European Review for Medical and Pharmacological Sciences
Insulin formulations – a review
A.M. GUALANDI-SIGNORINI, G. GIORGI
Department of Pharmacology “G. Segre”, University of Siena - Siena (Italy)
Abstract. – Although the improvement
on insulin therapy since it was first conceived,
it is still far from mimicking physiological se-
cretion of pancreatic b-cells and research to
find new insulin formulations and new routes
of administration continues. Human biosynthet-
ic insulin (rapid-acting, intermediate-acting and
long-acting), produced by recombinant DNA
technique, is currently available. The pharma-
cokinetic profile of rapid-acting insulin (regu-
lar) does not adequately reproduce the physio-
logical post-prandial insulin response. This has
led to the development of molecular analogues
with slight modifications that prevent the spon-
taneous polymerisation underlying delayed ab-
sorption. Fast-acting analogues such as
Lyspro and Aspart can be injected immediately
before the meal, inducing a very fast and sub-
stantial peak of insulin, similar to that pro-
duced by b-cells, but have the disadvantage of
short duration of action. For this reason, and
because of the difficulty of obtaining sufficient
basal insulin concentrations to control pre-
prandial blood glucose levels with current
long-acting insulins, analogues known as
Glargine and Detemir have been synthesized.
They have virtually no plasma peak and acts
for about 24 h. These characteristics make it
ideal to cover basal insulin requirement. With
insulin analogues, it also seems possible to
overcome the problem of intra- and inter-indi-
vidual variability in absorption after subcuta-
neous injection. This variability is directly pro-
portional to the duration of insulin action.
Research into new routes of administration has
led to production of inhaled insulin powder,
soon to become commercially available. Insulin
is absorbed through the lung alveoli. Trials to
evaluate efficacy and toleration have shown
that inhaled insulin has a similar kinetic profile
to the fast-acting injected analogue and can
therefore be used for mealtime requirement,
combined with a single daily injection of long-
acting insulin. Oral insulin is currently being
studied in type 1 diabetes prevention with
promising results.
Key Words:
Human insulin, Lyspro, Aspart, Glargine, Inhaled in-
sulin, Oral insulin.
2001; 5: 73-83
Introduction
In the history of insulin therapy some mile-
stones are present. In 1921, the young physician
Frederick Banting (1881-1941) and the forth
year medical student Charles Best (1899-1978),
working in the laboratory of Prof. McLeod in
the Toronto University, found the final link in a
series of studies begun in 1916 by other re-
searchers that guessed the pancreas secretetion
of substance, already named “insulin”, capable
to decrease blood glucose concentrations. The
young researchers isolated this pancreas extract
that “cured” hyperglycemia in diabetic dogs1-2
and in 1922 they successfully administered it
for the first time to a 14-year-old diabetic pa-
tient, Leonard Thompson3.
In 1923, the company Lilly began marketing
animal insulin. In 1928, the hormone was iden-
tified to be a protein. Since the insulin of
Banting and Best could not function for more
than 6 h, much research went into finding ways
of prolonging action. In 1936, Hagedorn noted
that addition of a basic protein, such as prota-
mine, to the insulin preparation, kept the hor-
mone in suspension at the injection site, delay-
ing absorption and prolonging its action4. In
1946, the first insulin “Isophane NPH”
(Neutral Protamine Hagedorn), obtained com-
bining insulin and protamine in stoichiometric
quantities (hence the term isophane from the
Greek equal and manifest) at neutral pH, was
marketed5. In 1952, the first Lente insulin, re-
tarded with zinc and without protamine, was
produced in Denmark6. In 1955, Sanger deter-
mined the exact formula of bovine insulin7. The
sixties saw the development of a radioim-
munoassay for insulin8. In the seventies, pro-
duction of mono-component insulins, obtained
by ion exchange chromatography and of mono-
peak ones, separated by Sephadex G50 col-
umn, began. High performance chromatogra-
phy made it possible to isolate an insulin 99%
pure from proinsulin and other islet hormones9.
73
 A.M. Gualandi-Signorini, G. Giorgi
Human Insulin
Human insulin is a globular protein with a
molecular weight of about 5,800 kd, consist-
ing of 51 aminoacid residues organised in two
polypeptide chains (A and B), linked by two
disulphide bonds. Chain A consists of 21
residues with an extra disulphide bond be-
tween A6 and A11; chain B consists of 30
aminoacids. Complete synthesis of the human
insulin molecule was achieved in 196610.
Insulin exists as a monomer only at low con-
centrations while it shows propensity to aggre-
gate into stable dimers at higher concentra-
tions, in aqueous solution at pH 2-8 and into
hexamers in the presence of zinc ions. The
hexamer, in which chain A constitutes much of
the polar surface, is almost spherical in struc-
ture, with a diameter of 5 nm and a height of
3.5 nm. Polymerisation of the hormone has
major pharmacological implications.
Before the eighties, insulin was extracted
from bovine or pig pancreas. Commercial
production of human insulin began in 1982.
This milestone in the history of diabetes is of
similar scientific relevance to the discovery of
insulin itself. Once it became clear that it was
impossible to obtain sufficient human insulin
for world requirements from cadavers, the
techniques for synthesising insulin were de-
veloped. Semisynthetic insulin was obtained
by an enzymatic method, in which trypsin
catalyses the substitution of alanine, in posi-
tion B30 of pig insulin, with threonine. The
insulin thus produced has the same
aminoacid sequence that the human one and
it was completely free of other pancreatic and
gastroenteric hormones11-12.
Genetic engineering has enabled industrial
synthesis of human insulin by the technique of
recombinant DNA. Fragments of DNA cod-
ing for chains A and B are inserted separately
in plasmids and then in a special strain of
Escherichia coli which acts as a microlaborato-
ry for in vitro synthesis. The insulin chains re-
leased into the medium are harvested (togeth-
er with other bacterial proteins) and reassem-
bled with the appropriate disulphide bonds13-
15
. To avoid contamination during the various
steps of the procedure, plasmids were pro-
duced with genetic material coding for the
proinsulin molecule, which is isolated from the
culture medium and deprived of its connecting
peptide16-18. Biosynthetic insulin can be also
74
produced by another technique. The proce-
dure exploits the yeast Saccharomyces cerevisi-
ae as a living laboratory by inserting purified
DNA that dictates the synthesis of a single
chain precursor different from proinsulin in
one of the yeast’s plasmids. The protein, pro-
duced by yeast continuously during fermenta-
tion, is harvested from the culture medium,
isolated by centrifugation and crystallisation,
converted to insulin ester by trypsin transpep-
tidisation and finally hydrolysed. This method
guarantees high purity12,19-20.
Human insulins currently on the market in-
clude rapid-acting (Regular), intermediate-
acting (NPH and Lente) and long-acting
(Ultralente) formulations. The former is a
clear, colourless, aqueous solution buffered at
neutral pH (7-7.8). Meta-cresol is added as
preservative, glycerol as tonic stabiliser, as
well as zinc chloride. Hexamers, made stable
by zinc ions, are the predominant quaternary
structure of pharmacological insulin; other
structures include dimers and tetramers 21.
The molecule tends to aggregate in the vial
and in the tissue where it is injected. The
hexamers need to be split for the insulin ab-
sorption from the subcutaneous injection site.
For this reason, Regular insulin enters the
general circulation after a lag phase about 30
min after injection. It achieves the plasmatic
peak at 2 to 4 h and lasts for 6 h.
Absorption of NPH is delayed by prota-
mine, a protein extracted from the nucleus of
fish sperm, where its role is to stabilise DNA.
The commercial form is Isophane-NPH in-
sulin, a white suspension of orthorhombic
crystals containing 0.9 molecules of prota-
mine and two atoms of zinc per hexamer5. In
the crystal, protamine regulates interactions
between dimers and hexamers. The vehicle is
water buffered at pH 6.9-7.5. Phenol or meta-
cresol are added as preservatives21. Insulin
crystals are insoluble in water and tend to
precipitate to the bottom of the vial, which
has to be tipped various times to resuspend
them before use22. NPH is longer-acting: in
fact its blood absorption begins 1.5 h after
subcutaneous injection; it has a peak plasma
concentration at 4 to 12 h and disappears
within 24 h. The “tail”, however, is relatively
ineffective. Although NPH has the most reg-
ular absorption of all intermediate- and long-
acting insulins, inter- and intra-individual
variability is high.
 Insulin formulations – a review
Lente, delayed without addition of prota-
mine, is obtained by precipitating the hor-
mone in the presence of zinc salts. When its
molar ratio with respect to insulin is greater
than one, the zinc ion reduces the solubility
of insulin in neutral solvent6. When an acid
solution of insulin is brought to pH 7.4 with
an excess of zinc ions, the resulting precipi-
tate is amorphous and has moderately de-
layed absorption after subcutaneous injec-
tion. This kind of insulin formulation is
known as Semilente, and was produced until
several years ago. If insulin is allowed to crys-
tallise at pH 5.5 before zinc is added and pH
corrected, the obtained formulation is known
as Ultralente. This insulin consists of crystals
insoluble in water which remain in suspen-
sion and are absorbed very slowly. A 3:7 mix-
ture of amorphous and crystalline insulin,
known as Lente, has an intermediate absorp-
tion profile. In commercial preparations,
methyl-parahydroxybenzoate is used as
preservative and the suspension is buffered at
pH 7-7.821. It appears in the circulation 2.5 h
after injection, has the peak plasma concen-
tration at 7 to 15 h and disappears by 20 h.
Both NPH and Lente insulins can be mixed
with Regular in the syringe. However, while
NPH and Regular conserve their pharmaco-
kinetic characteristics, Lente/Regular mixture
changes in time because the excess of zinc
ions binds part of the rapid-acting insulin,
transforming it into a form similar to
Semilente.
Long-acting insulin (Ultralente) is an aque-
ous suspension of zinc-insulin crystals at neu-
tral pH, of milky appearance21. It has an on-
set of action after 4 h, achieves a slight but
undesirable peak plasma concentration at 7 h
and sustains blood insulin levels approxi-
mately 8 to 20 h; it inconsistently mimics en-
dogenous basal secretion. Moreover its ab-
sorption is completely irregular23 and it can-
not be mixed with Regular insulin in the sy-
ringe because the action of the latter would
be excessively delayed.
Insulin Analogues
Since the nineteen-eighties, pharmacologi-
cal research has been concentrated on ana-
logues of human insulin, seeking to develop
molecules with minimal modifications and
appropriate kinetics. In 1996, an analogue in
which the aminoacids in positions B28 and 29
were inverted, was marketed24-26. The inver-
sion conferred the property of prompt ab-
sorption and hence fast action. The analogue
is known as Lyspro, from lysine and proline,
the aminoacids transposed. The handling of
the molecule prevents its natural tendency to
hexamers aggregation, since position B28 is
crucial for its spatial configuration27. Because
the interaction between B28 and B23, two
molecules of insulin align antiparallel28 form-
ing a non polar dimer which in turn aggre-
gates into hexamers by linking to zinc in
pharmacological preparations29. Since modifi-
cation of aminoacid residue B28 counteracts
polymerisation of insulin, the analogue may
be injected immediately before the meal, as it
acts in a few minutes. Lyspro maintains a
plateau for 1 to 2 h and disappears by 3 to 4
h. Need for availability of insulin analogue
was dictated by three pharmacokinetic de-
fects typical of Regular insulin:
1. Its relatively slow absorption causes
lacking reproduction of physiological
profile of the b-pancreatic secretion.
2. The comparison of the insulin curve in a
normal subject to that in a type 1 diabet-
ic patient (injected with Regular insulin)
shows the following figures: (a) the ab-
sence of the first peak, (b) the flat over-
all profile in the first two hours after the
meal (leading to early post-prandial hy-
perglycemia) and (c) the persisting
raised insulin plasma levels during post-
absorptive period.
3. This pattern may lead to pre-prandial
hypoglycemic events and conditions the
prescription of snacks for type 1 diabetic
patients.
A lot of Italian, European and American
trials carried out after experimentation and
consolidated use of insulin Lyspro30-37 indi-
cate that the post-prandial insulin profile af-
ter the analogue administration is very close
to the physiological one. Comparison with
the insulin profile after an injection of
Regular insulin (even when the patient waits
the prescribed 30 min before eating) con-
firms the higher validity of Lyspro to mimic
physiological glucose-stimulated insulin se-
75
 A.M. Gualandi-Signorini, G. Giorgi
cretion. In clinical practice, this phenomenon
should lead to more physiologic glycemia 90-
120 min after the meal. However, while the
short duration of action allows for better
control over post-prandial glucose levels, it
does not satisfy, alone, the basal insulin re-
quirement in diabetic patients with low en-
dogenous insulin reserve (type 1). In order to
use the fast-acting analogue in the daily ther-
apeutic protocol of the type 1 diabetic sub-
ject, a combination of Lyspro and intermedi-
ate-acting insulin is therefore necessary. On
the other hand, this imposes the use of the
syringe to mix insulins or the practice of a si-
multaneous double injection by the “pen”,
which is the simplest and more pleasant actu-
al method for self-administration of insulin.
Until Lyspro becomes available in “penfills”
with a premixed preparation of the analogue
(25, 50 and 75%) and intermediate-acting in-
sulin (neutral protamine Lyspro, NPL, 75, 50
and 25%) (expected soon), it cannot com-
pletely replace Regular insulin, but it can on-
ly coexist for a more a versatile use. For ex-
ample, it can be administered to type 2 dia-
betic patients requiring insulin due to sec-
ondary failure of oral hypoglycemic agents.
In this case it can be used at mealtimes, in as-
sociation to intermediate- or long-acting in-
sulin at bed-time, if necessary. The pharma-
cokinetic of Lyspro suggests that type 2 dia-
betics, characterised by lacking first peak of
insulin secretion, can mostly benefit from the
analogue. It may also be successfully used in
those patients with iatrogenic diabetes due to
steroids, in whom hyperglycemia occurs se-
lectively after meals, or in those patients
with diabetes associated to liver disease hav-
ing the same feature. It may be useful in type
1 diabetic subjects, educated to manage
“free” variations in diet, for spot correction
of occasionally glucose peaks, detected dur-
ing self-monitoring (well illustrated in the
“pizza, coca-cola and tiramisù” study) 36 .
Another application is in the first period af-
ter diagnosis, when a residual basal insulin
secretion is consistent. An interesting indica-
tion is provided by patients who have an un-
certain food intake (intercurrent pathologies,
gastroparesis diabeticorum…) In these sub-
jects, Lyspro can be injected immediately af-
ter the meal at a dose calculated on the basis
of the quantity of food eaten, without deteri-
orating post-prandial glucose levels.
76
In 2000, another analogue of human in-
sulin, Aspart, became available. This ana-
logue is obtained by substitution of the pro-
line residue in position B28 with an aspartic
acid residue38. This substitution eliminates
the interaction with glicine B23 that triggers
polymerisation. Monomer status is also aided
by repulsion between the aspartic acid
residue and the nearby glutamic acid B21,
both positively charged. Since the variation
introduced in Aspart is not close to the recep-
tor site, there is no change in receptor bind-
ing affinity39 or in the rate of intracellular dis-
sociation40-41 with respect to native insulin.
This manipulation has also solved the prob-
lem of affinity for IGF-1 receptors. The delay
in making Aspart available was due to the in-
tense mitogenic activity (due to excessive
affinity for the IGF-1 receptor) of the origi-
nally proposed analogue with aspartic acid in
B10 position39.
There are many advantages of therapy us-
ing fast-acting insulin analogues. The need to
limit post-prandial hyperglycemia, both in
type 1 and 2 diabetics, emerges from recent
studies with large cohorts, the results of
which unequivocally correlate poor metabolic
control with the development and progres-
sion of complications. Specifically, post-pran-
dial hyperglycemia seems a major factor for
the cardiovascular manifestations so frequent
in type 2 diabetics42-44. This is the rationale for
another indication of insulin therapy in type 2
patients, using analogues. Restoration of the
early insulin peak seems to be particularly
important. In fact, correcting hyperglycemia
90-120 min after the meal, it is possible to re-
duce late hyperinsulinemia and, as a conse-
quence, the insulin resistance highly related
to the development of cardiovascular compli-
cations45-46. Pre-prandial and nocturnal hypo-
glycemia can also be removed by fast-acting
insulin37. Finally, the possibility of injecting
the hormone immediately before the meal,
without having to wait 30 min, greatly im-
proves the quality of life, especially for young
patients who are dependent on injections
throughout their life. However, type 1 diabet-
ic subjects still require simultaneous adminis-
tration of intermediate- or long-acting in-
sulin.
The pharmaceutical formulation of fast-
acting insulin is the same as that of regular in-
sulin.
 Insulin formulations – a review
Long-Acting Analogues
All available intermediate- and long-acting
insulins (NPH, Lente, Ultralente) have two
major handling problems. The first is that all
the classical formulations have wide variations
in absorption between different individuals,
and even in different injection sites and at dif-
ferent moments in the same subject, in a man-
ner which is directly proportional to the dura-
tion of their action 23. This phenomenon is
small, but present and significant with Regular
insulin; it is greater with NPH, Lente and
greatest of all with Ultralente, which is rarely
used for this reason. The second problem is
their time-action profile. After a delay of a few
hours, their absorption gives rise to an insulin
peak and a lowering effect on blood glucose,
the major cause of nocturnal hypoglycemia,
often unawareness, which afflicts many in-
sulin-treated patients47. The peak is followed
at an interval of 7-8 h by a significant drop in
plasma insulin concentrations, not sufficient to
prevent morning hyperglycemia. An attempt
was made to correct this by giving long-acting
insulin at bedtime, between 10 and 12 pm, in-
stead of at mealtime, before dinner, together
Regular or analogue48. This strategy has im-
proved basal hyperglycemia in many subjects,
enabling a reduction in dose, and consequent-
ly a reduced risk of nocturnal hypoglycemia,
but is still far from successful in all cases.
The need for an ideal long-acting insulin, that
gives rise to a square wave of plasma concentra-
tion, or in other words, constant basal insulin
levels, has prompted research into long-acting
analogues. The aim of the research is to obtain
an insulin formulation, which can be combined
with a fast-acting analogue to ensure a closer
parallel to physiological b-cell secretion.
Candidate analogues for daily use are: (i)
soluble analogues, created by varying the
electrical charge of the insulin molecule and
hence its isoelectric point, which affects solu-
bility in vivo49 (insulin Glargine) and (ii) acyl
analogues in which a fatty acid molecule is
linked to an aminoacid residue so as to bind
with albumin in the blood, forming a “depot”
product that gradually releases insulin into
the tissues50 (insulin Detemir).
Glargine
In 2001, insulin Glargine has become com-
mercially available. This long-acting analogue
is produced from human DNA recombinant
insulin, modified by the addition of two argi-
nine residues, conferring a positive charge, to
the C-terminal (B30a and B30b) of the mole-
cule. This addition shifts the isoelectric point
(pI) from 5.4 to 6.7, making the molecule
more soluble at the acid pH of the vial and
less soluble at the neutral pH of subcuta-
neous site. The acidity of the medium in
which insulin is conserved dictated another
modification of the molecule, namely substi-
tution of asparagine in position A21 with
glycine. This prevents deamidation and con-
fers stability 51. It has been demonstrated 52
that Glargine interacts with insulin receptors
in a similar way to human regular insulin and
also that its dissociation is similar; it does not
have mitogenic effects due to activation of
IGF-1 receptors53.
Pharmacological studies54-55 have been car-
ried out with the iso- and euglycemic clamp
techniques to evaluate the action profile of
Glargine in normal and diabetic subjects. Its
absorption rate has been measured in type 2
diabetics and compared with that of insulin
NPH: the mean 25% disappearance times of
125
I-insulin from the injection site were 15 and
6.5 h for Glargine and NPH, respectively, and
residual radioactivity at 24 h were 54.4 and
27.9% 56. The area under the blood insulin
curve between 0 and 6 h after administration
of Glargine was significantly less than that of
NPH57. Maximum plasma concentration in
the first 4 h was 5.11 IU/l for Glargine and
10.8 IU/l for NPH58. A recent study57 in type
1 diabetic patients without endogenous in-
sulin reserve (C-peptide negative) confirmed
that the action of Glargine lasts 24 h and that
it gives no plasmatic insulin peak, whereas
NPH and Ultralente achieve significantly high
plasma levels a few hours after injection.
Moreover, NPH lasts only 12 h and the disad-
vantage of Ultralente is its enormous variabil-
ity in absorption. Compared to NPH,
Glargine therefore shows a constant increase,
followed by a stable plateau, which, in some
patients, lasts almost 24 h. All these features
indicate that the pharmacokinetical charac-
teristics of Glargine meet the criteria neces-
sary to ensure basal insulin requirement in
type 1 diabetics with negligible plasma C-
peptide concentrations. Many clinical trials
show that Glargine provides good control of
basal glycemic levels59-64.
77
 A.M. Gualandi-Signorini, G. Giorgi
Glargine insulin is a clear liquid (like fast-
acting insulin) with a pH of 4. After injection, it
forms a microprecipitate in the tissues, ensur-
ing slow and steady absorption. Unfortunately,
it cannot be mixed with fast-acting formula-
tions and this is a problem since the number of
daily injections increases when therapy proto-
col is based on the introduction of Glargine.
Stability of Insulin in
Commercial Preparations
Agents as heat, repeated and strong shak-
ing, exposure to hydrophobic surfaces (such
as drip attachments) cause changes in insulin
conformation leading to linear aggregation
and formation of insoluble fibrils65. This phe-
nomenon mainly affects intravenous infu-
sions (drips required in acute situations) and
continuous subcutaneous infusions by
minipump used for intensive insulin therapy.
Many measures have been devised to avoid
it, such as addition of glycerol66, albumin67,
patient’s serum68 or blood69, but none of them
have proved practical, physiological and safe.
At temperatures above 25° C, chemical
destabilisation due to deamidation or poly-
merisation may occur70. Current insulin for-
mulations ensure stability of the hormone be-
low 25° C for a month. Vials for daily use can
therefore be kept at room temperature and
supplies must be kept at 2-8° C, where their
physical and chemical characteristics are
maintained for a period of about 30 months.
Absorption
Insulin must be administered parenterally
because digestive enzymes destroy it. When
injected s.c., insulin must be split into
monomers before it enters circulation. The
lymphatic system only plays a secondary role
in absorption of the hormone. Less than 5%, a
clinically insignificant amount, is broken down
at the injection site. As already mentioned, the
problem of intra- and inter-individual variabil-
ity in insulin absorption is very significant and
can cause a variation of 10-52% in the amount
of insulin absorbed daily, responsible for ex-
cursions in glycemia by as much as 80%71. This
problem has not yet been solved.
78
Many factors influence the absorption rate
of insulin.
Injection site
The abdominal region offers faster and
more reproducible absorption (T/2=87 min)
than the arm (140 min), buttocks (155 min)
and thighs (165 min)72. This difference is of
clinical significance and is linked to regional
differences in blood flow. Absorption of in-
sulin monomers is correlated with available
capillary area, explaining why muscle con-
traction (which causes vasodilation) around
the site of a recent injection, significantly
speeds onset of the glucose lowering effect of
the hormone. Similarly, high room tempera-
ture or sauna facilitate Regular but not long-
acting insulin to be absorbed; the mechanism
involved may not be related to changes in
blood flow but rather to accelerated diffusion
in the tissues. The fast action of analogues is
maintained irrespective of injection site25.
Injection depth
If the injection is too deep and does not
distribute the insulin solution or suspension
in the subcutaneous tissue but rather in the
muscle, absorption is faster due to the greater
vascularisation of muscle with respect to sub-
cutaneous fat. If the injection is too superfi-
cial (in the derma), the process is slower and
incomplete.
Concentration
Injection of smaller volumes of insulin so-
lution, corresponding to higher concentra-
tions, improves absorption. The influence of
concentration has been eliminated by stan-
dardising all commercial preparations to 100
IU/ml.
Massage of injection site
Massage with cotton wool soaked with dis-
infectant should be avoided because it in-
creases the absorption rate by a mechanism
apparently not related to blood flow73.
Distribution and Elimination
Intravenous insulin has a half-life of 4 min-
utes. If administered subcutaneously, it is dis-
tributed in the plasma phase in the free form,
 Insulin formulations – a review
that is, not bound to proteins, as does en-
dogenous insulin, unless the organism has
produced a significant quantity of antibodies
against it. These antibodies act as a buffer
system, binding insulin with a capriciously re-
versible bond. In normal subjects, insulin has
a distribution volume of 85 ml/kg74. It reaches
specific receptors in target tissues. It is elimi-
nated by break down in the liver (60-80%),
kidney (10-20%), muscle and adipose tissue
(10-20%). A small amount is excreted in the
urine. Paradoxically, significant impairment
of renal function reduces its clearance more
than severe liver disease 75. The residence
time in subcutaneous tissue is brief for fast-
acting analogues, making their action profile
short. Subcutaneous deposit of Regular and
long-acting insulin is significant and is
achieved after repeated administrations. The
size of deposit is related to the number of in-
jections/die. For example, a dose of 40 IU/die
in two injections, is calculated to deposit 22-
60 IU; the same dose in multiple doses de-
posits 12-20 IU and continuous infusion de-
posits 1-14 IU76. This explains why patients
taking few doses/die are protected for longer
against rises in blood glucose levels, whereas
patients undergoing continuous infusion can-
not even tolerate brief interruptions without
developing ketosis.
Alternative Routes of
Insulin Administration
Since 1924 researches about alternative
routes of insulin administration (oral, nasal,
rectal, transdermal) were reported77-78, but
until recently no realistically useful prepara-
tions had been obtained. Of all these routes,
inhalation is the one most tested greatly. The
rationale behind inhalation is the passage of
protein molecules, including insulin, across
the mucosa of the lung alveoli, which have a
surface area of 100 m2. Insulin has shown a
particular aptitude for this route79. A phar-
macological formulation of the hormone for
inhalation will soon become commercially
available. It is a dry powder dosed in mg, in
blisters containing 1 or 3 mg of insulin in 5
mg of excipient (mannitol, glycine and sodi-
um citrate powder). The blisters are opened
and emptied directly into the inhalation ap-
paratus. One or two inhalations (2-6 mg) are
generally the appropriate dose for counter-
acting post-prandial hyperglycemia in a type
1 diabetic patient with poor endogenous in-
sulin reserve. Pharmacokinetical studies in
normal and diabetic subjects80 show that its
absorption rate is similar to that of injected
analogue. A plasma peak is achieved 5-60
min after inhalation, compared to 60-80 min
for Regular insulin. It is therefore adminis-
tered immediately before the three meals. Its
duration of action is intermediate between
analogue and Regular. A mean daily require-
ment of 12 mg is equivalent to 350 IU of solu-
ble insulin. The divergence between these
doses and those usually administered by in-
jection of soluble or suspended insulin is due
to a series of factors that cause dispersion.
1) The first loss of hormone is in the in-
halation apparatus, but 80-95% reaches
the oral cavity.
2) The second loss is the quantity that de-
posits on the mucosa of the mouth and
pharynx on the way to the alveoli.
3) Lung tissue metabolises part of the in-
sulin, which is not absorbed. The effi-
ciency of absorption is calculated at 20-
40%, in other words, 6-8 out of 10 in-
sulin molecules do not enter circula-
tion80-81.
Many trials have been conducted with type
1 and 2 diabetic patients, comparing tradi-
tional therapy with inhaled insulin at meal-
times associated to a subcutaneous Ultralente
injection at bedtime. All unequivocally found
the similarity in the hypoglycaemic effect of
the inhaled insulin in respect to subcutaneous
route82-85. In a 3-month study, inhalation was
so well tolerated and accepted that 80% of
patients chose to continue inhalation therapy
for a year 80. However there are still some
doubts as to whether this route is suitable for
all patients, that it is actually more practical
than subcutaneous injection and that it is not
subject to substantial interference by factors
such as smoking and intercurrent pathologies
of the airways81,84.
The oral route has been neglected for some
time because of the difficulty to keep insulin
intact in the gastric environment and to get it
across the barrier of the intestinal mucosa. It
is currently used in experimental studies for
79
 A.M. Gualandi-Signorini, G. Giorgi
the prevention of type 1 diabetes mellitus, ex-
ploiting its property of modifying the immune
response underlying insulitis by contact with
the tolerant mucosa of the digestive system86.
Moreover, at the annual meeting of the
American Society for Clinical Pharmacology
and Therapy, Still recently presented a hexyl-
insulin monoconjugate 2 (HIM2) in which an
alkyl-polyethylene-glycol group attached to
lysine B29 confers stability in the gastric envi-
ronment. The phase II trials of oral insulin
have been presented at American Diabetes
Association annual meeting82. Oral adminis-
tration of insulin would be a break-through
for obvious reasons of patient convenience
and also because it restores the primary role
of the liver as manipulator of insulin, a role
lost with the introduction of subcutaneous
administration. Under physiological condi-
tions, b-cell hormone is captured by the liver
which metabolises about 40% and then re-
leases the remaining 60% into the peripheral
circulation, whereas injected insulin is ab-
sorbed directly into the general circulation,
causing excessive impregnation of the periph-
eral tissues. This is a major aspect in which
insulin replacement therapy diverges from
what happens in nature.
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