Pharmaceutical compositions

Compositions containing a bile salt and a buffer such as a carbonate or bicarbonate salt which is adapted to buffer the gut to a pH of from 7.5 to 9 are capable of increasing the bioavailability of an active molecule whilst minimising the toxic side effects which are generally associated with bile salts.

This application is a continuation of PCT/GB95/02015 filed Aug. 25, 1995. 
The present invention relates to pharmaceutical compositions and, in 
particular to orally administrable compositions of proteins, peptides and 
other active molecules which are not generally easily absorbed from the 
gastrointestinal tract. 
Medical practice has for many years prescribed or advised the 
administration of many biologically active materials for the treatment or 
prophylaxis of a wide variety of diseases or conditions. One of the most 
well known, but by no means the only, prescribed biologically active 
proteinaceous material is insulin, which is used for the control of 
diabetes. Other biologically active proteinaceous materials include growth 
factors, interleukins and calcitonin and non-proteinaceous biologically 
active materials include oligonucleotides and polysaccharides. 
Possibly the easiest method of taking any medication is oral ingestion. 
Such route of administration, which may be by means of syrup, elixir, 
tablets, capsules, granules, powders or any other convenient formulation, 
is generally simple and straightforward and is frequently the least 
inconvenient or unpleasant route of administration from the patient's 
point of view. 
It is therefore unfortunate that most of these materials are very poorly 
absorbed when administered orally. Firstly, the preferred route of 
administration of proteinaceous medicaments and other biologically active 
materials involves passing the material through the stomach, which is a 
hostile environment for many materials, including proteins. As the acidic, 
hydrolytic and proteolytic environment of the stomach has evolved 
efficiently to digest proteinaceous materials into amino acids and 
oligopeptides for subsequent anabolism, it is hardly surprising that very 
little or any of a wide variety of biologically active proteinaceous 
material, if simply taken orally, would survive its passage through the 
stomach to be taken up by the body in the small intestine. 
It is possible to provide enteric coated formulations which are protected 
from the acid environment of the stomach but even so, relatively large 
molecules such as peptides and proteins are poorly absorbed by the small 
intestine. 
The result, as many diabetics can testify, is that many proteinaceous 
medicaments have to be taken parenterally, often by subcutaneous, 
intramuscular or intravenous injection, with all the inconvenience, 
discomfort and difficulties of patient compliance that that entails. 
This is not an isolated problem, as diseases needing control by the 
administration of proteinaceous material can be very widespread. Diabetes, 
for example, claims a large number of sufferers in many countries of the 
world and there are numerous other conditions which require treatment by 
administration of a proteinaceous compound or another macromolecule. 
Osteoporosis is another example of a condition which can be treated using 
a protein--in this case calcitonin--and protein growth hormones can be 
used to treat dwarfism. 
Clearly, therefore, there is a need for pharmaceutical formulations of 
proteins and other macromolecules which can be administered orally and 
which provide acceptable bioavailability of the active material. 
It is known that when pharmaceutically active compounds are administered 
together with certain bile salts, their bioavailability is increased. This 
is discussed by Kakemi et al, (Chem. Pharm. Bull, 18(2) 275-280 (1970)) 
who showed that the bioavailability of various substances could be 
increased by administration in combination with taurocholate or 
glycocholate. Further experiments by Kojima et al (Chem. Pharm. Bull, 
25(6) 1243-1248 (1977)) showed that sodium cholate is capable of 
increasing the absorption of various substances but that release of cell 
membrane components is also accelerated. 
In addition there are several other references in the prior art to the use 
of bile salts as absorption enhancers including GB-A-2244918 which teaches 
that the bioavailability of somatostatin can be improved when it is 
administered with a cholanic acid derivative such as chenodeoxycholic acid 
or ursodeoxycholic acid. 
It is believed that the bile salts improve absorption of biologically 
active materials because of their action on the cell membranes of 
epithelial cells. The cell membranes become more permeable, possibly 
because the detergent action of the bile salts removes lipids from cell 
membranes and causes them to become more fluid. One theory is that the 
increased permeability of the cell membrane enables the active materials 
to pass through the epithelial cells. Alternatively, it is possible that 
the cytoskeletal structure of the epithelial cells is modified as a result 
of changes in cytoplasmic levels of sodium or calcium arising from 
increased membrane permeability. This would result in alteration of the 
integrity of the tight junctions so that there are gaps between the cells 
through which the active materials can pass. Whatever the mechanism, it 
seems clear that the increased absorption arises from the increased 
permeability of the cell membranes. 
However the inclusion of bile salts in pharmaceutical formulations has 
always been a problem because they have unacceptably high toxicity when 
used in the amounts required for them to exert their bioavailability 
improving effect. It has been suggested that the reason for this is that 
the increase in the permeability of epithelial cells brought into contact 
with bile salts results in an increased flow of ions into and out of the 
cell. In order for cells to remain viable, the intracellular 
concentrations of ions such as sodium, potassium and calcium ions must 
remain within relatively narrow ranges and it may be that the increased 
ion flow caused by the presence of bile salts causes the intracellular ion 
concentrations to move outside the ranges within which cells are viable. 
Thus, the problem with the use of bile salts as absorption enhancers is 
that they have always been considered to have an unacceptably low 
therapeutic index. Throughout the present specification the term 
"therapeutic index" has been used to refer to the ratio 
toxic dose: absorption enhancing dose. 
It would thus be extremely useful to be able to reduce the toxicity and 
hence effectively to increase the therapeutic index of bile salts to the 
point where they could be used in pharmaceutical compositions without 
unacceptable toxic side effects. 
In a first aspect of the present invention there is provided a 
pharmaceutical and/or veterinary composition comprising a biologically 
active material, a bile acid or salt and an agent adapted to adjust the pH 
of the gut to a pH value of from 7.5 to 9. 
WO-A-9012583 teaches a pharmaceutical composition comprising an active 
agent, a bile salt and an additional component of bile. However, the only 
additional components suggested in this document as likely to be useful in 
such compositions are additional bile salts and biliary lipids, in 
particular phospholipids. Although bicarbonate (a preferred pH adjusting 
agent useful in the invention) is a component of bile, there is no mention 
of bicarbonate salts in WO-A-9012583 and it is clear that bicarbonate 
salts were not envisaged as being useful components of a composition 
containing a bile salt and a pharmaceutically active agent. Thus the 
effect of bicarbonate or other pH adjusting agents in effectively 
increasing the therapeutic index of bile salts was certainly not disclosed 
in this prior document. 
In the present invention it should be understood that the terms bile salt 
and bile acid are used inter-changeably because whether the salt or its 
conjugate acid is present will depend on the pH of the surrounding 
environment. Thus, solid formulations according to the invention may 
contain either a bile salt or a bile acid. However, the compositions as 
administered will often be of a pH such that if an acid is used, it will 
be converted into the salt form when in solution. 
When a bile salt is used in the compositions of the present invention, it 
is preferred that there is a soluble counter-ion present such as sodium or 
potassium. It is also possible to use, for example, an ammonium ion but 
this is less preferred. 
Bile salts are naturally occurring surfactants. They are a group of 
compounds with a common "backbone"0 structure based on cholanic acid found 
in all mammals and higher vegetables. Bile salts may be mono-, di- or 
tri-hydroxylated; they always contain a 3.alpha.-hydroxyl group whereas 
the other hydroxyl groups, most commonly found at C.sub.6, C.sub.7 or 
C.sub.12, may be positioned either above (.beta.) or below (.alpha.) the 
plane of the molecule. 
Within the class of compounds described as bile salts are included 
amphiphilic polyhydric sterols bearing carboxyl groups as part of the 
primary side chain. The most common examples of these in mammals result 
from cholesterol metabolism and are found in the bile and, in derivatised 
form, throughout the intestine. 
In the context of this specification, the term may also apply to synthetic 
analogues of naturally occurring bile salts which display similar 
biological effects, or to microbially derived molecules such as fusidic 
acid and its derivatives. 
The bile salt may be either unconjugated or conjugated. The term 
"unconjugated" refers to a bile salt in which the primary side chain has a 
single carboxyl group which is at the terminal position and which is 
unsubstituted. Examples of unconjugated bile salts include cholate, 
ursodeoxycholate, chenodeoxycholate and deoxycholate. A conjugated bile 
salt is one in which the primary side chain has a carboxyl group which is 
substituted. Often the substituent will be an amino acid derivative which 
is linked via its nitrogen atom to the carboxyl group of the bile salt. 
Examples of conjugated bile salts include taurocholate, glycocholate, 
taurodeoxycholate and glycodeoxycholate. 
The quantity of bile acid contained in a single dose of the formulation 
will vary depending on the particular bile acid chosen and the rate and 
extent to which that bile acid dissolves in the aqueous fluid contained in 
the intestine. For chenodeoxycholic acid, and most other bile acids, this 
is likely to be within the range 10 mg to 1 g, preferably between 20 mg to 
200 mg, and most preferably 30 mg to 100 mg. For deoxycholic acid, the 
maximum will generally not exceed 500 mg, in view of its slightly greater 
activity. 
The gut of many animals (particularly humans and other mammals) is 
naturally buffered to a pH below neutrality. Compositions of the invention 
comprise an agent adapted to adjust the pH of the gut to a pH of from 7.5 
to 9. The agent is "adapted" to adjust the pH either by its chemical 
nature or by the amount in which it is present or, usually, both. The 
optimum pH to which the gut is adjusted is in the range 7.8 to 8.3. 
While simple agents adapted to adjust the pH of the gut into the range 
specified above may be successfully used in the invention, it is preferred 
that the pH adjusting agent also has the capability of buffering the gut 
to a pH within the stated range. This can give a more long lasting effect, 
which may be desired in many circumstances. Also, a buffer has a greater 
capacity to accommodate the patient-to-patient variability of endogenous 
gut pH, as well as the viability of gut pH seen over time in any 
individual patient; in particular, a buffer can act as a safety barrier to 
ensure that the pH of the patient's gut is not radically changed outside 
safe limits during the administration of formulations of the invention. 
Two of the most favoured agents for adjusting the pH suitable for use in 
the invention, either separately or in combination, are carbonate and 
bicarbonate ions. In the discussion which follows, bicarbonate is referred 
to by way of illustration of the principles involved, but the same 
principles apply equally to other agents capable of exerting a similar 
effect on intestinal pH. 
The amount of agent necessary to adjust the pH to within the range 
contemplated is difficult to determine directly because the intestinal pH 
can vary between 5 to 7, and its aqueous content can vary, as can its 
intrinsic buffering capacity, which acts to maintain a low pH. 
Consequently considerations aimed at determining the appropriate quantity 
of agent controlling pH need to take into account a "worst-case" scenario. 
An indication may be obtained by observing the pH achieved upon adding a 
potential pH-adjusting agent to a 50 mM solution of MES (morpholino ethane 
sulphonic acid) adjusted to pH 6.0. Concentrations useful in the invention 
are those that result in the adjustment of the pH to within the range 7.5 
to 9; preferred concentrations result in an adjustment to within the range 
7.8 to 8.3. (For reasons which will be explained below, the weight amount 
of pH adjusting agent is generally that which produces the desired 
concentration in 10 ml liquid.) Values for pH attained are shown below for 
the preferred bicarbonate at different concentrations, dispersed in 
distilled water or in MES. 
______________________________________ 
Bicarbonate 
concentration 
(molarity) Water MES 
______________________________________ 
0.002 8.86 6.42 
0.004 8.67 6.58 
0.008 8.63 6.86 
0.016 8.57 7.14 
0.031 8.55 7.40 
0.062 8.49 7.68 
0.125 8.41 7.93 
0.250 8.30 8.14 
0.500 8.16 8.05 
1.000 8.02 7.96 
______________________________________ 
Hence, the minimum amount of bicarbonate useful in the invention in most 
cases is about 0.045M, which yields a 25 pH in the gut of about 7.5. This 
translates to about 40 mg sodium bicarbonate, for a 10 ml dispersion 
volume. 
A saturated solution of bicarbonate in water (&lt;2M) has a pH of 8.01. From 
this it will be seen that, regardless of the concentration of preferred 
bicarbonate administered, the pH will not rise above 9.0. Under certain 
circumstances, it may be that a concentrated solution of a salt of the 
bile acid itself has sufficient capacity to raise the pH to the 
appropriate level, although the effect will not be as strong as for the 
preferred bicarbonate. Hence, it is possible, although not always 
desirable, for the pH adjusting agent to be the bile salt itself, or a 
different bile salt. 
The greater the buffering capacity of the composition, the longer that the 
pH specified above will prevail in the gut and hence the longer the 
benefits of the invention will prevail. 
Two of the most favoured buffering agents suitable for use in the 
invention, either separately or in combination, are carbonate and 
bicarbonate ions. As already mentioned, the advantage of the compositions 
of the present invention is that they are capable of effectively 
increasing the therapeutic index of the bile salt present in the 
composition. The beneficial effect is present both for conjugated and 
unconjugated bile salts but the action of the carbonate or bicarbonate 
ions on conjugated bile salts is slightly different from the action on 
unconjugated bile salts. 
With conjugated bile salts, the presence of sufficient bicarbonate or 
carbonate has the effect of increasing cell membrane permeability during 
the exposure of cells to a given amount of bile salt without affecting 
cell viability. This means that the amount of bile salt needed to obtain a 
given increase in permeability is reduced and so the permeability of the 
cells can be increased without increasing the adverse effects of the bile 
salt. 
With unconjugated bile salts in the presence of carbonate or bicarbonate, 
cell permeability is not increased during exposure of the cell to the bile 
salt but, rather, the toxic effect on cells after exposure to bile salt is 
reduced. Again, this means that the amount of bile salt which can be 
administered without affecting the viability of the cells is increased in 
the presence of bicarbonate or carbonate ions. 
In general, the amount of pH adjusting agent will be such that when a unit 
dose of the composition is dispersed in the amount of liquid which would 
be present in the length of gut over which the composition would be 
distributed on administration to a patient, the pH adjusting agent 
concentration is at least about 0.01M, although a more precise estimate 
can be had by reference to the MES buffer test referred to above. Usually, 
and preferably for carbonate and bicarbonate, however, the concentration 
of bicarbonate or carbonate will be greater than 0.05M and it is most 
preferred that the concentration is at least about 0.1M although higher 
concentrations, for example up to 1M can be used. 
A typical length of the small intestine over which a composition of the 
present invention would be likely to be distributed would be 30 cm and the 
amount of liquid which would be present in that length of gut would be 
likely to be about 10 ml. However, it should be stressed that the choice 
of 30 cm as a suitable length is arbitrary and the invention is not 
intended to be limited to compositions which are distributed over this 
distance in the small intestine. There may be reasons why it would be 
desirable for a composition to be distributed over a shorter or a much 
longer period of time, for example a sustained release composition may 
disperse much more slowly and would therefore be distributed over a much 
longer length of the small intestine. Such compositions are familiar to 
those skilled in the art who would easily be able to determine the most 
suitable type of composition to meet a particular therapeutic requirement. 
If the composition were adapted to be dispersed over a longer or shorter 
length of the small intestine then the amount of water in which a unit 
dose of the composition would be dispersed to give a particular 
concentration would be accordingly greater or smaller. 
The unit dose which is dispersed in water to determine the minimum amount 
of bicarbonate required will be the dose which would be administered to a 
patient at any one time. For liquid formulations, this will be a 
predetermined amount calculated by a physician or pharmacist. For solid 
formulations, such as tablets or capsules, the unit dose will generally be 
a single tablet or capsule. However, there may be circumstances in which a 
patient would be required to take more than one tablet or capsule, for 
example if large doses of active substance are needed, and in this case 
the amount of bicarbonate may be divided between two or more tablets or 
capsules. 
It appears that an additional beneficial effect of the preferred carbonate 
or bicarbonate ions may arise because they can increase the solubility of 
the bile acids. In general bile salts start to be converted to their 
conjugate acid at pH of about 6.8 or below and the acid form is insoluble 
in aqueous solutions. Since the buffering agent has the effect of 
buffering the compositions of the invention to a pH of about 7.5 or above, 
the solubilised bile salt will be present rather than the insoluble bile 
acid. A solubilised bile salt will be able to act on the epithelial cells 
when in solution, whereas this may not be possible in the solid acid form. 
The higher the concentration of buffering agent, the more rapidly will a 
satisfactory pH be attained, resulting in more rapid dissolution of the 
bile acid or salt; this will result in a higher local concentration of the 
bile salt in solution, leading to greater efficacy in enhancing 
permeability to bioactive materials. 
It is also possible that the reason for the particularly beneficial effect 
of carbonate and bicarbonate ions is associated in some way with the fact 
that bicarbonate receptors are expressed on the surfaces of intestinal 
cells. However, the nature of this link, if indeed it exists, is not clear 
at present and, in any case, it should be stressed that the correctness or 
otherwise of this theory does not limit the effectiveness of the present 
invention. The results obtained using bicarbonate are generally superior. 
As discussed above, it is thought that one of the functions of the 
bicarbonate or carbonate ions is to ensure that the bile salt is in the 
soluble form. However, the presence of calcium ions increases the pH at 
which the salt becomes the prevalent form and below which insoluble bile 
acid will start to precipitate out of solution. This pH varies according 
to the particular bile salt but it is desirable to prevent precipitation 
of the bile acid. For this reason, it is often advantageous to include in 
the composition a chelator of calcium ions such as a salt of a di- or 
tri-carboxylic acid (for example a citrate salt), 
ethylenediaminetetraacetic acid (EDTA), ethylene glycol 
bis-(.beta.-aminoethylether)N,N,N',N'-tetraacetic acid (EGTA) or phytate 
and other polyphosphorus compounds. 
When a citrate salt is used as the calcium ion chelator, it is greatly 
preferred that it should be in a form soluble in water. The most suitable 
salts are therefore sodium and potassium citrate although in some 
circumstances ammonium citrate may also be used. 
The term "biologically active material" includes, in particular, 
pharmaceutically active proteinaceous materials. The proteinaceous 
material may be a pure protein, or it may comprise protein, in the way 
that a glycoprotein comprises both protein and sugar residues. 
The material may be useful in human or veterinary medicine, either by way 
of treatment or prophylaxis of diseases or their symptoms, or may be 
useful cosmetically or diagnostically. Examples of proteinaceous 
biological material which can be provided as orally or rectally 
administrable formulations in accordance with this invention include 
protein or peptide hormones or hormone releasing factors such as insulin, 
calcitonin and growth hormone, whether from human or animals or semi- or 
totally synthetically prepared, or other bioactive peptides such as 
interferons including human interferon alpha and interleukins including 
IL-1, IL-2, IL-3, IL-4 and IL-5. Analogues and active fragments of these 
or other proteins may also be used. 
It is particularly remarkable that the invention not only works, but works 
well, with insulin, as insulin is normally poorly soluble at the pH values 
contemplated by the present invention. It seems that the insulin is only 
able to disperse as a result of some unexpected interaction between 
insulin and bile salt which stabilises it in bicarbonate or other high pH 
solution. 
Biologically active materials may also be oligo-nucleotides such as 
antisense oligonucleotides and their analogues which may be useful for 
interfering with the replication of nucleic acids in virally infected or 
cancerous cells and for correcting other forms of inappropriate cell 
proliferation. Polysaccharides such as heparin are also suitable for use 
in the present invention as are combinations of one or more protein, 
nucleic acid or polysaccharide. 
The molecular weight of the biologically active material should preferably 
not be greater than about 20,000 Da. This is because, even with the 
increased permeability of the cells which comes about as a result of the 
use of the compositions of the present invention, it is no easy matter to 
achieve effective bioavailability with active molecules of greater size 
than this. In general, however, the smaller the size of the active 
molecule, the easier it becomes to deliver and it is therefore preferred 
that the biologically active molecule has a molecular weight of less than 
about 10,000 Da and, most suitably, less than about 5,000 Da. 
The amount of biologically active material present will naturally depend on 
its intrinsic potency. All that is necessary is that it is present in a 
sufficient amount that it manifests its desired activity when ingested. 
For pharmaceuticals, the amount administered will be under the guidance of 
the physician or clinician. 
Other excipients which may be present include lactose, employed as a 
filler, to ensure homogeneity of the composition, and to aid in handling 
of the preparation, Ac-di-sol.TM. (cross-carmellose sodium), a swelling 
agent which aids tablet disintegration, and polyvinyl pyrollidone, 
commonly used as a binding agent during granulation processes. 
The formulation will usually be in a solid or, exceptionally, liquid form. 
Solid forms are preferred because the active material can easily be 
prevented from being digested in the stomach by enteric coating, and it is 
much easier to ensure that the active ingredient(s) and the bile salt 
reach the small intestine both intact and contemporaneously; this is much 
more difficult to achieve with a liquid formulation. If the composition is 
formulated as a solid, which should, for a prolonged shelf life, be 
substantially dry, it may be in tablet, bolus, powder, granular or 
microgranular form and may also contain appropriate fillers or binders 
which are well known to those skilled in the art. Powders, granules or 
microgranules may be encapsulated. As mentioned above, either a bile acid 
or a bile salt may be used in a solid formulation. 
Liquids suffer from the disadvantages referred to above. However, if for 
any reason it is particularly desired to formulate a composition of the 
invention as a liquid, an enteric coated capsule is probably the best 
means of administration of the contents, which may be a syrup or elixir. 
The compositions may be formulated for rapid or sustained release or a 
combination of these two release forms may be used. The compositions can 
also be incorporated into immediate, delayed or pulsed release 
formulations. The amount of buffering agent included in the composition 
will be dependent upon the way in which the composition is formulated and 
the time taken for it to disperse in the gut and thus the amount of 
buffering agent included in a sustained release formulation will generally 
be much greater than the amount needed for a rapid release formulation. 
In general the compositions of the invention may be prepared simply by 
admixing the ingredients using techniques well known to those skilled in 
the art of preparing pharmaceutical formulations. 
The invention also relates to a method of improving the bioavailability of 
an active material, the method comprising coadministering to a patient an 
active material, a bile salt and an agent adapted to adjust the gut to a 
pH of from 7.5 to 9. 
Therefore, in a second aspect of the invention there is provided the use of 
a bile salt and an agent adapted to adjust the gut to a pH of from 7.5 to 
9 in the preparation of an agent for increasing the bioavailability of a 
biologically active material to be coadministered with the agent. 
Preferred features are as detailed for the first aspect of the invention, 
mutatis mutandis.

Preparation of Solutions for Use in Examples 
A neutral red stock solution was prepared at concentration of 0.5 mg/ml in 
DMEM (Dulbecco's Modified Eagle Medium) (pH 4.5) which was then diluted 
1:10 with TCM (Tissue Culture Medium) or Hanks Balanced Salt Solution 
(HBSS) before use. 
Hanks Balanced Salt Solution (HBSS) was prepared containing the following 
ingredients: 
______________________________________ 
g/L mM 
______________________________________ 
CaCl.sub.2.2H.sub.2 O 
0.19 1.26 
KCl 0.40 5.37 
KH.sub.2 PO.sub.4 
0.06 0.44 
MgCl.sub.2.6H.sub.2 O 
0.10 0.49 
MgSO.sub.4.7H.sub.2 O 
0.10 0.41 
NaCl 8.00 133.33 
NaHCO.sub.3 0.35 4.17 
Na.sub.2 HPO.sub.4 
0.48 3.38 
D-Glucose 1.00 5.56 
Phenol Red 0.01. 
______________________________________ 
A 0.1M solution of sodium bicarbonate in distilled water was prepared 
(buffer solution). Similar 0.1M solutions of sodium acetate, sodium borate 
and sodium carbonate in distilled water were also prepared. These are also 
referred to in the examples as buffers. 
Test solutions were prepared containing various concentrations of a bile 
salt in either HBSS or in one of the 0.1M buffer solution. The following 
bile salts were tested: 
Unconjugated Bile Salts: sodium cholate, sodium ursodeoxycholate, sodium 
chenodeoxycholate, sodium deoxycholate; 
Conjugated Bile Salts: sodium taurocholate, sodium glycocholate, sodium 
taurodeoxycholate, sodium glycodeoxycholate. 
A MTT stock solution was prepared at a concentration of 5 mg/ml in 
distilled water, which was then diluted 1:10 with TCM before use. 
The tests described in Examples 1 and 2 are well known tests and conform to 
British Standards (BS 5750). Caco-2 cells are well recognised by those 
skilled in the art to be the best available in vitro model for intestinal 
cells. 
EXAMPLE 1 
In Vitro Culture Experiments for Determination of Cytotoxicity and Cell 
Membrane Permeability Preparation of Cells 
Test plates were set up by aliquotting 200 .mu.l of a Caco-2 cell 
suspension containing 1.times.10.sup.5 cells/ml into each well of a 
96-well microtitre plate. The outermost wells were filled with 200 .mu.l 
saline alone, to counteract the effects of evaporation. The cells were 
incubated in high glucose Dulbecco's modified eagles medium (DMEM) tissue 
culture medium (TCM) at 370.degree. C., 5% CO.sub.2 in air, for 8 days, 
with feeding where necessary. The cells were then used for performance of 
either an initial insult or a recovery study. 
A. Assessment of Toxicity Upon Initial Insult 
Neutral red stain is actively taken up by viable cells. Cells with more 
permeable membranes will lose stain into the bathing medium and thus cell 
membrane permeability can be directly assessed after exposure to the test 
material by quantifying the stain remaining in the cell. In this 
procedure, therefore, cells are first stained and then incubated with test 
materials and measurements are made to determine the extent to which 
incubation with test materials has caused the stain to leak out of the 
cells during exposure. An exposure duration of 2 hours was used and the 
experiment was conducted according to the following protocol. 
TCM was removed from cell monolayers in each well and replaced with 200 
.mu.l of stain. The plates were incubated for 2 hours at 37.degree. C., 5% 
CO.sub.2, and then checked for even staining and normal morphology. Stain 
was removed from each well and replaced with 150 .mu.l of the appropriate 
buffer. Buffer was removed from wells in column 2 and replaced with 150 
.mu.l of test solutions at the highest concentration. 150 .mu.l of test 
solutions was added to each of wells in column 3, mixed gently by 
aspiration repeatedly, and 150 .mu.l transferred to wells in column 4. 
This process was repeated down the plate to obtain two-fold serial 
dilutions of test material along each row. One row on each plate was 
allocated to HBSS as a control, and one row for a buffer control if 
necessary. 
The cells were incubated for 2 hours at 37.degree. C. in 5% CO.sub.2. The 
stain was then aspirated from the wells and the plates incubated with 
shaking for 20 minutes in desorbing solution (100 .mu.l/well) consisting 
of 1% glacial acetic acid in 50% ethanol. 
In order to quantify the amount of stain remaining in the cells, 
absorbances were measured on a plate reader at 550 nm, and readings 
compared with buffer controls to assess leakage of the stain from the 
cells. 
The test is thus used as a measure of cell membrane permeability at the 
time of exposure to bile salts. 
B. Recovery from Toxic Insult 
Neutral red stain is actively taken up by viable cells and non-viable cells 
will not stain. Cells with increased cell membrane permeability will have 
a reduced stain content. In this variant of the procedure, cells are first 
incubated with test materials, then incubated with stain, and measurements 
are made to determine the extent to which incubation with the test 
materials has caused the cells to be unable to take up/retain neutral red 
after exposure. A 2-hour exposure period is used together with a 3-hour 
recovery period in optimal growth material during which stain is taken up. 
The following protocol was followed: 
TCM was removed from each well and replaced with 150 .mu.l of the 
appropriate buffer. 
Buffer was removed from wells in column 2 and replaced with 150 .mu.l of 
test solutions at the highest concentration. 
150 .mu.of test solutions was added to each of wells in column 3, mixed 
gently by aspirating repeatedly, and 150 .mu.l transferred to wells in 
column 4. This process was repeated down the plate to obtain two-fold 
serial dilutions of test material along each row. One row on each plate 
was allocated to HBSS as a control, and one row for a buffer control if 
necessary. The cells were incubated for 2 hours at 37.degree. C. in 
CO.sub.2. 
The supernatant liquid was then removed from cell monolayers in each well 
and replaced with 200 .mu.l of stain. 
The plates were incubated for 3 hours at 37.degree. C., 5% CO.sub.2. The 
stain was then aspirated from the wells and the plates were then incubated 
with shaking for 20 minutes in desorbing solution (100 .mu.l/well) 
consisting of 1% glacial acetic acid in 50% ethanol. 
Absorbances were measured on a plate reader at 550 nm, and readings 
compared with buffer controls to assess leakage of neutral red out of the 
cells after active uptake of the stain. 
The test is thus used as a measure of cell membrane permeability during the 
recovery period after exposure to bile salts. 
A comparison of the effects of bicarbonate and HBSS on cells treated with 
bile salts is set out in Tables 1 and 2 and FIGS. 2 and 3. The effects of 
HBSS and bicarbonate, acetate and borate buffers are compared in FIGS. 4 
and 5. 
It should be noted that in HBSS the proportions of the components have been 
adjusted so as to achieve pH of 7.0 to 7.2 when dissolved together in 
distilled water. In the small intestine, the pH will be lower than this. 
With 0.1M bicarbonate, in contrast, as may be seen from the table given 
earlier, the pH will be close to pH 8.0, and was confirmed to be so at the 
end of each of the experiments described here. 
EXAMPLE 2 
In Vitro Culture Experiments for Determination of Cytotoxicity Using MTT 
Staining for Mitochondrial Activity 
Preparation of Cells 
Test plates were set up by aliquotting 200 .mu.l of a Caco-2 cell 
suspension containing 1.times.10.sup.5 cells/ml into wells of a 96-well 
microtitre plate. The outermost wells on each plate were left blank, and 
filled with 200 .mu.l saline, to counteract the effects of evaporation. 
The cells were is incubated in high glucose DMEM tissue culture medium 
(TCM) at 37.degree. C., 5% CO.sub.2 in air, for 8 days, with feeding when 
necessary. 
Test for Mitochondrial Activity 
MTT is a tetrazolium salt which is converted by mitochondrial 
dehydrogenases in viable cells to form an insoluble purple crystal. Dead 
cells would not effect this conversion. Crystal formation can be 
quantified colorimetrically and used to asses mitochondrial activity and 
therefore cell viability. The preparation of MTT stock solution, test 
solutions and buffer solution is described above. 
In this procedure, cells are first incubated with test materials and then 
incubated with MTT and measurements are made to determine the extent to 
which incubation with test materials has altered the mitochondrial 
activity of the cells. A two hour exposure period is used together with a 
three hour recovery period during which time staining is conducted. 
TCM was removed from each well and replaced with 150 Al of the appropriate 
buffer. 
Buffer was removed from wells in column 2 and replaced with 150 .mu.l of 
test solutions at the highest concentration. 
150 .mu.l of test solutions was added to each of wells in column 3, mixed 
gently by aspirating repeatedly, and 150 .mu.l transferred to wells in 
column 4. This process was repeated down the plate to obtain two-fold 
serial dilutions of test material along each row. One row on each plate 
was allocated to Hanks Balanced Salt Solution (HBSS--See Example 1) as a 
control, and one row for a buffer control if necessary. The cells were 
incubated for two hours at 37.degree. C. in 5% CO.sub.2. 
The cells were incubated for 2 hours at 37.degree. C. in 5% CO.sub.2. The 
supernatant liquid was then removed from cell monolayers in each well and 
replaced with 200 .mu.l of MTT. 
The plates were incubated for 3 hours at 37.degree. C., 5% CO.sub.2, and 
the wells examined to assess the level of staining by eye. The stain was 
then aspirated from the wells and the plates were incubated with shaking 
for 20 minutes in desorbing solution (100 .mu.L/well) consisting of 0.1M 
HCl in anhydrous isopropanol. 
Absorbances were measured on a plate reader as the difference between 550 
and 650 nm, and readings compared with buffer controls to assess change in 
mitochondrial activity. A reduction in staining below that for controls 
indicates a reduction in mitochondrial activity, and is highly indicative 
of a toxic action of materials in the incubation medium. 
RESULTS 
The results of Examples 1 and 2 are set out in Tables 1 and 2, and FIGS. 1 
to 5 which illustrate the effects exerted by conjugated and unconjugated 
bile acids in the presence of high pH buffers, using bicarbonate as a 
particular example. 
TABLE 1 
______________________________________ 
Maximum Concentration of Unconjugated Bile Salt 
(mg/mL) Tolerated by Caco-2 Cells In Vitro in 
the Presence of Either Bicarbonate or HBSS 
Neutral Neutral 
MTT 
Test Red Red Viability 
Bile Salt Medium (During) (After) 
(After) 
______________________________________ 
Cholate HBSS 0.313 0.313 0.313 
Bicarb. 0.625 1.25 2.5 
Ursodeoxycholate 
HBSS 1.25 0.313 0.625 
Bicarb. 0.313 1.25 1.25 
Chenodeoxycholate 
HBSS 0.078 0.03 0.125 
Bicarb. 0.04 0.125 0.25 
Deoxycholate 
HBSS 0.04 0.01 0.01 
Bicarb. 0.078 0.04 0.156 
______________________________________ 
TABLE 2 
______________________________________ 
Maximum Concentration of Conjugated Bile Salt 
(mg/mL) Tolerated by Caco-2 Cells In Vitro in 
the Presence of Either Bicarbonate or HBSS 
Neutral Neutral 
MTT 
Test Red Red Viability 
Bile Salt Medium (During) (After) 
(After) 
______________________________________ 
Taurocholate 
HBSS 1.25 5 5 
Bicarb. 0.313 5 5 
Glycocholate 
HBSS 1.25 2.5 2.5 
Bicarb. 0.625 2.5 2.5 
Taurodeoxycholate 
HBSS 0.313 0.313 0.313 
Bicarb. 0.039 0.313 0.313 
Glycodeoxycholate 
HBSS 0.313 0.313 0.313 
Bicarb. 0.078 0.313 0.313 
______________________________________ 
Tables 1 and 2 show the results of the experiments described in Examples 1 
and 2. The first columns of the tables list the bile salts which were 
present in the test media and the test media themselves are set out in the 
second column. The test medium was either HBSS or the 0.1M sodium 
bicarbonate solution. 
The results for the neutral red assays during and after exposure to bile 
salt give the maximum concentration of bile salt in mg/mL at which the 
permeability of the cell remains unaffected compared with cells incubated 
in medium with no bile salt) . The results in the MTT column represent the 
maximum concentration of bile salt at which the viability of the cell 
remained unaffected after incubation. 
Table 1 gives the results for the unconjugated bile salts and shows that, 
in each case, the figures in the Neutral Red (After Exposure) and MTT 
column are higher for the bile salts administered with bicarbonate than 
for those administered with HBSS although there is no significant 
difference in the figures in the Neutral Red (During Exposure) column. 
This indicates that the permeability of the cells during incubation with 
the bile salt solution is not greatly changed in the presence of 
bicarbonate ions, but that a great deal more bile salt is needed for a 
toxic effect to be apparent in the cells after incubation. Effectively 
this means that cells can be exposed to a much greater amount of bile salt 
when in the presence of bicarbonate ions before a toxic effect on the 
cells becomes apparent. Because it is possible to increase the amount of 
bile salt, the permeability of the cells after incubation can also be 
increased without affecting the viability of the cells. 
Table 2 gives the results for the conjugated bile salts and shows that, in 
each case, the figures in the Neutral Red (During Exposure) column are 
increased when the bile salt is administered in the presence of 
bicarbonate while the figures in the Neutral Red (After Exposure) and MTT 
columns are the same. This indicates that much less bile salt is needed to 
increase the permeability of the cells when they are incubated with bile 
salts in the presence of bicarbonate than when bicarbonate is not present. 
The results also show that the permeability and viability of the cells 
after incubation is not affected by the presence of the bile salt. Thus, 
in the presence of bicarbonate ions, it is possible to increase the 
permeability of cells during incubation with conjugated bile salts without 
increasing the bile salt concentration and thus without adversely 
affecting the viability of the cells. 
FIGS. 1 to 3 are graphical representations of the results presented in 
Tables 1 and 2. FIG. 1 shows the effect of bicarbonate on the viability of 
cells incubated with bile salt solutions as measured by the MTT viability 
test. For conjugated bile salts, it can be seen that the presence of 
bicarbonate does not affect the viability of the cells after incubation 
with the bile salt but for the unconjugated bile salts, the concentration 
of bile salt with which cells can be treated without affecting their 
viability is considerably increased in the presence of bicarbonate. 
FIG. 2 shows the results for cell permeability as measured by the neutral 
red assay both during and after incubation with conjugated bile salts. It 
can be seen that during exposure, the amount of bile salt needed to 
increase the permeability of cells is considerably lower in the presence 
of 0.1M bicarbonate than in the presence of HBSS. However, after exposure, 
the permeability of the cells returns to normal. This means that for a 
given amount of bile salt, cell permeability can be significantly 
increased during exposure. However, after exposure, the viability of the 
cells remains unchanged with reference to control (cells incubated with 
bile salts and HBSS with no bicarbonate). 
FIG. 3 is similar to FIG. 2 but in this case the results for unconjugated 
bile salts are presented. In this case it can be seen that bicarbonate has 
no clear cut effect on the permeability of the cells during exposure to 
the bile salt but that after exposure, the maximum concentration of bile 
salt which can be administered without causing an increase in cell 
permeability is increased and thus the cell permeability is significantly 
decreased in the presence of bicarbonate. 
FIG. 4 is a plot showing the percentage release of stain in the neutral red 
recovery assay after exposure of stained cells to bile salt at different 
pH values, using chenodeoxycholate as an example. This plot shows quite 
clearly that much less stain is released from cells which have been 
incubated at pH above 7.5, and this is an indication that the prospects 
for both short- and long-term recovery of the cells are much better under 
conditions of elevated pH. Similarly, FIG. 5, which is a plot showing the 
percentage reduction in mitochondrial activity after incubation with 
chenodeoxycholate in different buffers, indicates that a marked reduction 
in toxicity is achieved under conditions of elevated pH. 
The pH 7.0 medium was prepared using HBSS containing 3% (v/v) 1M HEPES 
(hydroxyethyl propylidene ethane sulphonic acid) solution adjusted to pH 
7.0. The pH 7.5 medium was prepared using HBSS containing 50 mM 
TRIZMA.TM., and adjusted to pH 7.5. The pH 8.0 medium was prepared using 
HBSS containing 50 mM TRIZMA.TM., and adjusted to pH 8.0. The pH 8.5 
medium was prepared using HBSS containing 50 mM Tricine, and adjusted to 
pH 8.5. The results also indicate that this phenomenon is observed with 
0.1M bicarbonate at pH 8.0, but not with HBSS alone at pH 7.0. 
Confirmation that the concentrations of bicarbonate used do indeed have the 
desired effect in the gut milieu is obtained in the in vivo experiments 
reported in the following examples. 
EXAMPLE 3 
In vivo Efficacy Study--Salmon Calcitonin 
Pigs (large white.times.large white/landrace, male, 65 kg) were employed 
for the study and were surgically prepared ten days before commencement by 
implantation, under anaesthesia, of two catheters in the dorsal aorta via 
the median saphenous arteries. A cannula of approximately 1.6-2mm internal 
diameter was implanted in the jejunum. Animals were provided with water ad 
lib, fasted overnight, and fed twice daily in the morning and evening. On 
days of treatment, the morning feed was withheld until after the taking of 
the last blood sample. 
Dosing of salmon calcitonin (sCT) was administered by instillation via the 
indwelling cannula into the jejunum, followed by flushing with warm 
sterile water. 8 ml blood samples were removed at various times before and 
after dosing, and divided into two portions. One portion was allowed to 
clot at room temperature, and the serum removed and stored at -20.degree. 
C. until ready for use. The other portion was transferred to heparinised 
tubes to which 4000 kiu aprotinin was added, and the plasma decanted after 
centrifugation and stored at -20.degree. C. Serum calcium levels were 
measured colorimetrically after complexation with the calcium-sensitive 
chelating agent arsenazo III. 
Nine animals were used in the study, and formulations were administered on 
three separate days, each separated by at least one day, on a random 
three-way crossover basis, so that all pigs received all three treatments. 
Each of the treatments consisted of 625 iu of sCT dissolved in 2 ml of 
phosphate-buffered saline at pH 7.5, either alone (-o-), together with 25 
mg of sodium chenodeoxy cholate (-.DELTA.-), together with 50 mg of sodium 
chenodeoxy cholate (-.box-solid.-), or with 50 mg of sodium chenodeoxy 
cholate where the pH was subsequently brought down to 6.5 with 
hydrochloric acid (-x-) . The results are shown in FIG. 6 and are 
expressed as variations in plasma calcium concentration with time due to 
calcitonin, from which it can be seen that the efficacy of bile salts in 
enhancing uptake of calcitonin is dependent on the concentration of bile 
salt present, and the pH at which it is administered. 
EXAMPLE 4 
In vivo Efficacy Study Insulin 
Pigs prepared in an identical manner to that described in Example 3 were 
employed. Blood samples taken were analysed for glucose using standard 
enzyme-based methodology. 
Animals were dosed with a solution of 200 iu insulin in 2 ml (at pH 5 to 
bring the peptide into solution) , or with insulin formulated as a solid 
dose, either with or without sodium bicarbonate. Each dose contained 300 
mg of either formulation, suspended rapidly in 2 ml of phosphate-buffered 
saline immediately prior to administration. The composition of the two 
formulations is listed below on a weight-for-weight basis. 
______________________________________ 
Without bicarbonate (wt/wt) 
With bicarbonate (wt/wt) 
______________________________________ 
Bovine insulin 
2.4 Bovine insulin 2.4 
Chenodeoxycholic acid 
16.7 Chenodeoxycholic acid 
16.7 
Lactose 74.9 Lactose 66.6 
Ac-di-sol 3.0 Ac-di-sol 3.0 
Polyvinyl pyrollidone 
3.0 Polyvinyl pyrollidone 
3.0 
pH &lt;7.0 Sodium bicarbonate 
8.3 
pH &gt;7.5 
______________________________________ 
The results are shown in FIG. 7, and are expressed as variations in plasma 
glucose concentration with time due to insulin, from which it can be seen 
that the efficacy of the bile acid chenodeoxycholic acid in enhancing 
uptake of peptide across the gut is markedly improved by the presence of 
sodium bicarbonate in the solid dose formulation.