Rapid-Acting and Human Insulins: Hexamer Dissociation Kinetics upon Dilution of the Pharmaceutical Formulation
Rapid-Acting and Human Insulins: Hexamer Dissociation Kinetics upon Dilution of the Pharmaceutical Formulation
Klaus Gast 0 1
Anja Schüler 0 1
Martin Wolff 0 1
Anja Thalhammer 0 1
Harald Berchtold 0 1
Norbert Nagel 0 1
Gudrun Lenherr 0 1
Gerrit Hauck 0 1
Robert Seckler 0 1
0 Sanofi-Aventis Deutschland GmbH , Industrial Park Höchst D-65926 Frankfurt , Germany
1 Physical Biochemistry, University of Potsdam , Karl-Liebknecht-Str. 24-25 Golm, D-14476 Potsdam , Germany
Purpose Comparison of the dissociation kinetics of rapidacting insulins lispro, aspart, glulisine and human insulin under physiologically relevant conditions. Methods Dissociation kinetics after dilution were monitored directly in terms of the average molecular mass using combined static and dynamic light scattering. Changes in tertiary structure were detected by near-UV circular dichroism. Results Glulisine forms compact hexamers in formulation even in the absence of Zn2+. Upon severe dilution, these rapidly dissociate into monomers in less than 10 s. In contrast, in formulations of lispro and aspart, the presence of Zn2+ and phenolic compounds is essential for formation of compact R6 hexamers. These slowly dissociate in times ranging from seconds to one hour depending on the concentration of phenolic additives. The disadvantage of the long dissociation times of lispro and aspart can be diminished by a rapid depletion of the concentration of phenolic additives independent of the insulin dilution. This is especially important in conditions similar to those after subcutaneous injection, where only minor dilution of the insulins occurs. Conclusion Knowledge of the diverging dissociation mechanisms of lispro and aspart compared to glulisine will be helpful for optimizing formulation conditions of rapid-acting insulins.
circular dichroism; dissociation kinetics; insulin analog; light scattering; rapid-acting
One of the great challenges for biotechnological research is
the optimum substitution of the natural regulation of the
active insulin concentration in blood lost in diabetic patients by
appropriate insulin analogs and advanced injection
techniques. This comprises both the establishment of the basal
level of insulin by long-acting insulins and the fast adjustment
of the instantaneous insulin concentration in accordance to
food uptake by rapid-acting insulins (RAI) (
). The present
work relates to the second category of insulins. Specifically,
we have compared the dissociation kinetics of the three RAI
analogs lispro, aspart, and glulisine and regular human insulin
(HI) from the oligomeric storage state in formulation into the
monomeric state directly by measuring changes in the average
The presence or fast adoption of the monomeric state is the
most important precondition for the instantaneous action of
insulin since only this state is biologically active. However,
regular HI is monomeric only at very low concentrations (<
10−5 M in the presence of Zn2+ ions). Therefore, recombinant
analogs with reduced stability of oligomers have been
developed for rapid-acting purposes. On the other hand,
monomeric insulins do not fulfil the conditions of long-term storage
required for drug products (
). To ensure sufficient shelf life,
insulin in formulation must be in a compact oligomeric
(essentially hexameric) state that is resistant to chemical
degradation and toxic aggregation as well. High insulin
concentration and the presence of allosteric ligands,
such as Zn2+ and phenolic compounds, favor the
population of the hexameric state. Consequently, two
opposing requirements, high stability of insulin in formulation
and fast dissociation after subcutaneous injection play an
important role for the development of RAI.
Insulin monomers consist of an A-chain (21 amino acid
residues) and a B-chain (30 amino acid residues) linked by
two disulfide bonds. Most information about association states
of insulins in solution were originally obtained from
hydrodynamic investigations at low pH (
) and neutral pH (
High-resolution X-ray studies revealed the existence of
different conformations of subunits within the hexameric assemblies
depending on environmental conditions. Spectroscopic
investigations supported the assumption that specific oligomeric
structures identified in solution are consistent with the
corresponding high-resolution crystal structures (8).
Nearly all insulin formulations contain phenolic substances
as antimicrobial preservatives. First indications for their
influence on insulin structures came from differences in the
crystallization behavior. This effect was also clearly shown for
insulin in solution by Wollmer et al. (
). Subsequent X-ray
) demonstrated explicitly the changes from hexamers
in the 2 Zn2+-structure to the structure specific in the presence
of phenol. At the same time, known canonic hexamer
structures of insulin in the absence and presence of allosteric
ligands, such as bivalent metal ions (Zn2+, Co2+), lyotropic
anions (Cl−, SCN−), and phenolic ligands (phenol, m-cresol)
were classified as T6, T3R3, and R6 (
). The R6-hexamer
in the presence of phenol was reported to be the
thermodynamically most stable species. More structural details of the R6
human insulin hexamer in solution were provided by NMR
spectroscopy and restrained molecular dynamics (
solution structure resembles the X-ray structure. However,
the extension of the α-helix in the B-chain, a characteristic
of the R6 state, is shorter in the solution structure.
Lispro was the first formulated RAI (Humalog®) on the
market. The primary structure of lispro differs from that of
HI by an inversion of the amino acids Pro28 and Lys29 in the
sequence of the B-chain. The crystal structure obtained under
formulation conditions displays a T3R3f conformation (
However, there is evidence that the structure in formulation
is rather the R6 conformation (
). This should be expected
since the formulation contains both Zn2+ ions and m-cresol at
sufficiently high concentrations.
The R6 conformation was indeed observed in crystals of the
RAI aspart grown in the presence of either phenol or m-cresol
). In the case of aspart, Pro28 of the B-chain is substituted by
Asp. Formulations of Aspart (NovoRapid®) contain equal
amounts of phenol and m-cresol (Supplemental Table I).
Recently, the pharmacokinetics and pharmacodynamics of
insulin aspart could be improved using formulations (called
fasteracting insulin aspart) containing the additional excipients
niacinamide and L-arginine (
The amino acid sequence of the third RAI glulisine differs
from the HI sequence at positions 3 and 29 of the B-chain.
Asn3 is substituted by Lys and Lys29 by Glu. In contrast to
Humalog® and NovoRapid®, formulations of glulisine
(Apidra®) contain no Zn2+ (Supplemental Table I). In the
absence of Zn2+ ions, specific differences in the solution
structure and the dissociation behavior compared to
Zn2+-stabilized insulin structures can be expected. To the best of our
knowledge, the high-resolution structure under conditions
corresponding to Apidra® formulation has not been
published so far. Known structural and functional properties of
glulisine have been reviewed by Becker (
Direct investigations of the kinetic changes of the
association state in terms of the mass of oligomers have not been
published so far. Techniques yielding the desired information
(light, x-ray, and neutron scattering, hydrodynamic
investigations including size exclusion chromatography) are
slow in general and require relatively high protein
concentrations. Up to now, information about the dynamics of insulin
assemblies and the kinetic transitions between different
oligomeric states was obtained only using indirect (mostly optical
spectroscopic) signals (
). Particularly, very different
rates of dissociation of T6 hexamers (
) and R6 hexamers
) have been observed.
The purpose of our studies is a comparison of the
dissociation behavior of the three RAI and HI. Specifically, we
wanted to elucidate biophysical properties of the insulins
potentially important for their dissociation and absorption after
injection, but we do not aim at modeling or simulating the
corresponding processes under in vivo conditions.
We initiated oligomer dissociation by changing both,
insulin concentration and solvent composition (medium) and we
monitored structural changes using static light scattering
(SLS), dynamic light scattering (DLS), and circular dichroism
(CD). The most direct measure of the association state is the
average molecular mass, which we obtained from static light
scattering. To further characterize initial, transient and final
states, we used DLS to determine hydrodynamic Stokes radii
(RS) and we measured CD signals. Combination of
hydrodynamic dimensions, RS, with the corresponding masses gives an
idea of the compactness of insulin oligomers or aggregates in
different conformational states. The near-UV CD data yield
information about extent and time dependence of changes in
tertiary structure. This is of particular importance, as the
nearUV CD spectra can be used to distinguish between T6, T3R3
and R6 conformations of insulins (
Two main experimental schemes were used. Scheme I is
characterized by a substantial dilution of the insulins with an
appropriate solvent facilitating pronounced changes in the
association state. To reach this, we have rapidly diluted RAI
formulations with PBS. This simple experimental scheme
provides information about basic differences concerning the
dissociation kinetics and the final association states and on the
influence of formulation excipients. This type of investigations
yields the Bspeed limit^ of the dissociation process and may be
considered to reflect the dilution conditions after intravenous
Scheme II is derived from the conditions after
subcutaneous injection. In this case, we study the dissociation processes
at insulin concentrations corresponding to an only two-fold
dilution of U100 formulations and at various m-cresol
concentrations, decreased stepwise from the formulation
concentration. This scheme may be considered to simulate the
conditions in the bolus formed after injection, where a continuous
disappearance of phenolic additives is expected to occur after
an initial moderate dilution of the insulin formulation. The
rapid diffusion of phenolic additives after injection is
considered as an essential factor influencing insulin dissociation
kinetics and the entire absorption process in the literature
MATERIAL AND METHODS
Materials and Sample Preparation
For studies on insulins under formulation conditions we have
used the marketed U100 formulations Humalog® (Lilly,
I n d i a n a p o l i s , U S A ) , N o v o R a p i d ® ( N o v o N o r d i s k ,
Bagsveard, Denmark), as well as Apidra® and Insuman
Rapid® (Sanofi-Aventis, Frankfurt, Germany). The
compositions of the formulations are shown in Supplemental Table I.
Lyophilized samples of the insulins (lispro, aspart, glulisine,
and HI) and solvents to the corresponding formulations
(placebos) were provided by Sanofi-Aventis (Frankfurt,
Germany). PBS (10 mM buffer) was prepared from tablets
(Calbiochem, Germany). Glycerol (85%), phenol and
mcresol were provided from Roth (Germany). ZnCl2 was
supplied from Merck (Germany). All chemicals were of analytical
grade. Solvents were prepared using Milli-Q water (Merck
Millipore, Germany) and were filtered and degassed after
preparation using 0.45 μm Millicup filter units (Merck
Millipore, Germany). In special cases, dialysis against the
corresponding solvent using dialysis tubing (MW cut-off 3500,
Spectra/Por©, Spectrum Laboratories, USA) was done
ensuring the specified solvent conditions. Dialysis was always
done, if stock solutions with high insulin concentrations were
used. For measurements in Zn2+-free PBS, EDTA was added
to the stock solutions after dissolving the drug substance, in
order to remove Zn2+ completely. Afterwards, the samples
were dialysed against PBS.
Samples for light scattering were filtered either through
0.1 μm Whatman-Anotop filters (VWR, Germany) directly
into 3-mm-pathlength micro-fluorescence cells (105.251-QS,
Hellma, Germany) immediately prior to use or were subjected
to ultracentrifugation. Usually, about 500 μl were centrifuged
at 75000 g for 30 min. An amount of 80–150 μl of the
supernatant was quickly transferred into the sample cell. Peptide
concentrations were determined photometrically using a
specific absorption A(276 nm, 1 cm pathlength, 1 mg/ml) of 1.08.
Choice of Dilution Media and Dilution Jumps
An ideal dilution medium has to combine two requirements:
to be suitable for the chosen biophysical methods and to be
consistent with physiological environments. The first step
during application of insulins is the injection into the
subcutaneous tissue (ST). Therefore, the initial solvent change is a
mixture of formulation with the interstitial fluid (IF). IF (see
Supplemental Table II) is not a suitable medium because of
the high protein content. Thus, one has to select a substitute,
which is similar to the IF at least concerning essential solvent
properties as ionic strength, kinds of ions and pH. In addition
to Na+ and K+ the IF contains the bivalent metal ions Mg2+
and Ca2+ that could affect the association state of insulins, as it
was shown for Ca2+ (
). Therefore, we tested possible
influences of MgCl2 and CaCl2. The presence of 0.6 mM
MgCl2 had practically no effect on the association state of
the investigated insulin analogs. However, dilution of
formulations with PBS containing additionally CaCl2 (final
concentration 1.6 mM after mixing) led to a rapid formation of huge
amounts of large aggregates in the case of lispro and aspart
(data not shown). This impedes the use of simulated IF
composition, since it cannot be applied to all analogs in the present
study. Therefore, we decided to use PBS as an adequate
dilution medium for a comparison of the dissociation behavior
of the RAI analogs.
The technique of mixing formulation with dilution
medium strongly determines the feasibility of dissociation
experiments as well as the relation of the results to physiological
processes. From a biophysical point of view, the applied
changes in the solution conditions must lead to clearly
measurable changes in the association state towards the
biologically active monomeric state. This was realized by scheme I,
which uses sufficiently large protein concentration jumps
caused by dilution with solvent (PBS). By adding particular
amounts of excipients to the solvent, concentration changes of
excipients upon dilution can be compensated or modified in
order to measure the influence of excipients on dissociation.
The dilution factor is limited by the sensitivity of the
biophysical methods. Taking into account the concentration of U100
insulin formulations of 3.5 g/l and a low-concentration limit
for light scattering investigations of insulins of 0.1–0.2 g/l, a
dilution factor of 20 is feasible. We have mostly used 20-fold
dilution to determine the maximum speed of dissociation
during one-step dilution experiments. Furthermore, this scheme is
useful to measure the influence of excipients on the
dissociation kinetics and the final association state after dilution. In
order to vary the final concentrations of excipients over a wide
range we have applied other dilution factors and modified
formulations with higher insulin concentrations in some cases.
In vivo, the immediate dilution factor upon subcutaneous
injection is assumed to be only about two (
). Such a weak
protein dilution does not considerably change the association
state. However, the decrease in the concentration of excipients
due to fast diffusion can alter the association state rather
quickly in an alternative way. This is of particular relevance
for phenolic additives. Their influence will be considered in
more detail in the case of m-cresol. In order to take into
account this pathway, we have used experimental scheme II (Fig.
11) to imitate twofold dilutions of U100 formulations together
with a subsequent decrease in the concentration of m-cresol.
This procedure is based on the presumption that the final
equilibrium state and the kinetics after dilution depend only
on final solution conditions and on identical initial states prior
to dilution. The reference condition for the experiments is
insulin U100 twofold diluted with PBS. Following scheme II,
the insulin concentration is kept constant while the m-cresol
concentration is lowered stepwise. Scheme II uses modified
formulations (U900) with nine-fold higher insulin
concentration (32 g/l) and an m-cresol concentration of 29 mM, which
are 18-fold diluted with PBS containing defined amounts of
m-cresol. The identity of the initial states in U900 and U100
conditions was tested in equilibrium light scattering and CD
experiments. The m-cresol concentration can thus be lowered
stepwise down to 1.6 mM (Fig. 11). In order to further
decrease the m-cresol concentration, we have also used dialysis
against media with the corresponding low m-cresol content.
Circular Dichroism (CD)
CD measurements in the near-UV region were done in a
Jasco J-715 spectropolarimeter equipped with a
Peltierthermostat controlled cell holder using quartz cuvettes with
appropriate pathlengths between 0.1 mm and 10 mm
(Hellma, Germany). The instrument was calibrated using
1S-(+)-10-camphorsulphonic acid (
). After baseline
correction, measured ellipticities θ were converted into
meanresidue ellipticities [θ] using the mean-residue weights of
113.6 g/mol for HI and lispro, 114.0 g/mol for aspart, and
114.2 g/mol for glulisine, respectively. The CD
measurements in formulations and diluted formulations were
complicated considerably by strong absorption of the phenolic
excipients. Because both, the concentrations of phenolic additives
and the insulins were varied, it is difficult to specify general
detection limits. Kinetic experiments were triggered by
manual mixing within the sample cells.
Equilibrium and Kinetic Measurements of Static and Dynamic Light Scattering
Simultaneous static and dynamic light scattering
measurements were done with the same instrument at a scattering
angle of 90°. The custom-built apparatus, equipped with a
diode-pumped continuous wave laser (Millennia IIs,
Spectra-Physics) and a high quantum yield avalanche
photodiode, has been described in detail (
). Instead of using a
commercially available stopped-flow mixing device (
manual mixing (dead time about 10 s) was preferred in the present
work. Manual mixing ensures stable solution composition,
particularly during long-term experiments.
A primary data accumulation interval Tacc = 8 s was used
for all SLS and DLS experiments. Tacc defines the time
resolution that is consistent with the dead time and yields
reasonable (short) time averages of the mean scattering intensity
I(Tacc) and the time-autocorrelation function ACF(Tacc) of
the fluctuations in the instantaneous scattering intensity.
Hundreds of pairs of I(Tacc) and ACF(Tacc) were stored
transiently before calculating kinetic and equilibrium data. Fig. 2
shows an example of an original data trace of scattering
intensity. Translational diffusion coefficients D were obtained from
the measured autocorrelation functions using the program
). CONTIN yields intensity distribution
functions I(D), which can be calculated without further
assumptions concerning the morphology of the particles. Diffusion
coefficients were converted into Stokes radii via the
StokesEinstein equation RS = kBT/(6πηD), where kB is Boltzmann’s
constant, T is the temperature in Kelvin, and η is the solvent
viscosity. In our work, the hydrodynamic quantities are
presented in terms of RS. Viscosities were measured using an
Ubbelohde-type viscometer (Viscoboy-2, Lauda, Germany).
Apparent molecular masses, Mapp, were calculated
from the relative excess scattering intensity, Iexc,rel defined
as Iexc , rel = (Isolution − Isolvent)/Ireference, where Isolution, Isolvent
and Ireference are the scattering intensities of solution,
solvent, and reference scatterer (toluene, in our case),
respectively. Mapp is related to Iexc,rel by Mapp = kopt⋅Iexc , rel/c,
where c is the peptide concentration and kopt is an
optical constant depending on physical quantities of the
scattering experiment as scattering angle, wavelength,
reference sample, refractive index n of the solution
and refractive index increment (dn/dc) of the proteins.
A (dn/dc) of 0.19 ml/g was used for all insulins in the
individual solvents. The size distribution obtained with
CONTIN was additionally used to separate the
scattering intensity of insulin in the association equilibrium
from that of other scattering particles.
The notation Mapp must be used for the following reasons.
Extrapolation to zero insulin concentration to eliminate the
influences of intermolecular interactions (virial effects) on the
true molecular mass is not adequate because we wanted to
study the association equilibrium of the insulins. However,
significant virial effects in addition to changes caused by shifts
in the association state became evident only at concentrations
above 10 g/l under the investigated solvent conditions.
Possible influences on the measured association state will be
discussed in particular cases.
Furthermore, only the term Mapp is correct, as the
measured mass is mostly an average over different association
states. It is even more appropriate to present relative apparent
molecular masses Mrel = Mapp/Mmon. Mmon is the monomer
mass calculated from the amino acid sequence.
Use of Power Laws for Estimations of Compactness
Data on the association states of the different insulins are
derived from the apparent molecular masses measured by SLS.
Stokes radii measured by DLS depend on both the state of
association and the shape of oligomers, particularly the
packing of the monomeric subunits within the oligomer. The
Stokes radius is not unequivocally related to the association
General information about the compactness of oligomers
can be obtained from the relation between Mapp and RS. This
can be done using known scaling laws for the link between
hydrodynamic properties (e.g. RS) and the corresponding
mass for particular structural classes (
). The general form
of such a scaling law is RS = a.Mb. The coefficients for any
structural type can be obtained from a sufficiently large
number of related pairs M, RS. For proteins such scaling
coefficients have been obtained for the globular (compactly folded)
and the fully unfolded state (proteins lacking disulfide bonds at
high denaturant concentrations) (30). A plot of RS versus M in
double-logarithmic scale for both cases is shown in Fig. 7 (solid
and dashed lines). This representation is particularly useful to
classify the overall structural type of a protein, peptide or
peptide aggregate with unknown high-resolution structure in
solution. The positions of the corresponding data pairs M, RS
in the diagram provide information about the molecular
dimensions of the molecule or the molecular assembly.
Equilibrium States of Rapid Acting Insulins and Human
Before measuring the dissociation kinetics of the insulins,
equilibrium association states under various conditions, initial states in
formulations and final states after dilution were characterized by
equilibrium measurements. The association equilibria under
near physiological (PBS with no added Zn2+) and formulation
solvent conditions are shown in Fig. 1a and b, respectively.
Although the results in Fig. 1a indicate a strong tendency of all
insulins to dissociate with decreasing concentration in saline
solution, the differences caused by modifications in the amino acid
sequence are evident. The higher stability of oligomeric
structures in formulation solvents becomes obvious by comparing the
association states in Fig. 1a and b.
The association states after mixing different amounts of
formulation and PBS are shown in Fig. 1c. The concentration
dependence differs considerably from that in PBS and cannot be
considered as a simple association equilibrium, as both insulin
concentration and solvent composition are changed by mixing
formulation and buffer. The association states at insulin
concentrations of 0.17 mg/ml marked by a dashed vertical line
correspond to the final states that are approached during kinetic
experiments using 1:20 dilution jumps. The values of Mrel close to
one indicate that all RAI approach the monomeric state under
those dilution conditions. The association state of HI after 1:20
dilution is still far away from the monomeric one. The large
difference between the states of HI in PBS (Fig. 1a) and after
dilution of the formulation with PBS (Fig. 1c) demonstrates the
strong Zn2+ binding and consequently the higher stabilization of
oligomers in the case of HI.
Fig. 1d shows the corresponding changes in RS after
dilution of formulations with PBS. The dissociation of oligomers is
also indicated by the decrease in RS. However, Stokes radii
cannot be directly related to the number of monomers
forming the oligomers. Nevertheless, Stokes radii are useful
to estimate the compactness of oligomeric states in particular
Kinetics of Changes of Relative Molecular Masses
Initially, all systematic kinetic light-scattering experiments
were done using 1:20 dilution jumps of U100 formulations
with PBS. Figure 2 shows the original data of the temporal
changes in the light-scattering intensity observed for
Humalog®. The changes in scattering intensity level
off after about 40 min. The intensity pattern is typical
of the majority of experiments. Note the low excess
scattering of the solution over that of the solvent. The
low excess scattering and the unavoidable contribution
of spurious scattering were the main problems involved
in these investigations. A special data treatment
procedure had to be applied to derive relative masses,
Mrel = Mapp/Mmon, from the primary data.
The changes in Mrel of the RAI and HI after 1:20 dilution
jumps are shown in Fig. 3. Between 10 and 20 original data
acquisition intervals had to be accumulated in order to
improve the (S/N) ratio of the intensity time-autocorrelation
functions. The loss in the number of data points had to be
accepted in favor of obtaining size distributions allowing the
correct estimation of the scattering from insulin oligomers.
Interestingly, dissociation of insulin glulisine into monomers
is complete within the dead time of the experiment. In
contrast, insulins lispro and aspart dissociate much more slowly.
Both insulins approach a quasi-monomeric state with
comparable rates. Some dissociation of human insulin also occurs on
the same time scale of a few minutes. However, the final
dissociation steady state reached by HI is far away from the
monomeric one, as expected from the equilibrium studies.
All kinetic traces could be fitted by a sum of one exponential
and a constant background within the experimental error
(solid lines in Fig. 3).
Fig. 3 Changes of the average relative molecular masses Mrel after 1:20
dilution jumps of formulations with PBS, pH 7.4, T = 23°C. The large open
symbols at t = 0 indicate the initial Mrel in formulation measured in equilibrium
experiments. The continuous lines show single-exponential fits to the
experimental data (for results see Table I).
Near-UV CD Spectra in Equilibrium States and Kinetic
Changes of the Ellipticities at Characteristic
The equilibrium near-UV CD spectra of the RAI and HI in
formulation, after 20-fold dilution with PBS and in PBS
are shown in Fig. 4. Knowledge of the changes in
nearUV CD accompanying the dissociation process is
indispensable in order to distinguish different types of
Fig. 4 Near-UV CD spectra
obtained for (a) Humalog®, (b)
NovoRapid®, (c) Apidra®, and (d)
Insuman Rapid®. All figures
additionally contain the respective
spectra after 1:20 dilution of the
formulations with PBS, pH 7.4,
T = 23°C (light blue) and of the
insulin dissolved in PBS, pH 7.4,
T = 23°C (dark blue, RAI only). The
arrows indicate the expected signal
changes at 255 nm (a, b, d) or
275 nm (c) during kinetic
hexamers and to estimate the time scales of changes in
tertiary structure. For example, strong negative
ellipticities in the wavelength range between 250 and 265 nm
are typical of insulin in the R6 state observed in the
presence of both Zn2+ and phenolic additives (
Furthermore, consideration of equilibrium CD spectra
is important for the choice of wavelengths for
monitoring the kinetic CD changes. According to the initial and
final spectra, the kinetic traces were recorded at 255 nm
for the formulations Humalog®, NovoRapid® and
Insuman Rapid® and at 275 nm in the case of
The time course of the CD signal at the selected
wavelengths after 1:20 dilution jumps is shown in Fig. 5. Some
features resemble the dissociation kinetics observed in
lightscattering experiments. Again, very fast changes
completed within the experimental dead time were observed
for Apidra®. The changes in the cases of Humalog®
and NovoRapid® clearly include a slow component.
Insuman Rapid® shows also slow, but only weak
changes within the accessible time range. Interestingly,
remarkable dead time amplitudes have been measured in
the case of Humalog®, NovoRapid® and Insuman
Rapid®, which do not have its counterpart in the
changes of relative masses. As in the case of light
scattering the kinetic records could be fitted by one
exponential and a constant.
Summary of Standard Dilution Data, Dependence of the Dissociation Rates on the Final Concentration of Excipients
Characteristic parameters of the kinetic transitions for the
1:20 dilution are summarized in Table I. Aspart and lispro
approach a final state that is largely monomeric indicated by
Mrel ~ 1.5, while glulisine is completely dissociated (Mrel = 1).
There are only small differences between the time constants
obtained for lispro and aspart. Additionally, the reaction times
obtained by light scattering and CD agree within the
experimental error. In the case of human insulin, the reaction time
constants are of the same order of magnitude as for lispro and
aspart. The difference between the time constants obtained
for HI by CD and light scattering is probably due to the large
experimental errors caused by the small kinetic amplitudes
and the low (S/N) ratio of the kinetic records.
Since phenolic additives stabilize the hexameric states of
insulins considerably, systematic investigations of the influence
of phenolic excipients on the dissociation kinetics were done
up to final excipient concentrations of 4 mM. The results are
shown in Fig. 6. The influence is remarkable, for both insulin
lispro and aspart. The same increase of the concentration of
phenolic additives had no measurable effect on the fast
dissociation of glulisine hexamers (data not shown).
Fig. 6 Dependence of the observable dissociation time τ on final
concentration of phenolic additives after dilution for lispro and aspart. Humalog®
contains 29 mM m-cresol, while NovoRapid® contains 16 mM m-cresol and
16 mM phenol. The methods for variation of final concentration are described
in Materials and Methods. For the calculations of τ, we have used the light
scattering results and fitted the concentration dependence by an empirical
power law τ(c) = τ0 + a*cb .
The influence of further additives appeared to be
negligible. Results of investigations on the influence of excess zinc are
summarized in Table II. No influence was found concerning
the presence of glycerol (data not shown).
Consequences of the Absence and Presence of Zn2+
and Phenolic Additives on Insulin Association States
and Dissociation Kinetics
The investigations aim to understand the different
self-association/dissociation properties of these insulins combined with
the influence of relevant excipients. The results show the
influence of specific modifications of the formulation conditions
of lispro, aspart, glulisine and HI on the association states and
the dissociation kinetics.
Figure 7 shows the effect of removing Zn2+ from the
formulations of Humalog® and NovoRapid® on the average
molecular mass and the Stokes radius. This was done by
adding EDTA, thus bringing the formulation conditions of
Humalog® and NovoRapid® closer to that of Apidra®. In
both cases, the stable and compact hexameric state dissociates
slowly (kinetic data not shown) into smaller assemblies
according to the average mass. Remarkably, this is not accompanied
by a measurable decrease in the hydrodynamic dimension for
Table I Summary of Kinetic Data
after 1:20 Dilution of Formulations
with PBS According To Scheme I
state in formulation
Table II Influence of Zn2+ on
final Zn2+ concentration after
monomer molar ratio
Dissociation times determined from CD data, the higher Zn2+ concentrations were produced by adding Zn2+ to the
both insulins. Accordingly, the presence of Zn2+ is crucial for
the formulations Humalog® and NovoRapid® in order to
obtain compact oligomeric states. These general observations
render further modifications of Humalog® and NovoRapid®
Figs. 8 and 9 demonstrate differences and similarities
between insulin glulisine and HI by adding or removing Zn2+.
According to Fig. 8a, removal of Zn2+ transforms HI from a
structure in a phenol stabilized hexamer (see also the spectrum
of aspart in formulation) to a state that is characterized by a
CD spectrum similar to insulin glulisine in Apidra®
formulation. On the other hand, addition of Zn2+ to the Apidra®
formulation converts glulisine into a structure with a
nearUV CD spectrum nearly identical to that of aspart in
NovoRapid® formulation (Fig. 8b).
Fig. 9a and b show the consequences of the same changes
on the association state and the dissociation kinetics. After
complete removal of Zn2+ from the Insuman Rapid®
formulation, HI dissociates into monomers as fast as glulisine in
Apidra® (Fig. 9a). However, agglomeration of HI into
structures larger than hexamers is observed under the modified
starting conditions. On the other hand, addition of Zn2+ to
Apidra® supports the formation of larger assemblies in
formulation and brings the dissociation behavior close to that of
HI in Insuman Rapid® (Fig. 9b).
To get further structural information on the state of
glulisine in Apidra®, we have done additional experiments.
Figure 10 gives evidence for significant changes in the
association equilibrium of glulisine, if Zn2+ is added to the
formulation, and shows the effect of the inverse procedure in the case
of HI, namely the removal of Zn2+ from the formulation
In the presence of Zn2+ and at concentrations below 2 mg/
ml, oligomers of both insulins exhibit similar stabilities
according to the association equilibria. However, insulin
glulisine shows a tendency to form larger assemblies
above 2 mg/ml. HI does not form such structures under
the same conditions.
In the absence of Zn2+ and low concentrations, insulin
glulisine and HI populate association states of nearly the same
size. Remarkably, the size of glulisine assemblies levels off
close to that of hexamers at high concentrations, whereas the
size continuously increases in the case of HI. Differences
between the large oligomeric structures of glulisine and HI will
be considered in more detail in the discussion.
The compactness of the investigated insulins in their
formulations is compared in Fig. 7 by plotting the Stokes radii
versus the average mass in double logarithmic scale. For better
illustration, we have shown the lines corresponding to the
scaling equations between mass and Stokes radius for
unfolded and folded proteins. All insulins are essentially as compact
as typical globular proteins under their particular formulation
conditions. Notably, this holds also for glulisine, despite a
possibly different structural organization of hexamers compared
to the R6 states of HI, insulin lispro and insulin aspart (see
Impact of m-Cresol on the Dissociation Behavior of Lispro under Subcutaneous Injection-like Conditions
The results presented in the previous section have
demonstrated the importance of the concentration of phenolic additives
for the dissociation rates of the insulins lispro and aspart. Since
the disappearance rate of phenolic compounds in the ST is
rather high, we have applied the experimental scheme II (Fig.
11) to study the influence of decreasing m-cresol concentration
on the dissociation process of lispro after twofold dilution of
Humalog® U100 with PBS. Humalog® was chosen, because
lispro approaches an essentially monomeric state in the
absence of Zinc and phenolic additives at concentrations as high
as 1.75 g/l (see Fig. 1a). In addition, we also tested whether a
decrease in m-cresol concentration changes the association
state of glulisine under similar conditions.The experiments
with Humalog® demand a fast solvent exchange leaving the
insulin concentration nearly constant. This is not trivial if one
is interested in measuring the kinetics of dissociation. A
method to study at least equilibrium states is dialysis against media
with varying m-cresol concentration, but otherwise
unchanged solvent composition. In order to circumvent the
problems involved in measuring kinetic data, we have
developed a strategy allowing both kinetic and equilibrium
experiments, which is based on scheme II described in materials and
methods. The entire experimental strategy is shown in Fig. 11.
A1 and A2 denote the initial conditions with18-fold dilution or
dialysis, respectively. B1 and B2 are the final conditions,
which differ only in the concentration range of m-cresol.
The attainable concentration range for B1 is above
After each dilution step from A1 to B1, we have measured
the kinetics of dissociation in terms of the light scattering
intensity, Stokes radius, and CD signal at 255 nm and the final
equilibrium data. The time constants (τ1) for approaching the
equilibrium states calculated using single-exponential fits are
given in Table III. Mrel of the final association states at
different m-cresol concentrations obtained by either dilution or
dialysis are shown in Fig. 12a and Table III. Results from
dilution and dialysis are separated by the dashed line in Fig.
12a. The values of Mrel at 0 and 29 mM m-cresol are those
attributed to PBS and U100 formulation, respectively (cf. Fig.
1a and b). Table III also contains the equilibrium Stokes radii
of lispro in the B states. Comparable results at 1.6 mM
mcresol confirmed the equivalence of the two pathways A1 →
B1 and A2 → B2 (Fig. 12a, b and Table III).
Fig. 10 Influence of Zn2+ on the association behavior of glulisine in placebo
to Apidra®. The association equilibrium is strongly shifted towards hexamers
at low concentrations. Second, formation of assemblies larger than hexamers
becomes evident at concentrations above 2 mg/ml. This behavior is totally
different from that of HI in the presence of Zn2+. For comparison, we have
also shown the association equilibria of HI in PBS with and without Zn2+.
Fig. 12b shows the specific ellipticities at 255 nm in the final
states B. Again, the values at 0 and 29 mM m-cresol are those
corresponding to PBS and U100 formulation, respectively.
It is instructive to compare the CD signals and Mrel at low
m-cresol concentrations. While the CD signal hints at a
preferential population of the monomeric state, Mrel remains
rather large (close to 4). Furthermore, there are practically no
changes in RS (see Table III). Both, the values of Mrel and
RS point to the existence of an oligomeric state. Thus, it was
essential to investigate the kinetic stability of the association
states B using 1:10 dilution jumps under constant solvent
condition (Fig. 11, B → C).
Mrel after additional 1:10 dilution is shown in Fig. 12a. The
10-fold dilution was chosen in order to reach the same final
insulin concentration as in 20-fold dilution experiments of
U100 using scheme I. Note that the final solvent conditions
resulting from scheme I and II setups are different. The
measured time constants (τ2) and the corresponding
equilibrium values of Mrel and RS are given in Table III.
For comparison, we have investigated the influence of
mcresol on the association state of glulisine. As the transitions
are always fast in the case of glulisine (see Fig. 3), we have
measured only Mrel and RS in equilibrium states at different
m-cresol concentrations. Mrel changed from 4.1 to 3.0 upon
lowering the m-cresol concentration from 14.5 mM down to
1.9 mM (data not shown). The values are between those,
which could be expected as limits from the data shown in
Fig. 1a and c for PBS and diluted formulation, respectively.
The focus of this work is to compare the dissociation kinetics of
commercially formulated rapid-acting insulins and human
insulin. A description of the differences should be helpful to
reveal influences and basic mechanisms that are important
for an understanding of the entire absorption process of
insulins in the patient after injection.
The pharmacokinetic behavior of marketed RAI
formulations has been studied demonstrating that insulin glulisine is
absorbed slightly faster compared to insulin lispro and insulin
). Surprisingly, the differences in dissociation
kinetics observed in our experiments are significantly more
pronounced. This underlines that the absorption of an RAI is not
solely governed by its dissociation behavior. We additionally
demonstrate that the amount of phenolic compounds present
in solution strongly influences insulin dissociation times at
conditions expected after subcutaneous injection. However, for a
complete understanding of the RAI pharmacokinetics, a
thorough analysis of all processes involved in the absorption
process is essential.
*too fast to measure τ properly
**n.d. due to very small signal changes
Estimated errors are about 8% for Mrel, 2% for RS and 20% for τ. In the cases these errors are exceeded, values are given in parentheses
Comparison of Specific Equilibrium Association States of RAI and HI
The concentration dependences observed under formulation
conditions and in PBS yield the association equilibria, which
are measures of the stability of oligomeric structures, under
the limiting conditions of sufficiently high concentrations of
stabilizing ligands and of their virtual absence.
The formation of classical oligomeric structures in PBS
(Fig. 1a) is restrained because of the absence of ligands like
Zn2+ ions (
). All RAI form considerably smaller
assemblies than HI particularly at higher insulin concentrations.
Moreover, the tendency to associate in the absence of Zn2+
is different for the various RAI. These observations
demonstrate that the particular modifications in the primary
structure result in altered subunit interactions. The association
state at low concentrations is closest to the monomeric one
in the case of insulin lispro. This is in agreement with previous
Here, we use the notation ‘assemblies’ for all types of
oligomers, for which the structural arrangement of subunits (i.e.,
the quaternary structure) is not fully verified. We refrain from
modeling the experimental data using association equilibria
between specific oligomers, as they exist in the presence of
Zn2+. It has been shown for equilibrium centrifugation (
and static light scattering data (
) obtained for bovine insulin
in the absence of Zn2+ that the results can be fitted by simple
isodesmic infinite or enhanced isodesmic infinite
selfassociation models. The simple isodesmic infinite association
model is based on a stepwise addition of monomers to each
existing oligomer with identical association constant.
However, these association schemes can be ruled out, if an
upper limit of oligomer size is approached above a
characteristic insulin concentration.
Such a behavior was indeed observed for lispro, aspart and
HI in formulation (Fig. 1b). Lispro, aspart and HI are in the
hexameric state throughout the investigated concentration
range. The conformation of the hexamers is assumed to be
R6, since both Zn2+ and phenolic additives are present in all
formulations. The corresponding near-UV CD spectra (Fig.
4), particularly the strongly negative ellipticities between 251
and 255 nm, support this assumption.
The association equilibrium of glulisine in its formulation
lacking Zn2+ is very different and resembles its association
equilibrium in PBS. The stabilization of larger assemblies
due to the presence of m-cresol is clearly visible, but the
Fig. 12 Plots of (a) Mrel and (b)
[θ]255 of lispro versus m-cresol
concentration after processing of
samples according to experimental
scheme II. Mrel was determined at
lispro concentrations of U50
(circles) and U5 (crosses). Values at
0 mM and 29 mM m-cresol
represent insulin dissolved in PBS
and formulation, respectively. The
dashed lines separate data obtained
by dialysis and dilution.
assemblies of glulisine in its formulation are much less stable
than the oligomers of lispro, aspart and HI in their respective
formulations containing Zn2+.
Notably, the average size of glulisine assemblies approaches
that of hexamers at the concentration used for U100
formulations (c = 3.5 mg/ml, Fig. 1b) and remains essentially
constant at higher peptide concentrations (Fig. 10). Additionally,
the compactness of glulisine assemblies under formulation
conditions is practically indistinguishable from that of the
other insulins in their formulations (Fig. 7). Thus, although
isodesmic infinite association may be valid for HI in the
absence of Zn2+, insulin glulisine seems to form well-ordered
hexameric assemblies in the presence of m-cresol, but in
absence of Zn2+. As the CD spectra of glulisine assemblies (Fig.
8) clearly differ from those observed for all investigated
insulins in the presence of Zn2+ and phenolic ligands, the
conformation of glulisine hexamers must be different from the R6
state. High-resolution NMR studies would be required to
decide, whether it is similar to or different from the well-known
T6 state of HI.
Fundamental Differences in the Dissociation Rates of the Various Insulins
The results of the kinetic light scattering experiments (Fig. 3)
not only show clear differences between HI and the RAI, but
also between the individual RAI. According to the rates and
amplitudes of the kinetic changes, we may classify the insulins
investigated here into three categories, as illustrated in Fig. 13:
RAI type I, RAI type II and HI. RAI type I stands for the
dissociation behavior of insulin glulisine, whereas insulin lispro
and insulin aspart are assigned to type II. The initial structures
of HI, lispro and aspart are specified as R6 hexamers based on
their typical near-UV CD spectra (black triangles in Fig. 13).
The initial structure of insulin glulisine symbolized by an
assembly of gray triangles in Fig. 13 is predominantly hexameric,
and is labeled with T6*. The difference in tertiary structure
compared to the other insulins is evident from the near-UV CD
spectrum. On addition of Zn2+, T6* hexamers can be converted
into R6 hexamers comparable to that of HI as indicated by the
changes in near-UV CD, dissociation kinetics and association
equilibrium at low concentrations (Figs. 8b, 9b and 10).
However, these R6 oligomers must differ somewhat from their
counterparts in the case of HI, lispro and aspart. This is revealed
by the formation of larger assemblies (probably di-hexamers etc.)
at high glulisine concentrations (Fig. 10). Remarkably, this type of
assemblies disappears rapidly within the experimental dead time
of the dissociation kinetics (Fig. 9b) reflecting their disassembly
prior to the slow hexamer dissociation.
The other arrays of gray triangles in Fig. 13 represents
allosteric hexamers, in general. The particular conformation
(R6, T3R3 or T6) will be a matter of further discussion. The
arrays of open triangles in the right interval symbolizes T6
hexamers or putative tetramers and dimers being in
equilibrium with T6 hexamers. Other types of oligomers are shown
as assemblies of monomers (open circles).
While the association states of insulin lispro, insulin aspart,
and HI remain essentially unchanged within the experimental
dead time of 10 s, glulisine immediately attains a completely
monomeric state. The rates of changes in the association state
observed in the second phase as illustrated in Fig. 13 for lispro,
aspart, and HI are similar (Table I). Insulin lispro and aspart
approach the monomeric state less completely (Mrel = 1.5,
Table I) than insulin glulisine. The large final state of HI
(Mrel = 4.2, Table I) is in accordance to that measured in
equilibrium experiments (Fig. 1c) and underlines the high
stability of the hexamers.
Equivalent to the observed kinetic changes in the
association state, similar rates were observed also for the CD signals
of lispro, aspart, and HI within the observable interval (Fig. 5,
Table I). In contrast to the corresponding changes in the
association state, significant dead time amplitudes were
observed, particularly in the case of aspart. The changes of the
CD signal of glulisine occurred entirely within the dead time
of the experiment.
Influence of Phenolic Additives and the Mechanisms of Slow Hexamer Dissociation
The influence of formulation excipients on the rates of the
transitions is essential for understanding the dissociation
mechanisms in detail. An increase in the concentration of
excipients did not drive the dissociation constants of glulisine
into the observable interval, confirming that the association
mechanism of glulisine differs from that of the other RAI.
Consequently, systematic studies of the effect of excipients
on the rate of dissociation have been done for lispro and aspart
We have studied the influences of phenolic additives, excess
Zn2+ and glycerol that are the major constituents of
formulations (Supplemental Table I). Glycerol (data not shown) and
excess Zn2+ (Table II) had practically no influence on the slow
dissociation of both lispro and aspart. However, a
considerable influence of m-cresol and equimolar mixtures of phenol
and m-cresol was found for both lispro and aspart. The
concentration dependence of the measured dissociation time
constants is shown in Fig. 6, which can be described by an
empirical equation of the form τ(c) = τ0 + a ⋅ cb, where c is the total
molar concentration in mM and a, b, and τ0 are fit
parameters. τ0 can be considered as dissociation time in the absence of
phenolic additives and reflects the dissociation kinetics of T6
hexamers. We obtained τ0 = 0.27 min and τ0 = 0.32 min for
lispro and aspart, respectively. The values of τ0 are in good
agreement with the results of lifetime measurements (
under comparable conditions. We found no dependence on
insulin concentration of the dissociation time at high
concentrations of phenolic additives. However, a subtle decrease in τ
with decreasing insulin concentration at low concentration of
m-cresol was found (see Table II).
In the light of earlier publications, the long dissociation
times and the strong influence of phenolic excipients on the
dissociation rates are not surprising. Birnbaum et al. (
measured the rate of bivalent metal ion extraction from
Co(II)-R6 hexamers of lispro and HI. Hassiepen et al. (
reported results on kinetic lifetimes of T6, T3R3 and R6
hexamers. Interestingly, the lifetimes were about one minute
for T6, but as large as hours for R6 hexamers. Further
information about the kinetic stability of the Zn(II)- and
Co(II)insulin hexamer complexes has been obtained using the
chromophoric chelator 2,2′,2″-terpyridine as a kinetic probe
). Furthermore, these authors have additionally
discussed the relation between kinetic and thermodynamic
stability in the presence of phenolic ligands and the high
kinetic barrier for the transition to T6.
Altogether, these results have been obtained using elegant
but indirect approaches to measure oligomer dissociation.
Taken together, those results and our direct measurements
of oligomer size indicate that the association state of type II
RAI existing after the dead time (Fig. 13, gray symbols) is still
the R6 state. The measured dissociation is then a slow
transition from the R6 to the T6 state. After this rate-limiting step,
the less stable T6 states dissociate rapidly into monomers. A
fast dissociation of lispro after removal of phenol was also
discussed by Ciszak et al. (
) to explain why the formulated
analog exhibits rapid time action after injection despite its
hexameric conformation in formulation. HI, however, is still
in an association equilibrium shifted towards T6 hexamers at a
comparably reduced concentration of phenolic ligands.
The proposed dissociation kinetics characterized by a
ratelimiting transition from R6 to T6 hexamers, followed by a
faster transition towards monomers, is reflected in the slow
changes of the CD signals. However, the interpretation of
the observed dead time amplitudes in the CD signal at
255 nm of lispro and aspart (less pronounced in the case of
HI) is not straightforward. The specific ellipticities
approached within the dead time in this wavelength region
are comparable with those typical of T3R3 hexamers. This
could be interpreted as a transient population of T3R3 states
on the transitions between R6 and T6 as it has been discussed
frequently. Since conformational transitions from T3R3 to T6
states are slow, the additional population of a T3R3 state is not
in conflict with the observed slow changes in CD and light
scattering signals. However, there is a discrepancy concerning
the fast rate of population of the T3R3 state right after dilution
of formulation, which must be very high according to the dead
time CD amplitudes. This is extremely unlikely since high
kinetic barriers between R3 and T3 are expected in any case
because of the conformational switch in the N-terminus of the
B-chains for R6 → T3R3 and T3R3 → T6 as well (
Changes of the CD signals in the wavelength range
between 250 and 255 nm are expected to result from specific
variations in the environment of PheB1 (
). An alternative
explanation is possible concerning rather unspecific effects
caused by the drop (mostly 20-fold) in the concentration of
phenolic compounds during the applied dilution jumps.
Indeed, the concentrations of phenolic substances used in
formulation and in our experiments for measuring the effects on
dissociation rates are so high that mass action effects of
binding can be expected (
). This is also supported by our
observation that the dissociation rates depend on the total
concentration of phenolic additives and practically not on the molar
ratio [phenol]/ [insulin] at high concentrations of phenolic
additives. The changes in CD could result from changes in
the packing density of the tertiary and quaternary structures
upon binding of phenolic additives. Such a mechanism was
already discussed by Rahuel-Clermont et al. (
precondition for further discussion of the dead time CD changes is to
assemble complete CD spectra from single kinetic records at
Impact of Phenolic Additives on the Dissociation
Behaviour under Subcutaneous Injection-like
For a rapid absorption of insulin after subcutaneous injection,
a fast dissociation of insulin hexamers is necessary (
disappearance rate of phenolic additives is supposed to be one
rate-limiting factor in this process (
). Yet, it is not known,
how the acceleration of dissociation proceeds in detail and
how different insulin analogs respond to a decrease in the
concentration of phenolic additives. In the following, we will
discuss the results of twofold dilution of lispro (Humalog®) as
a prototype for the behavior of the RAI type II insulins. The
results concerning the RAI type I insulin glulisine have
confirmed the expected small effect of decreasing concentrations
of m-cresol on the association state and do not demand
Kinetic changes upon lowering the m-cresol concentration
were monitored by measuring the time courses of the light
scattering intensity and the CD signal at 255 nm (see
Table III). Detectable changes of the association state could
be observed only when the concentration was lowered at least
down to 7.3 mM m-cresol, where we obtained time constants of
τ1,LS = 25 min and τ1,CD = 32 min. Further dilution down to
3.5 mM led to a nearly 10-fold decrease in the dissociation time
constants revealing a steep concentration dependence in this
range. Thus, right after injection at m-cresol concentrations of
14.5 mM the dissociation times must be in the order of hours.
At m-cresol concentrations below ~2 mM, lispro is able to
dissociate quickly within a few minutes. Accordingly, the
dissociation rate should be no longer a bottleneck for the
absorption process. Under these conditions, both type I and type II
RAI dissociate rather fast. This explains why the extremely
faster dissociation kinetics of glulisine is only marginally
translated into the pharmacokinetic profile.
Interestingly, the concentration of about 2 mM is close to
the midpoint of the transition of the CD signal at 255 nm,
which indicates the transition from the hexameric R6 state to
the monomeric state (Fig. 12b). The transition to the
monomeric state appears to be almost complete at the lowest
mcresol concentration of 0.4 mM used in our studies (Fig. 12b).
This was supported by the CD spectrum (data not shown),
which was essentially identical to that typical of the
monomeric state of lispro (Fig. 4a).
In contrast to these observations, Mrel does not
considerably decrease with m-cresol concentration. Particularly at
0.4 mM m-cresol, lispro is still in an association state far from
the monomeric one. This contradiction hints to the formation
of an alternative type of assemblies, which involves
monomeric subunits with the structure of isolated monomers. The
formation of higher order assemblies upon depletion of phenolic
additives has been described also by Teska et al. (
association states may resemble rather colloidal assemblies
than discrete (classical) insulin oligomers.
However, irrespective of the structure of the intermediate
association states at low m-cresol concentrations, their ability
to dissociate is more important. For this purpose, we have
done additional 1:10 dilution experiments with all B state
samples (Fig. 11). Below 1.6 mM m-cresol, lispro is
preferentially in the monomeric state (Fig. 12a). The equilibrium state
is approached with τ2 < 5 min (Table III). This means that the
formation of these transient association states does not
considerably slow down the dissociation process.
The situation is different at high m-cresol concentrations.
At 7.3 mM, Mrel changes only from 5.2 to 4.1 with τ2 =
57 min. This means that the majority of lispro molecules
remains still in the original R6 state and only a small fraction
In summary, with our experimental strategy we were able
to determine dissociation rates and the attained equilibrium
association states at discrete m-cresol concentrations.
However, without information about in vivo distribution rates
of phenolic additives, we cannot predict actual dissociation
times of insulins in the IF.
It is tempting to speculate, which additional factors other
than the prolongation of dissociation rates by phenolic additives
are time limiting for absorption. According to our results (Fig.
1c), the decrease in insulin concentration itself must be equally
important by shifting the equilibrium towards dimers and
monomers. It has to be taken into account that the decrease
in the instantaneous insulin concentration due to diffusion
within the subcutaneous tissue and by penetration into capillaries is
a particular driving force for dissociation thus accelerating the
dissociation in a feedback-loop manner. In this context, faster
acting insulin formulations must improve the transition
efficiency of insulins from ST into the bloodstream. Accordingly,
additional excipients could be tested for this purpose as recently
shown by Heise et al. (
) in the case of aspart. It would be
interesting to investigate if the used excipient nicotinamide
(probably responsible for acceleration) also alters the absorption
properties of other RAI formulations.
Our investigation is the first direct comparison of the inherent
dissociation properties of marketed fast-acting insulins.
Despite the essentially accelerated dissociation of all three
RAI compared to HI, there are distinctive differences between
glulisine (type I RAI) and lispro and aspart (type II RAI). From
a biophysical point of view, these are mainly based on the
different kinetic stabilities of the insulins at their specific
The superior dissociation behavior of glulisine is based on
its specific oligomeric state in formulation lacking Zn2+. The
low kinetic stability of this essentially hexameric state
compared to that of the R6 state existing in formulations
containing both Zn2+ and phenolic additives ensures very fast
dissociation. The time constants are shorter than our experimental
dead time of 10 s under all conditions obtained after dilution
The dissociation behavior of type II RAI is affected by the
high stability of the hexameric R6 state in the formulations
containing Zn2+ and phenolic additives. However, an
acceleration of dissociation is attainable by fast depletion of the
concentration of phenolic additives right after dilution.
Similar conditions exist after subcutaneous injection due to a
fast diffusion of phenolic additives. Our investigations with
Humalog® revealed that a tenfold reduction in m-cresol
concentration decreases the dissociation time from about one
hour to one minute. Consequently, also the dissociation of
type II RAI becomes fast enough not to be the limiting factor
of the absorption process.
A further acceleration of insulin absorption is obviously
regulated by other factors as diffusion in the ST or the
transition from ST into blood capillaries. The decrease in the actual
insulin concentration directly influences the dissociation
equilibrium and is thus an additional driving force for dissociation.
In this context, our results will be helpful to optimize future
formulation conditions for rapid-acting insulins.
ACKNOWLEDGEMENTS AND DISCLOSURES
This work was supported by grants from Sanofi-Aventis
The authors thank Nadin Jahnke for her skillful support
during data acquisition and evaluation. The authors declare
that they have no conflict of interest.
Open Access This article is distributed under the terms of the
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medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
1. Zaykov AN , Mayer JP , DiMarchi RD . Pursuit of a perfect insulin . Nat Rev Drug Discov . 2016 ; 15 ( 6 ): 425 - 39 .
2. Brange J , Owens DR , Kang S , Vølund A . Monomeric insulins and their experimental and clinical implications . Diabetes Care . 1990 ; 13 ( 9 ): 923 - 54 .
3. Jeffrey PD , Coates JH . An equilibrium ultracentrifuge study of the self-association of bovine insulin . Biochemistry . 1966 ; 5 ( 2 ): 489 - 98 .
4. Pekar AH , Frank BH . Conformation of proinsulin. A comparison of insulin and proinsulin self-association at neutral pH . Biochemistry . 1972 ; 11 ( 22 ): 4013 - 6 .
5. Goldman J , Carpenter FH . Zinc binding, circular dichroism, and equilibrium sedimentation studies on insulin (bovine) and several of its derivatives . Biochemistry . 1974 ; 13 ( 22 ): 4566 - 74 .
6. Richards JP , Stickelmeyer MP , Flora DB , Chance RE , Frank BH , DeFelippis MR . Self-association properties of monomeric insulin analogs under formulation conditions . Pharm Res . 1998 ; 15 ( 9 ): 1434 - 41 .
7. Jeffrey PD , Milthorpe BK , Nichol LW . Polymerization pattern of insulin at pH 7.0 . Biochemistry . 1976 ; 15 ( 21 ): 4660 - 5 .
8. Wollmer A , Rannefeld B , Johansen BR , Hejnaes KR , Balschmidt P , Hansen FB . Phenol-promoted structural transformation of insulin in solution . Biol Chem Hoppe Seyler . 1987 ; 368 ( 8 ): 903 - 11 .
9. Derewenda U , Derewenda Z , Dodson EJ , Dodson GG , Reynolds CD , Smith GD , et al. Phenol stabilizes more helix in a new symmetrical zinc insulin hexamer . Nature . 1989 ; 338 ( 6216 ): 594 - 6 .
10. Kaarsholm NC , Ko HC , Dunn MF . Comparison of solution structural flexibility and zinc-binding domains for insulin, proinsulin, and miniproinsulin . Biochemistry . 1989 ; 28 ( 10 ): 4427 - 35 .
11. Chang X , Jorgensen AM , Bardrum P , Led JJ . Solution structures of the R6 human insulin hexamer . Biochemistry . 1997 ; 36 ( 31 ): 9409 - 22 .
12. Ciszak E , Beals JM , Frank BH , Baker JC , Carter ND , Smith GD . Role of C-terminal B-chain residues in insulin assembly - the structure of hexameric Lys(B28)pro(B29)-human insulin . Structure . 1995 ; 3 ( 6 ): 615 - 22 .
13. Bakaysa DL , Radziuk J , Havel HA , Brader ML , Li S , Dodd SW , et al. Physicochemical basis for the rapid time-action of Lys(B28)pro(B29)-insulin: dissociation of a protein-ligand complex . Protein Sci . 1996 ; 5 ( 12 ): 2521 - 31 .
14. Whittingham JL , Edwards DJ , Antson AA , Clarkson JM , Dodson CG . Interactions of phenol and m-cresol in the insulin hexamer, and their effect on the association properties of B28 pro -> asp insulin analogues . Biochemistry . 1998 ; 37 ( 33 ): 11516 - 23 .
15. Heise T , Zijlstra E , Nosek L , Rikte T , Haahr H . Pharmacological properties of faster-acting insulin aspart vs insulin aspart in patients with type 1 diabetes receiving continuous subcutaneous insulin infusion: a randomized, double-blind, crossover trial . Diabetes Obes Metab . 2017 ; 19 ( 2 ): 208 - 15 .
16. Heise T , Hövelmann U , Brøndsted L , Adrian CL , Nosek L , Haahr H . Faster-acting insulin aspart: earlier onset of appearance and greater early pharmacokinetic and pharmacodynamic effects than insulin aspart . Diabetes Obes Metab . 2015 ; 17 : 682 - 8 .
17. Becker RH . Insulin glulisine complementing basal insulins: a review of structure and activity . Diabetes Technol Ther . 2007 ; 9 ( 1 ): 109 - 21 .
18. Koren R , Hammes GG . Kinetic study of protein-protein interactions . Biochemistry . 1976 ; 15 ( 5 ): 1165 - 70 .
19. Rahuel-Clermont S , French CA , Kaarsholm NC , Dunn MF . Mechanisms of stabilization of the insulin hexamer through allosteric ligand interactions . Biochemistry . 1997 ; 36 ( 19 ): 5837 - 45 .
20. Birnbaum DT , Kilcomons MA , DeFelippis MR , Beals JM . A s s e m b l y a n d d i s s o c i a t i o n o f h u m a n i n s u l i n a n d Lys(B28)pro(B29)-insulin hexamers: a comparison study . Pharm Res . 1997 ; 14 ( 1 ): 25 - 36 .
21. Hassiepen U , Federwisch M , Mulders T , Wollmer A . The lifetime of insulin hexamers . Biophys J . 1999 ; 77 ( 3 ): 1638 - 54 .
22. Wollmer A , Rannefeld B , Stahl J , Melberg SG . Structural transition in the metal-free hexamer of protein-engineered B-13 Gln insulin . Biol Chem Hoppe Seyler . 1989 ; 370 ( 9 ): 1045 - 53 .
23. Rasmussen CH , Røge RM , Ma Z , Thomsen M , Thorisdottir RL , Chen JW , et al. Insulin aspart pharmacokinetics: an assessment of its variability and underlying mechanisms . Eur J Pharm Sci . 2014 ; 62 : 65 - 75 .
24. Coffman FD , Dunn MF . Insulin-metal ion interactions: the binding of divalent cations to insulin hexamers and tetramers and the assembly of insulin hexamers . Biochemistry . 1988 ; 27 ( 16 ): 6179 - 87 .
25. Hill CP , Dauter Z , Dodson EJ , Dodson GG , Dunn MF . X-ray structure of an unusual Ca2+ site and the roles of Zn2+ and Ca2+ in the assembly, stability, and storage of the insulin hexamer . Biochemistry . 1991 ; 30 ( 4 ): 917 - 24 .
26. Kelly SM , Jess TJ , Price NC . How to study proteins by circular dichroism . Biochim Biophys Acta Proteins Proteomics . 2005 ; 1751 ( 2 ): 119 - 39 .
27. Gast K , Noppert A , MullerFrohne M , Zirwer D , Damaschun G . Stopped flow dynamic light scattering as a method to monitor compaction during protein folding . Eur Biophys J . 1997 ; 25 ( 3 ): 211 - 9 .
28. Provencher SW . Contin - a general-purpose constrained regularization program for inverting noisy linear algebraic and integralequations . Comput Phys Commun . 1982 ; 27 ( 3 ): 229 - 42 .
29. Wilkins DK , Grimshaw SB , Receveur V , Dobson CM , Jones JA , Smith LJ . Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques . Biochemistry . 1999 ; 38 ( 50 ): 16424 - 31 .
30. Uversky VN . What does it mean to be natively unfolded? Eur J Biochem . 2002 ; 269 ( 1 ): 2 - 12 .
31. Bolli GB , Luzio S , Marzotti S , Porcellati F , Sert-Langeron C , Charbonnel B , et al. Comparative pharmacodynamic and pharmacokinetic characteristics of subcutaneous insulin glulisine and insulin aspart prior to a standard meal in obese subjects with type 2 diabetes . Diabetes Obes Metab . 2011 ; 13 ( 3 ): 251 - 7 .
32. Heise T , Nosek L , Spitzer H , Heinemann L , Niemoller E , Frick AD , et al. Insulin glulisine: a faster onset of action compared with insulin lispro . Diabetes Obes Metab . 2007 ; 9 ( 5 ): 746 - 53 .
33. Blundell T , Dodson G , Hodgkin D , Mercola D. Advances in Protein Chemistry . Academic Press 1972. Chapter Insulin: The structure in the crystal and its reflection in Chemistry and Biology; 26 : 279 - 402 .
34. Attri AK , Fernández C , Minton AP . pH-dependent self-association of zinc-free insulin characterized by concentration-gradient static light scattering . Biophys Chem . 2010 ; 148 ( 1-3 ): 28 - 33 .
35. Kang S , Brange J , Burch A , Vølund A , Owens DR . Subcutaneous insulin absorption explained by insulin's physicochemical properties: evidence from absorption studies of soluble human insulin and insulin analogues in humans . Diabetes Care . 1991 ; 14 ( 11 ): 942 .
36. Teska BM , Alarcon J , Pettis RJ , Randolph TW , Carpenter JF . Effects of phenol and meta-cresol depletion on insulin analog stability at physiological temperature . J Pharm Sci . 2014 ; 103 ( 8 ): 2255 - 67 .