Clinical trial with monocomponent lente insulins

Diabetologia, Dec 1973

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 X 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 radioimmunoelectrophoresis. 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, immunogenic 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.

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Clinical trial with monocomponent lente insulins

Prof. Dr. B. Bruni Ospedale Maria Vittoria Via Cibrario 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; Berson 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 Like Components). 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 I-Ieding (1972) ; normal 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. Federlin (1971) . 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 treatment. 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. Results Immunological Situation 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 abovementioned type. Clinical Situation 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 mild reactions. Complications such as infections, 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 immunologieM situation. I n s u l i n Requirement. No correlation could be 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. Conclusions 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 activity. Our data agree with the preliminary results reported in 37 cases b y K o r p and Levett (/973) ; and in 48 cases, b y Andreani et al. (1972) . 4. The course of clinical conditions (such as insulin hyposensitivity, insulin allergy, insulin lipoatrophy, Total IRI (~UlmO 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 immunological state. 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 state. 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 dose. 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. IgG binding (mU/ml) Total IRI (~.U/1 O0 ml) 9 743.~_ 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. 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B. Bruni, M. D'Alberto, M. Osenda, C. Ricci, G. L. Turco. Clinical trial with monocomponent lente insulins, Diabetologia, 1973, 492-498, DOI: 10.1007/BF00461695