co. St. Louis, Mo. 
USA. 
1-ethyl-3-(dimethylaminopropyl)carbodiimide HCl (EDAC) was obtained from 
BIORAD Labs, California, while N,N'dicyclohexylcarbodiimide (DCC) was 
purchased from Fluka. 
PREATION 1 
Monocarboxyl-Derivative of VB12 
The acid derivative of VB12 can readily be prepared by hydrolysing native 
VB12 for 72 h in 0.4M HCl at room temperature. The reaction is stopped by 
passing the hydrolysate down an ion-exchange column of DOWEX AG1-X8. The 
flow through containing the monoacid VB12 is lyophilized and resuspended 
in 0.2M pyridine and adjusted to pH9.05 with 1M ammonium hydroxide. The 
solution is then passed down a Sephadex QAE A25 previously equilibrated 
with 0.2M pyridine and the monoacid eludated with a gradient from 0.4M 
pyridine to 0.4M, 0.16M acetic acid. The fractions containing the purified 
mono-acid are pooled and lyophilized.

The following examples illustrate preferred embodiments of the invention 
and should not be construed as limiting thereon. 
The monocarboxyl VB12 can be covalently crosslinked to any amino containing 
compound by the use of a suitable carbodiimide. 
EXAMPLE 1 
VB12-BSA 
VB12-BSA complex was formed by mixing an equal weight of COOH-B12 with BSA 
in distilled water, the pH was adjusted to 6.5 with 1M NaOH and an equal 
weight of solid EDAC was added to the solution and allowed to react 
overnight. Free, unreacted COOH-B12 was removed by chromatography of 
Sephadex G-25, followed by repeated ethanol precipitation of the VB12-BSA 
complex. 
EXAMPLE 2 
VB12-Lys-6-LHRH 
Monocarboxyl VB12 plus 1.5 equivalents of n-hydroxysuccinamide were 
dissolved in cold (4.degree. C.) dimethyl formamide (DMF). To this 
solution was added 1.1 equivalents of dicyclohexylcarbodiimide (DCC) in 
DMF. The solutions were warmed to room temperature and allowed to react 
for 1 hour. Lys-6-LHRH dissolved in DMF containing triethylamine was added 
and allowed to react overnight. The resultant complex was separated from 
the free reactants by chromatography on Sephadex G-25 followed by reverse 
phase HPLC. 
EXAMPLE 3 
VB12-Neomycin 
The total acid hydrolisate of VB12 was adjusted to pH6.5 with NaOH, an 
equal weight of neomycin sulfate was added to the solution followed by an 
equal weight of EDAC. The conjugation was allowed to proceed overnight 
after which the conjugate was separated from unreacted reagents by 
chromatography on G-25 and reverse phase HPLC. 
All reactions and purification procedures were monitored by thin layer 
chromatography. The degree of VB12 substitution of BSA was determined by 
spectrophotometric scanning of the conjugate using O.D.278 extinction 
values of 0.6 and 11.5, for 1 mg/ml solutions of BSA and VB12, 
respectively, and an O.D.361 of 20.4 for VB12. 
Female C57B1/6J mice (18-22 g) were obtained from the Animal Resources 
Centre (Perth, Western Australia). All mice received conjugate 
preparations in 0.5 ml of 0.1M carbonate/bicarbonate buffer pH9.5 using a 
specially prepared feeding needle. Mice were fed on days 0 and 14. On day 
21 the mice were bled from the orbital plexus. Antibody titres of serum 
were determined by ELISA using alkaline phosphatase conjugated anti-mouse 
serum. 
EXAMPLE 4 
Stimulation of serum antibodies following oral administration of VB12-BSA 
complex 
The possible potential for VB12 deliver protein molecules, covalently 
linked to it, from the intestine to the circulation was investigated. The 
immune response generated to this complex was compared to that generated 
by the protein fed alone or together with VB12, or to the protein injected 
intramuscularly. 
As seen in Table 1, feeding mice with microgram quantities of bovine serum 
albumen (BSA) or Fowl gamma-globulin (FGG) coupled to VB12 resulted in the 
stimulation of significant serum antibody responses to the BSA or FGG 
respectively. Feeding of either protein in similar amounts or in a 50 fold 
excess either mixed with VB12 or without VB12 resulted in the stimulation 
of no anti-BSA or anti-FGG antibodies. Feeding of these VB12-protein 
complexed was also capable of stimulating good cellular immunity (as 
measured by the footpad assay for DTH) 
TABLE 1 
______________________________________ 
Immune response to orally presented 
VB12-BSA or VBI2-FGG complex 
Oral Serum Antibody 
Footpad 
Immunogen Response* Response+ 
______________________________________ 
BSA (50 .mu.g) &lt;4 0 
BSA (2500 .mu.g) 
&lt;4 nd 
VB12 &lt;4 0 
VB12 + BSA &lt;4 0 
VB12 - BSA 1351 .+-. 198 
17.3 .+-. 5 
FGG &lt;4 0 
VB12 + FGG &lt;4 0 
VB12 - FGG 1584 .+-. 647 
23.3 .+-. 6 
FGG + FCA s.c. 16504 .+-. 3047 
27.4 .+-. 4 
______________________________________ 
*The reciprocal of the antiserum dilution that gave an ELISA reading of 
0.5 afer 45 min. at 37.degree. C. on day 21 after initial feeding. Each 
value represents the mean of 15 mice .+-. 1 standard deviation. Mice 
received two feedings of antigen (50 .mu.g) on days 1 and 14. On day 21 
mice were bled from the retro orbital plaxus and the antibody titres 
measured by ELISA as described previously (RussellJones et al., 1984). 
Each protein molecule was substituted with an average of 5 VB12 groups. 
+Footpad swelling was measured in mm using a microcaliper. All groups 
received a 50 .mu.g priming dose of antigen followed by challenge with 10 
.mu.g of the immunizing antigen in the right foot and 10 .mu.g of 
ovalbumen in the left footpad. Swelling was measured after 24 h. 
EXAMPLE 5 
Oral delivery of VB12-LHRH as a means of stimulating ovulation 
Although a number of hormones as oestrogen and progesterone are actively 
absorbed upon oral administration, there are may other which have little 
effect when given per os. Noteable amongst these hormones is the peptide 
hormone luteinizing hormone releasing hormone (LHRH), or gonadotrophin 
releasing hormone (GnRH). This hormone is normally secreted by the 
anterior pituitary and is responsible for the control of release of 
luteinizing hormone (LH) and follicle stimulating hormone (FSH). 
Parenteral injections of LHRH have previously been shown to be effective 
in stimulating FSH and LH release, however orally presented LHRH has 
little effect. Many studies have been performed on varying the sequence of 
LHRH, with the result that a number of agonists and antagonists have now 
been identified. Perhaps one of the most powerful agonists identified to 
date is the D-Lys-6 analogue of LHRH (D-Lys-6.LHRH). As the epsilon amino 
group on the lysine of this analogue is readily accessible for peptide 
cross-linking it was decided to use the DCC method to link monocarboxyl 
VB12 to D-Lys6.LHRH and to test it's efficacy upon oral administration. 
The D-lys-6 analogue of LHRH was synthesized by us and purified by reverse 
phase HPLC. The purified analogue was coupled to monocarboxyl VB12 using 
DCC as described in Example 2. The conjugated product was purified by 
Sephadex G-25 chromatography in 10% acetic acid, followed by HPLC 
Chromatography. 
Mature C57B1/6J female mice were treated in the following fashion: On day 0 
all mice received a subcutaneous (s/c) superovulating dose of pregnant 
mare serum gonadotraphin (PMSG) to stimulate the growth of ovarian 
follicles. After 48 hours mice received various doses of LHRH, Lys-6-LHRH 
or saline. On day 3 mice were sacrificed and examined for ovulation. 
Ovulation was assessed by examining for the presence of corpora 
haemorrhagica on the ovaries using a stereoscopic microscope at 80.times. 
power. 
The results below show that by coupling Lys-6-LRH to VB12 it is possible to 
deliver the analogue orally and to still observe a biological effect as 
exemplified by it's ability to stimulate ovulation in developing 
follicles. The inability of this preparation to exert it's effect when 
injected intravenously presumably reflects the rapid clearance of free 
VB12 when it is not complexed to transcobalamin II. 
TABLE 2 
______________________________________ 
Demonstration of the biological activity of Lys-6-LHRH 
Treatment Number of mice 
Day 0 Day 2 ovulating 
______________________________________ 
PMSG 8IU Lys-6-LHRH 50 .mu.g iv 
3/4 
PMSG 8IU LHRH 50 .mu.g iv 
1/4 
PMSG 8IU Saline 250 .mu.l iv 
0/4 
PMSG 4IU Lys-6-LHRH 50 .mu.g iv 
3/3 
PMSG 4IU LHRH 50 .mu.g iv 
1/3 
PMSG 4IU Saline 250 .mu.l iv 
0/3 
______________________________________ 
TABLE 3 
______________________________________ 
Demonstration of the ability of VB12 
to deliver the Lys-6-LRH orally 
Treatment Number of mice 
Day 0 Day 2 ovulating 
______________________________________ 
PMSG 8IU 
VB12-Lys-6-LHRH 
50 .mu.g iv 
0/5 
PMSG 8IU 
VB12-Lys-6-LHRH 
50 .mu.g/os 
3/5 
PMSG 8IU 
LHRH 50 .mu.g/os 
1/5 
PMSG 8IU 
Saline 250 .mu.l/os 
0/5 
______________________________________ 
TABLE 4 
______________________________________ 
Dose response to orally presented VB12-Lys-6-LHRH 
Treatment Number of mice 
Day 0 Day 2 ovulating 
______________________________________ 
PMSG 8IU 
VB12-Lys-6-LHRH 
50 .mu.g/os 
4/5 
PMSG 8IU 
VB12-Lys-6-LHRH 
25 .mu.g/os 
3/5 
PMSG 8IU 
VB12-Lys-6-LHRH 
12 .mu.g/os 
5/5 
PMSG 8IU 
VB12-Lys-6-LHRH 
6 .mu.g/os 
2/5 
PMSG 8IU 
LHRH 50 .mu.g/os 
1/5 
PMSG 8IU 
LHRH 25 .mu.g/os 
1/5 
PMSG 8IU 
LHRH 12 .mu.g/os 
0/5 
PMSG 8IU 
LHRH 6 .mu.g/os 
0/5 
PMSG 8IU 
HCG 10 IU iv 5/5 
PMSG 8IU 
Saline 250 .mu.l/os 
0/5 
______________________________________ 
EXAMPLE 6 
A number of drugs including the antibiotic, neomycin, are highly effective 
antibiotics when injected parenterally, however they are completely 
ineffective when given orally as they cannot be transported across the 
intestinal epithelium. It was therefore decided to see if VB12 could act 
as a carrier for an antibiotic (neomycin) which normally has no effect 
upon a systemic infection when the antibiotic was given orally. 
Neomycin was covalently linked to VB12 as described in Example 3 and fed to 
mice infected with S. typhimurium. 
Oral administration of neomycin, or neomycin plus VB12 was not able to 
eliminate systemic infection with S. typhimurium. When neomycin was 
coupled to VB12, however, a significant quantity of the conjugate was 
transported across the intestinal epithelium and was capable of 
eliminating a systemic Salmonella infection. Table 3 shows that mice 
infected with S. typhimurium could be saved by either feeding 
VB12.neomycin conjugate (1 mg total dose) or by the i.m. injection of 
neomycin or VB12.neomycin (both 1 mg total dose). All other treatments 
failed to prevent death due to infection. In addition, the extent to which 
orally presented VB12.neomycin was capable of clearing infective particles 
from the liver and spleen of experimental animals suggests that, at least 
for this dosage, VB12.neomycin is comparable to an i.m. injection of 
neomycin alone or the neomycin.VB12 conjugate (Table 5). 
TABLE 5 
______________________________________ 
Bactericidal properties of VB12-neomycin conjugates 
Suvivors (day 10) 
Treatment Route Number Percentage 
______________________________________ 
Saline oral 0 0 
Neomycin oral 0 0 
VB12 oral 0 0 
Neomycin + VB12 
oral 0 0 
Neomycin - VB12 
oral 2 100 
Saline i.m. 0 0 
Neomycin i.m. 2 100 
Neomycin - VB12 
i.m. 2 100 
______________________________________ 
Male C57B1/6J mice (/group) were fed 1.times.10.sup.6 S. typhimurium on day 
0. On day 3 mice received either saline, VB12, VB12+neomycin 
(Neomycin+VB12), VB12 coupled to neomycin (Neomycin-VB12), or neomycin 
alone. A total dose of 1 mg was administered as five smaller doses each 
separated by 12 hours. Neomycin was coupled to VB12 and the conjugate 
purified as outlined in Example 3. 
It is possible to covalently couple VB12 to proteins (FGG and BSA), 
hormones (LHRH) and antibiotics (neomycin) and to utilize the natural 
active uptake mechanism for VB12 to transport these molecules from the 
lumen of the gut into the systemic circulation while retaining full 
immunogenicity and/or biological activity of the molecules coupled to 
VB12. The importance of these findings lie in the potential use of VB12 as 
a specific carrier of highly potent hormones, antibiotics and vasoactive 
peptides which currently must be repeatedly administered by injection at 
considerable costs and inconvenience. 
INDUSTRIAL APPLICABILITY 
The present invention provides a simple and novel technique for the 
specific oral presentation of various molecules previously incapable of 
being transported across the gut in significant amounts or in producing a 
significant systemic immune response upon oral feeding of various 
antigens. These antigens would not normally elicit an immune response when 
fed unless very large quantities of antigen were administered. Similarly 
various active molecules which are normally only poorly absorbed from the 
intestine can be covalently linked to VB12 and so render them susceptable 
to intestinal uptake. 
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