Clinical trial with monocomponent lente insulins
Prof. Dr. B. Bruni
Ospedale Maria Vittoria
Clinical Trial with Monocomponent Lente Insulins PreliminaryReport
B. Bruni 0
M. ])'Alberto 0
M. Osenda 0
C. Ricci 0
G.L. Turco 0 1
0 Ospedale Maria Vittoria-Cliniea Medica, Centre di Studi Fisico-Biologici, Universit~ di Torino
1 With the technical assistance of B.C. Brossa, A. Bachiorlini , A. Giolitti
Summary. The immunogenicity of pork Monocomponent (MC) Lente insulin (Monotard| was studied for more than one year in a clinical trial series of 43 insulin-dependent diabetics in comparison with Monospecies (MS) Lente pork insulin (5 crystallized, not monocomponent). The antigenicity was estimated by determination of total extractable serum insulin by radioimmunoassay and of I-125 insulin-binding of the serum IgG measured by radioimmunoeleetrophoresis. MC Lente insulin was nonimmunogenic in newly diagnosed diabetics: at the end of the observation, the antibody titre was generally under the level of detection. Long-term diabetics transferred to MC Lente insulin from conventional insulin preparations showed a marked reduction in the high initial levels of insulin antibodies, sometimes with a multiphasic pattern; low initial levels remained unchanged at the end of the trial. MS Lente insulin was found to be, even if weakly, immunogenie in newly diagnosed diabetics. In long-term insulin-treated diabetics transferred to MS Lente insulin a tendency to reduction in the high antibody starting level was often observed. Diabetic manifestations such as high insulin requirement, insulin allergy, insulin lipoatrophy and diabetic microangiopathy showed -- in some instances -- a clinical course independent of immunological modifications. Clinical control of diabetes achieved both with MC and MS Lente insulins was rated as good. There was no ketosis or severe hypoglycaemia, and the daily insulin requirement was reduced in several cases. of the preparation "with little or no immunogenic activity when used for therapy". This concept was based on extensive studies in rabbits and humans.
Monocomponent lente insulin; monospecies lente insulin; insulin immunogenieity; insulin antibodies; high insulin requirement; insulin allergy; insulin lipoatrophy; diabetic microangiopathy; clinical control of diabetes
"Immunogenicity" of an insulin preparation is
currently defined as the ability of the insulin to induce
the formation of humeral and cellular antibodies in
animals or in insulin-treated patients.
Since the papers of l~aynaud and Lacroix, 1925;
Barral and Roux, 1931 ;
Bernstein et al., 1938
Wasserman, Broh-Kahn and Mirsky, 1940
; Jorpes, 1940;
Arquilla and Stavitsky, 1956
; Moinat, 1957;
and Yalow, 1957
, the central problem concerns the
question of whether the insulin molecule has, 19er se,
even weak immunogenie properties or whether these
properties are related to protein "impurities" contained
in commercial preparations.
The experimental work and the first clinical
investigations of Schlichtkrull and associates
(1968-1972) led to the conclusion t h a t :
a) the insulin molecule is not immunogenie; b) the
production of antibodies in subjects treated with
conventional insulin preparations is not solely due to
species differences, but rather to other peptides related
to insulin which contaminate the crystalline extracts
of beef or pork insulin (mainly the a- and b-Steiner
components : Pro-Insulin Like Components and Insulin
The term "Monocomponent insulin" (MC-Insulin)
is used b y Sehlichtkrull, 1972 to designate Sanger's
insulin purified from a- and b-components and resolved
from e-Steiner's component, to become the sole protein
a) newly diagnosed insulin-dependent diabetes,
never previously treated with insulin;
b) long-term, conventionally insulin-treated
diabetes presenting clinical conditions such as high insulin
requirement, insulin allergy, insulin lipoatrophy, and
diabetic mieroangiopathy, which m a y possibly
accompanied b y an increased rate of antibody formation.
An additional aim of the investigation was to get
information about the quality of clinical control of the
diabetes as achieved with MC or MS Lente insulin.
Material and Methods
Since December 1970 and up to August 1972, 43
insulin-dependent diabetic subjects of both sexes, aged
5 to 65 years, were submitted to t r e a t m e n t with Novo
MC Lente and Novo MS Lente insulins. The patients
were divided in the following groups :
Group 1 : 6 new insulin-requiring diabetics (never
previously treated with insulin) treated with MC Lcnte.
Group 2:5 new insulin-requiring diabetics treated
with MS Lente.
Group 3:18 long-term diabetics, previously treated
with conventional types of insulin, transferred to MC
Lente. The preferential criteria for selection were
clinical conditions as listed above.
Group 4 : 1 4 long-term diabetics, previously treated
with conventional types of insulin, transferred to MS
Lente. Selection criteria as in group 3.
Cooperative patients were hospitalized for a
fortnight for full examination and therapy. T h e y were on
a daily diet of 2000--2500 kcal (20% proteins, 40%
fats and 40% carbohydrates) and returned for
oncemonthly control after the hospitalization.
The immunogenicity of the insulin preparations
was estimated b y determination of:
1. total extractable serum insulin b y
radioimmunoassay (total I R I ) according to
values: 0 - - 2 5 ~U/ml;
2. specific insulin-binding capacity of serum IgG
(IgG binding) according to the
radioimmunoelectrophoresis method of Hein Christiansen (1973). 30 ~l of
a 1-125-pork insulin solution (20 mU/ml, specific
activity 20 mCi/mg) was mixed with patient's serum
and barbital buffer in equal proportion and incubated
overnight at -~4 C. 5 ~1 of the mixture was applied to
the holes of an agarose gel plate containing commercial
preparation of antihuman IgG from rabbits. The plate
was then transferred to the electrophoresis chamber;
electrophoresis was run at 2 - - 3 V/cm during 15--20 h.
"Cigar-shaped" precipitates containing the insulin
bound to the precipitated IgG fraction, were cut out
and transferred to a well gamma counter for counting
the radioactivity of IgG-bound insulin. Limits of
sensitivity: normal sera showed IgG binding values of
about 0.05 mU/ml; diabetic sera were accepted as
positive at values above 0.10 mU/ml. Autoradiography
of the precipitates was used only to get a qualitative
evaluation of the presence and location of the tracer of
the precipitates. Radioinsulin and antisera were
supplied b y Novo Research Institute, Copenhagen.
This method has proved suitable ranking of the sera
for routine clinical purposes
(Sch]ichtkrull et al., 1972;
Andreani et al., 1972)
. As for the binding of
Iodinelabelled insulin: cf.
The usual clinical and biochemical data were
recorded on standard trial case record sheets: history,
age, sex, duration of diabetes, previous treatment,
occurrence of other endocrine diseases, fasting blood
sugar (true glucose: rag/100 ml), M-value (during
hospitalization), urine glucose excretion (g/24 h),
proteinuria, ketonuria, body weight, clinical remarks
on complications, diet, and the current insulin
At the beginning of t r e a t m e n t and during periods
of readjustment, MC or MS Actrapid insulins were used
occasionally as a supplement to or a substitute for the
t r e a t m e n t with MC or MS Lente.
Group 1: New patients treated with 16--36 Units
of MC Lente daily showed, after 300--500 days of
treatment, zero or barely detectable IgG insulin binding
values (0.05--0.10 mU/ml). In 3 patients, however,
IgG binding reached maximal levels of 0.4 mU/ml
during the early period of treatment. The total I R I
displayed practically the same pattern (Fig. 1).
Group 2: New patients treated with 12--32 Units
of MS Lente daily showed, after the same period of
observation, a rise in the IgG binding values above the
level of detection up to 1.10 mU/ml, with a
corresponding pattern of total I R I (Fig. 2).
Group 3: A marked drop in high initial levels of
IgG binding values was observed in the majority of
patients after the transfer from conventional to MC
Lente insulin. The high starting levels of total I R I also
fell. As Fig. 3 shows, a multiphasic pattern with
transient increases -- especially in total I R I -- is
evident in some eases. After about 400 days, values
tend to diminish even if not quite to the normal level.
Cases with low initial antibody levels, did not have
relevant variations at the end of the trial.
Group 4: In patients treated with conventional
insulin and transferred to MS Lente, the levels of IgG
binding and total I R I , which were initially high, also
tended to decrease, as shown in F/g. d. At various points
during the study, however, more or less evident peaks
of IgG binding and I R I values were noted. Low IgG
binding and total I R I starting levels remained
unchanged or presented intercurrent rises of the
The clinical-metabolic control of diabetes after a
year or more on MC and MS Lente insulins was rated
good in all cases. Fasting blood sugar did not exceed
200 mg/100 ml; glueosuria was within 20--25 g/24 h,
except for one case; M-values during hospitalization
were under 15--20; ketonuria was generally absent;
n o episodes of keto-acidosis or hypoglyeaemic shock
I n s u l i n Lipoatrophy. After a year of t r e a t m e n t w i t h
occurred. E v e n i n cases with a r a p i d drop i n a n t i b o d y
MC L e n t e there was a complete absence of l i p o a t r o p h y
titres, h y p o g l y e a e m i a was reported o n l y i n the form of
i n the sites of i n s u l i n a d m i n i s t r a t i o n . I n two p a t i e n t s
there was a n almost complete remission of l i p o a t r o p h y
surgery a n d p r e g n a n c y h a d no o u t w a r d effect on the
caused b y the previous t r e a t m e n t . One p a t i e n t
excourse of the diabetes. A n i m p r o v e m e n t in the
subperienced a fall i n t o t a l I R I a n d I g G b i n d i n g ; the other
jective state was f r e q u e n t l y recorded.
h a d low s t a r t i n g a n t i b o d y levels.
The following clinical details deserve special
conc a n t changes.
sideration because of their possible relation to the
I n s u l i n
No correlation could
established between the b e h a v i o u r of a n t i b o d y level
a n d the change i n the daily i n s u l i n dose. Nevertheless,
a r e d u c t i o n i n i n s u l i n r e q u i r e m e n t i n groups 3 a n d 4
was possible as shown i n Table 1.
p r e l i m i n a r y results.
The following conclusions m a y be d r a w n from our
1. The association of the two m e t h o d s employed
I n s u l i n Allergy. Prior i n s u l i n allergy of the
imme(Total II%I a n d 1-125-IgG binding) seems to be suitable
diate local a n d generalized t y p e disappeared in 3 eases
for assessing t h e level of circulating i n s u l i n a n t i b o d y
after transfer to MC Lente. A related fall i n the serum
as a clinical routine.
i n s u l i n antibodies was f o u n d i n 2 cases.
2. Monocomponent Lente pork insulin was found
to be "non immunogenie" in newly diagnosed
insulindependent diabetics at the end of a t r e a t m e n t period
for more t h a n one year; slight transient antigenie effect
was occasionally observed only in the initial phase of
therapy. The disappearance or the reduction in the
level of circulating antibodies in patients transferred
to MC Lente insulin after years of t r e a t m e n t with
I n comparison to MC Lente insulin, MS Lente
insulin seems to possess a slightly greater immunogenie
Our data agree with the preliminary results reported
in 37 cases b y K o r p and Levett (/973) ; and in 48 cases,
Andreani et al. (1972)
4. The course of clinical conditions (such as insulin
hyposensitivity, insulin allergy, insulin lipoatrophy,
I I r , , , , " I ' ]
60 IfO lgO ~40 ~00 ~60 4~ 480 ~-0
60 120 lgO 240 300 360 420 4~0 ~0
conventional insulin preparations agrees with the
general impression of an i m p o r t a n t change in the
3. Monospeeies Lente pork insulin appears to be,
even if weakly, immunogenie as demonstrated in cases
of newly diagnosed insulin-dependent diabetes. I n
some cases of long-term diabetes, previously treated
with conventional insulin preparations, a downward
trend in serum a n t i b o d y levels after transfer to MS
Lente was observed.
and diabetic mieroangiopathy) was in several instances
independent of modifications in the immunological
5. A good clinical-metabolic control of diabetes,
even in cases of brittle diabetes, v e r y difficult to
compensate before the trial, was obtained in nearly all the
cases of our series, with a reduction of the daily insulin
60 120 tSO 240 300 360 420 ~0 r~O
60 ~0 ~.gO ~40 300 ~0 4~0 490 ~0
do~s oF treofrnen~
Acknowledgement. We are greatly indebted to I)r. Sc.
J. Schlichtkrull; and to Dr. H. Fornet and Coworkers of
the Novo Research Institute, Copenhagen, for their helpful
support during these studies.
(~.U/1 O0 ml)
60 NO IgO 240 300 3eo 4~ 4go ~40
60 I~o ego 24o ~00 360 4~0 450 gO
days off t r e ~ , ~ e r r
diabetics compared with clinical data. Diabetologia 7,
474 (1972) Abstr.
t t e i n Christiansen, Aa. : Radioimmunoeleetrophoresis in
the determination of insulin binding to IgG.
Methodological studies, t i o r m . Metab. Res. 5, 147--154 (1973)
Korp, W., Levett, R . E . : Erfahrungen m i t
Monokomponenten-Insulin. Wien. klin. Wsehr. 85, 326--330 (1973)
Jorpes, J . E . : I~eerystallized insulin for diabetic patients
with insulin allergy. Arch. intern. Ned. 83, 363--371
Moinat, P. : A quantitative estimation of antibodies to
exogenous insulin in diabetic subjects. Diabetes 7,
R a y n a u d , M., Lacroix, A.: Un eas d ' a n a p h y l a x i e a
l'insuline. Essai de pathog6nie des oed6mes insuliniens.
Bull. See. reed. Hop. Paris 49, 831--832 (1925)
Schliehtkrull, J., Munck, O., Jersild, M. : The M value. An
index of blood sugar control in diabetics. Acta reed.
seand. 177, 95--102 (1965)
I [ L ]
I120 19Q ~40 300 360 420 480 S40
Andreani , D. , Iavicoli , M. , Colletti , A. , Menzinger , G. , Maltarello , C. : Esperienze sul t r a t t a m e n t o del diabete con le insuline di tipo Monocomponente (MC) e Monospecie (MS) . Folia endocr. (Roma) 25 , 516 -- 539 ( 1972 )
Arquilla , E.R. , Stavitsky , A.B. : Evidence for the insulindirected specificity of rabbit anti-insulin serums . J. elin. Invest . 35 , 467 -- 473 ( 1956 )
Barrel , P. , Roux , J. : L'insuline constitue-t-elle-m~me u n antig@ne sp~cifique ? S@ances Soc. Chim. Biol . 106 , 292 -- 296 ( 1931 )
Bernstein , C. , Kirsner , J.B. , Turner , W.J. " Studies on anaphylaxis with insulin . J. Lab. din. Med . 32 , 938 -- 945 ( 1938 )
Berson , S.A. , Yalow , R. S. : Studies with insulin-binding antibody . Diabetes 6 , 402 -- 405 ( 1957 )
Deckert , T. , Grundahl , E. : The antigenicity of pig insulin . Diabetologia 6 , 15 -- 20 ( 1970 )
Fankhauser , S. , Michl , J. : New possibilities to avoid the formation of insulin-antibodies in diabetic patients . Diabetologia 7 , 478 ( 1972 ) Abstr .
Federlin , K. : Immunopathology of insulin . Berlin-Heidelberg-New York: Springer Verlag 1971
I-Ieding , L. G. : Determination of total serum insulin (IRI) in insulin-treated diabetic patients . Diabetologia 8 , 260 -- 266 ( 1972 )
Sehlichtkrull , J. , Brange , J. , Ege , It., Hallund , O. , i e - ding, L.G., JSrgensen, K. , Markussen , J. , Stahnke , P. , Sundby , F. , V61und , A. : Proinsulin and related proteins . Diabetologia 6 , 80 ( 1970 ) Abstr .
Schliehtkrull , J. , I-[eding, G., Hein Christiansen , Aa., V61und , A. : Immtmologieal aspects of insulin therapy . Lecture held at the 7th Acta Endoerin. Congr . Copenhagen J u l y 1971
Sehliehtkrull , J. , Brange , J. , Hallund , O. , Hein Christiansen , Aa., I-Ieding , L.G. , JSrgensen, K . t t . : t t i g h l y purified insulin for therapy. Lecture held at the 8 . @me R@union Ann. Ass. Suisse du Diab. Lausanne, J a n u a r y 1972
Schliehtkrull , J. , Brange , J. , Hein Christiansen , Aa., Hallund , O. , tteding , L.G., J6rgcnsen, K. I-I.: Clinical aspects of insulin-antigenicity . Diabetes 21 , Suppl . 2 , 649 -- 656 ( 1972 )
Steiner , D.F. , Hallund , O. , Rubenstein , A. , Cho , S. , Bayliss , C. : Isolation a n d properties of proinsulin, intermediate forms, and other minor components from crystalline bovine insulin . Diabetes 17 , 725 -- 736 ( 1968 )
Wasserman , P. , Broh-lKatm, R.I-I. , Mirsky , I . A . : The antigenic property of insulin . J. Immunol . 28 , 213 -- 224 ( 1940 )