Intermediate-acting Insulin Preparations:NPH and Lente

TORSTEN DECKERT

Physicochemically, NPH and lente insulins differ in size of crystals, content of protamine and zinc, andoften in species composition, since lente always contains beef insulin. The duration of the hypoglycemiceffect of lente insulin seems to be longer than 24 h, whereas that of NPH insulin does not exceed 24 hwhen given in amounts of 0.2-0.3 U/kg body wt. Moreover, NPH and lente insulins differ in theirability to form stable mixtures with neutral insulin solutions, since only NPH insulin can be mixed withregular insulin without changing the specific course of effect of regular insulin. Highly purified porcineNPH and the lente-like porcine insulin preparation, Monotard, do not seem to differ regarding sideeffects (lipoatrophy, immunogenicity). However, highly purified lente insulin (containing beef insulin)seems to be more immunogenic than highly purified porcine NPH insulin, DIABETES CARE 3- 623-626,SEPTEMBER-OCTOBER 1980.

The most widely used intermediate-acting insulinpreparations are NPH and lente insulins. It isoften asked whether there is any difference be-tween them in their course of action, reproducibil-

ity of resorption, mixability with regular insulin, and side ef-fects, particularly immunogenicity. To answer these ques-tions, the known characteristics of these two insulin prepara-tions are briefly reviewed.

HISTORICAL BACKGROUND

NPH (neutral protamine Hagedom) insulin is a further de-velopment of Hagedorn's protamine insulin1 made byKrayenbiihl and Rosenberg at Nordisk Insulinlaboratorium,Denmark, in 1946.2 They found that insulin and protaminebrought together in isophane proportions (the condition inwhich neither insulin nor protamine is found in excess) atneutral pH, in the presence of small amounts of zinc andphenol and/or cresol, will precipitate in oblong tetragonalcrystals without leaving any regular insulin in the superna-tant.

Lente insulin (zinc insulin preparation) was invented byHallas-Mtfller et al. in 1952 at the Novo company, also inDenmark.3 The prolongation of effect is based on the factthat the solubility of insulin at neutral pH in acetate buffercan be reduced by a surplus of zinc. Furthermore, it can bemodified by the physical state of zinc insulin and by properusage of the solubility characteristics of beef insulin.

PHYSICOCHEMICAL CHARACTERISTICS

NPH insulin preparations are suspensions of small insulinprotamine crystals of the same size (Figure 1). The volume ofthe crystals may vary between 1 and 20 fim3. The crystals aresuspended in a phosphate buffer at neutral pH. The lente in-sulin preparations are suspensions of about 70% large rhom-bohedral beef zinc insulin crystals (ultralente) and 30%amorphous porcine zinc insulin particles (semilente). Thevolume of the ultralente crystals is about 200- 1000 fxm3.The mixture is suspended in acetate buffer at neutral pHcontaining a surplus of zinc.

Bottles containing NPH as lente insulin have to be in-verted several times before the preparation is drawn up intothe syringes before injection. However, because the zinc in-sulin crystals are larger and heavier than the NPH crystals,lente insulin will sedimentate faster than NPH insulin aftershaking the suspension, which might have some practicalimportance. The content of protamine and zinc in NPH andlente insulins is given in Table 1.

Besides these major differences, the chemical differencesdepend on the species and the purity of the insulin used. Theinsulin used in manufacturing NPH insulin is beef or porkinsulin or a mixture of both. Lente insulin is always a mix-ture of beef and pork insulin, since the ultralente part oflente insulin is made from beef insulin and the semilente partof lente insulin is made from porcine insulin. Porcine lente-like insulin is available as Monotard insulin (Novo, Copen-

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INTERMEDIATE-ACTING INSULIN: NPH AND LENTE/TORSTEN DECKERT

(a)

(b)

FIG. I. Photomicrograph of (a) NPH suspension and (b) lente insulin suspension at room temperature. 1.0 cm =20 /u,m.

hagen, Denmark). The species specificity seems to play a rolein the immunogenicity of insulin, since insulin preparationscontaining beef insulin have been shown to induce insulinantibodies more easily than insulin preparations containingonly porcine insulin.11

The purity of the insulin component varies over a widerange. During the last years there has been a developmenttoward insulin preparations of higher purity, since it hasbeen shown that several side effects (for example, lipoat-rophy, insulin resistance, insulin allergy, and the develop-ment of several humoral antibodies) depend largely on thepurity of the insulin preparation.4*5 The purity of NPH as

well as lente insulin preparations is expressed in suffix as sin-gle peak, single component, monocomponent (MC), or rareimmunogenic (RI). Single peak means that insulin has beenisolated by gel filtration, which separates molecules accord-ing to their size (i.e., insulin, arginine insulin, desamidoin-sulin, ethylester insulin, and small amounts of other poly-peptides). Single component, monocomponent, and rareimmunogenum (RI) means that insulin has been furtherpurified by chromatography, which also separates moleculesof the same molecular size and therefore contains less impuri-ties. As well, NPH made from porcine insulin as lente insu-lin of highly purified quality is available. However, for docu-

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INTERMEDIATE-ACTING INSULIN: NPH AND LENTE/TORSTEN DECKERT

TABLE 1Content of protamine and zinc in NPH and lente insulins

ProtaminePhysical state pH Zn (mg/100 U) (mg/100 U)

NPHLente

Crystalline70% crystalline30% amorphous

7.37.3

0.020.20

0.3-0.40

menting purity, techniques such as gel filtration andchromatography are insufficient. The purity of insulin shouldbe further specified by radioimmunologic determination ofthe relevant contaminants [i.e., a-components, proinsulin,glucagon-like materials, pancreatic polypeptide (PP), vaso-active intestinal peptide (VIP), somatostatin] by well-docu-mented techniques. Also, the purity of protamine, zinc, andother additives should be stated. Otherwise, the purity ofNPH and lente insulins cannot be compared.

ABSORPTION FROM SUBCUTANEOUS TISSUE

bsorption studies with NPH and lente insulins havebeen done either by following the disappearanceof radioactivity from subcutaneous tissue afterthe injection of labeled NPH or lente insulin or

by following the plasma insulin concentration after subcuta-neous injection of these preparations in patients with onlyminor or no endogenous insulin secretion. However, thesame techniques have not been used and the results thereforeare not strictly comparable.

The most extensive studies with radioactive-labeled NPHinsulin have been done by Ktflendorf et al.6 and with labeledlente insulin by Binder et al.7'8 The differences betweenNPH and lente insulin, according to their results, are shownin Table 2. Twenty-four hours after injection in the femoralregion, remarkably more radioactivity is found at the injec-tion site after lente insulin compared with NPH insulin.

The disappearance of radioactivity at the injection siteafter NPH insulin followed first-order kinetics,6 whereaslente was absorbed in a biphasic curve,7 probably becauselente insulin is a mixture of amorphous and crystalline zincinsulin.

The reproducibility of day-to-day absorption in the samepatient, measured as the variation coefficient of the timeuntil 40-50% of the maximal radioactivity disappeared, was26% for NPH insulin6 and about 35% for lente insulin.7

Thus, the reproducibility of the resorption of both insulin

TABLE 2Differences in absorption between NPH and lente insulins

Percent of injected insulin at injection site after:

10 h 24 h 36 h

NPHLente

4070

1040

025

preparations seems to be in the same range. It must, how-ever, be mentioned that the experiments with lente insulinwere\done with non-highly purified insulins.

After subcutaneous injection of NPH insulin in reasonabledoses, plasma insulin concentration increases significantlyafter 2 h and is maximal after 5 h.6 After lente insulin incomparable amounts, the time for significant initial incre-ments to occur is not known. The maximal plasma insulinconcentration seems to be reached 7 h after the injection oflente insulin.9 Twenty-four hours after the injection of NPHinsulin, the plasma insulin concentration is not higher thanbefore the injection. Twenty-four hours after lente insulin,plasma insulin still seems to be elevated.10

COURSE OF ACTION

The time curve of the blood glucose lowering action of NPHand lente insulins (as given by Nordisk Insulinlaboratoriumand the Novo Company in 1978) is shown in Table 3. How-ever, randomized crossover experiments against placebo infasting diabetic subjects have only been done by K^lendorf etal. with NPH insulin.6 They found that the starting actionbegan 120 min after the injection, and that the maximal ac-tion occurred 5.5 h after the injection, 30 min after themaximal plasma insulin concentration had been reached.From Rasmussen et al.'s experiments in nonfasting diabeticsubjects, it seems that the maximal effect after lente was firstachieved after 10 h.9

The porcine lente-like preparation Monotard appears tohave a shorter effect than lente insulin. Our crossover ex-periments on maturity-onset diabetic subjects using highlypurified porcine NPH insulin and the highly purified porcinelente-like preparation Monotard did not indicate differencesin the blood glucose lowering effects between these two prep-arations (Figure 2). Reproducibility of the blood glucoselowering effect of NPH insulin in maturity-onset diabeteswas shown to be excellent.6

It can be concluded from resorption studies, studies ofplasma insulin concentration after subcutaneous injection,and studies of the blood glucose lowering effects of NPH andlente that lente has a more protracted action than NPH insu-lin.

MIXABILITY

Regular insulin can be mixed with NPH or lente insulin inthe same syringe in every ratio. However, the course of effectwill change with lente/regular mixtures depending on the

TABLE 3Time curve of action

NPHLente

of NPH and

Onset

1*42Vi

lente insulins

Course of effect

Maximum

4-12

(h)

End

2424 Vi

Data from K0lendorf et al.6 and Binder.7 Data from Nordisk Insulinlaboratorium and Novo Company, 1978.

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INTERMEDIATE-ACTING INSULIN: NPH AND LENTE/TORSTEN DECKERT

100--

80-

60-

40-

20-

per cent of initialblood glucose (m* SEM )

T injection

x NPH (Retard RI®)

o Lente like (Monotard®)

n= 12

^N-f+Hnu-——-—Hi

13 15 17 19 21

TIME

FIG. 2. Blood glucose lowering effect in percentof initial capillary blood glucose (initial = meanof blood glucose at 0700, 0800, and 0900 h)after highly purified porcine NPH insulin (Re-tard RI) and the highly purified porcine Iente-like preparation (Monotard) given subcutane-ous^ (at the arrow) in the femoral region.Randomized crossover experiment.

ratio between lente and regular insulin in the syringe,10 pre-sumably because the surplus of zinc in the supernatant oflente insulin will react with regular insulin and change it to asemilente-like preparation. This is not the case when mixingregular insulin with NPH. Stable mixtures of regular and in-termediate-acting insulin can only be prepared with NPH in-sulin.6

IMMUNOGENICITY

The immunogenicity of NPH and lente insulin de-pends largely on the purity and species of the insu-lin used. Preparations containing beef insulin aremore immunogenic than insulin preparations

made of porcine insulin of comparable purity.11 However, byusing lente insulin of monocomponent purity (lente preparedfrom highly purified beef and highly purified porcine insu-lin), formation of antibodies can be avoided in some in-stances,4 but not to the same extent as with highly purifiedporcine insulin.11 Highly purified porcine NPH insulin LeoRetard RI (Nordisk) and the highly purified porcine lente-like insulin preparation Monotard (Novo) are of very lowimmunogenicity. Antibody formation against proinsulin,12

glucagon, VIP, PP, and probably somatostatin can beavoided.13 Antibodies against protamine have not been de-scribed, but in a few patients with insulin allergy, positivecutaneous reactions were seen against highly purified insulinas well as protamine.14 It is not known whether the insulinpreparation plays a role in the development of late diabeticcomplications; probably this cannot be substantiated.

From Steno Memorial Hospital, DK-2820, Gentofte, Copenha-gen, Denmark.

REFERENCES1 Hagedorn, H. C , Jensen, B. N., Krarup, N. B., and Wod-

strup, I.: Protamine insulinate. JAMA 106: 177-80, 1936.

2 Krayenbiihl, C., and Rosenberg, T.: Crystalline protamine in-sulin. Rep. Steno Hosp. (Kbh.) 1: 60-73, 1946.

3 Hallas-Mtfller, K., Jersild, M., Petersen, K., and Schlichtkrull,J.: Zinc insulin preparations for single daily injection. JAMA 150:1667-71, 1952.

4 Schlichtkrull, J., Pingel, M., Heding, L. G., Brange, J., andJ^rgensen, K. H.: Insulin preparations with prolonged effect. In In-sulin II. Hasselblatt, A., and Bruchhausen, F. v., Eds. Berlin,Springer-Verlag, 1975, pp. 729-77.

5 Deckert, T., Andersen, O. O., and Poulsen, J. E.: The clinicalsignificance of highly purified pig-insulin preparations. Diabetologia10: 703-08, 1974.

6K«ilendorf, K., Aaby, P., Westergaard, S., and Deckert, T.:Resorption, effect and side effects of highly purified porcine NPH-insulin preparations (Leo®). Eur. J. Pharmacol. In press.

7 Binder, Chr.: Absorption of Injected Insulin. Copenhagen,Munksgaard, 1969.

8 Faber, O. K., Lauritzen, T., Binder, Chr., Mouridsen, H. T.,and V^lund, Aa.: Comparison of absorption and clinical effects ofInsulin Monotard® and Insulin Novo Lente®. Ugeskr. Laeg. 137:2510-14, 1975.

9 Rasmussen, S. M., Heding, L. G., Parbst, E., and V«ilund,Aa.: Serum IRI in insulin-treated diabetics during a 24/hour period.Diabetologia JJ: 151-58, 1975.

10 Schlichtkrull, J.: The absorption of insulin. Acta Paediatr.Scand. [Suppl.] 270: 97-102, 1977.

11 Chance, R. E., Root, M. A., and Galloway, J. A.: The im-munogenicity of insulin preparations. Acta Endocrinol. (Kbh.)Suppl. 205: 185-99, 1976.

12 Falling, I., Jerwell, J., Aagenaes, 0., and Aarseth, S.: Anti-bodies to insulin and proinsulin, metabolic control, and insulindose, in diabetics changing to highly purified insulins. Diabetologia12: 390, 1976.

13 Bloom, S. R., West, A. M., Polak, J. M., Barnes, A. J., andAdrian, T. E.: Hormonal Contaminants of Insulin from Gut Hor-mones. Bloom, S. R., Ed. Edinburgh, Churchill Livingstone, 1978,pp. 318-22.

14 Rosenthal, A.: Genetic control of insulin antibody formation.Juvenile Diabetes Foundation International Workshop on Insulin.New York, 1978.

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