Diabetes Care

Volume 22 Supplement 2
Improving Prognosis in Type 1 Diabetes
Proceedings from an Official Satellite Symposium
of the 16th International Diabetes Federation Congress

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Long-Acting Insulin Analogs

Ralf H. Rosskamp, MD

Once daily injection of existing intermediate/long-acting insulin preparations does not provide a 24-h basal insulinemia in most patients. High variability, pronounced insulin peaks, and (as a result) a high risk of nocturnal hypoglycemia only poorly simulate normal physiology. One principle to prolong insulin action is the shift of the isoelectric point of insulin towards neutral. One example is HOE 901, which shows in healthy volunteers a constant peakless profile over the entire 24-h clamp period. In 4-week trials in comparison to NPH insulin, significant lower fasting plasma glucose levels were achieved with lower rates of nocturnal hypoglycemia. Another principle to prolong insulin action is the use of soluble fatty acid acylated insulins that are bound to albumin after absorption. The combination of long- and short-acting insulins might provide the tools towards the final goal of achieving sustained normoglycemia in diabetic patients.

Diabetes Care 22 (Suppl. 2):B109–B113, 1999

Initially, treatment with insulin consisted of subcutaneous injections shortly before meals (1). However, to avoid multiple injections, a number of attempts were made early on to prolong the duration of action by using substances to retard absorption, e.g., gum arabic (1923), lecithin (1923) or oil suspensions (1925) (2). These attempts failed mainly because of poor stability or side effects at the local injection site. The first successful insulin preparation with a prolonged action was protamine insulin (3). The principle of prolonging the duration of action of insulin was to make the insulin less soluble at the neutral pH of the tissue fluid by forming a stable complex of insulin with a basic compound. When injected as a suspension, the duration of action was roughly twice as long as that of regular unmodified insulin. In an attempt to find a substitute for protamine, which is made primarily from fish sperm, a synthetically produced substance, surfen, was discovered and commercially introduced (4). In contrast to crystalline NPH suspensions, the surfen preparations were clear acidic solutions. Another way to obtain protracted insulin preparations was the so-called lente insulins series, preformed amorphous or crystalline suspensions obtained by addition of small amounts of zinc ions (140–160 µg/ml) (5).

All of these attempts to prolong the duration of action of insulin in the past were directed to finding insulins able to cover the insulin requirements of a diabetic patient with one daily injection. Ironically, this resulted overall in less sufficient metabolic control than in the early days of insulin therapy using multiple short-acting insulin injections. Meanwhile, the link between metabolic control and the development of microvascular late complications has been firmly established (6) and has led to a renewed interest in long-acting insulins on the basis of a new therapeutic concept. This time, the objective is not avoidance of multiple injections but the imitation of the physiological secretion of insulin in healthy subjects while in a fasting state. Additional mealtime insulin requirements are taken care of by short-acting insulin bolus injections before meals. This basal–bolus concept is the cornerstone of any kind of intensified insulin treatment, which leads to a significantly better metabolic control than one or two insulin injections per day can provide.

DISADVANTAGES OF EXISTING INTERMEDIATE/LONG-ACTING INSULIN PREPARATIONS— Ultratard HM is the longest-acting human insulin, but its duration of action is too short in some patients to allow a once-daily injection (7,8). The most widely used intermediate-acting human insulin is NPH insulin, which also has to be administered twice daily in many cases to provide a 24-h basal insulinemia. However, because regular insulin has a duration of action of more than 8 h in some patients, three mealtime injections during the day can compensate for the insulin waning effect of a single NPH injection at night. With the introduction of the short-acting monomeric lispro insulin, the too-short duration of action of NPH insulin has become obvious. In a study in 66 type 1 diabetic patients treated with lispro insulin at mealtime, NPH insulin injections were based on blood glucose determinations before main meals. After 5 months, there was a 43% increase in the NPH dose, and the number of NPH injections increased from a mean of 1.4 to 3.1 per day compared with the previous regimen using regular insulin before meals (9).

The number of injections of NPH insulins might not be so important to achieve a constant 24-h basal insulinemia, apart from patient comfort and convenience. However, the action profile of NPH, with its peak action observed 5–7 h after injection (10), might be much more of a concern. This means that NPH injected at 10:00 p.m. would result in its maximum hypoglycemic action at 3:00–5:00 a.m., a time when insulin requirements are low. Indeed, nocturnal hypoglycemia occurs in a disproportionately high number, ~50%, of all hypoglycemia events in type 1 diabetic patients (11,12). A decrease in the nighttime NPH dose might overcome nocturnal hypoglycemia but as a consequence emphasizes the relative insulin deficiency between 5:00 and 8:00 a.m. at a time where insulin sensitivity decreases (13). The well-known overall effect of this NPH shortcoming is morning fasting hyperglycemia.  The diurnal blood glucose profile of diabetic patients with insulin therapy varies considerably and can be accounted for to a large extent by the high intrasubject and intersubject variability of absorption (14,15). The variability is more pronounced with intermediate- and long-acting insulin formulations (16,17) than with short-acting insulins, with an overall range of variation of 25–50% (18). One reason for the higher variability of absorption with long-acting insulins is that insulin suspensions have to be thoroughly mixed before injection. Otherwise, sedimentation of insulin crystals in the vials and inhomogeneities in the suspension occur, and different amounts of insulin get injected. Another reason might be that insulin injected as a suspension of crystals might be trapped in tissue pores, unable to diffuse toward a capillary vessel, and undergo degradation over time. Insulin solutions are much more likely to be distributed homogeneously at the injection site, and chances of availability at a capillary surface are much higher (19). It appears clear that large fluctuations in insulin absorption associated with the physical characteristics of the insulin product likely contribute to a large extent to the high frequency of nocturnal hypoglycemia. It can be speculated that a long-acting insulin in a clear solution might therefore have less variability of absorption from the injection site.

"Frosting" phenomenon is another problem, especially with NPH insulin formulations, as it results in a loss of insulin potency in the vial (20,21). "Frosting" is due to freezing or overheating of the vials, causing the insulin to precipitate on the walls of the vials, giving them a "frosty" appearance. Therefore, patients are required to check the homogenous milky appearance of their NPH insulin before injection.

An additional but rare problem can occur when protamine sulfate is given to neutralize the effect of heparin. Anaphylactic reactions have been reported in diabetic patients (22).

DEVELOPMENT OF LONG-ACTING INSULIN ANALOGS—To overcome the shortcomings of existing intermediate- and long-acting insulins, an alternative insulin should be able to allow once-daily dosing without a pronounced peak (ideally, square wave profile) and should be in a clear solution to reduce variability from the injection site. One common principle for prolongation of action is followed in the design of many insulin analogs: shifting the isoelectric point of human insulin from pH 5.4 toward more neutral by adding positively charged amino acids to make the insulin less soluble at the neutral pH of the injection site. With gene technology, changes can be applied within the insulin molecule itself, whereas with NPH an organic basic protein is added to the insulin formulation. In addition, such analogs can be administered as clear solutions at a slightly acidic pH instead of as preformed crystal suspensions. Therefore, the mechanism and morphology of subcutaneous depot formation and the mechanism of insulin delivery may be more similar to that of the so-called surfen insulins.

One of the analogs attempting to meet this target product profile was B27Arg, A21Gly, B30Thr-NH2 human insulin, also called NovoSol Basal. Using radioactive absorption technique, NovoSol Basal was shown to have less intra-individual variation than Ultratard HM but no significantly different inter-individual subject variation (23). NovoSol Basal disappeared from the injection site by first-order kinetics with a clearly longer T50 than did Ultratard HM. In a 1-month treatment period, the same fasting blood glucose could be reached with one daily injection of this new insulin analog plus three daily injections of regular insulin in comparison with Ultratard HM plus regular insulin. This effect was only reached by using a nearly two times higher dose of the new insulin analog (24). These results indicate that NovoSol Basal had reduced bioavailability, but no further data are available in literature.

B31Arg, B32Arg human insulin is the first example where shifting of the isoelectric point toward neutral resulted in a protracted duration of action (25). However, as with NovoSol Basal, bioavailability was prominent in the case of preformed crystals, but clear acidic solutions exhibited only a weak depot effect comparable to that of NPH. A further substitution of asparagine in position A21 with glycine led to HOE 901 (Fig. 1), an insulin analog being developed by Hoechst Marion Roussel. X-ray crystallography demonstrated that changes of the amino acid sequence of HOE 901 also altered the association properties of this insulin, making its hexamer structure more stable (26). In animal models, the addition of low amounts of zinc further prolonged the duration of action. Therefore, in the first human trials, different zinc amounts were employed.

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Figure 1Primary structure of HOE 901.

STUDIES WITH HOE 901 IN HEALTHY VOLUNTEERS— To address the biological potency of HOE 901, two formulations of HOE 901 with either 15 or 80 µg/ml zinc in a dose equimolar to human recombinant insulin were administered intravenously to nine healthy male volunteers and compared with intravenous injection of 5 IU of human insulin. The insulin (Fig. 2) and the blood glucose profile (Fig. 3) were not different among treatments. The results demonstrate the same biological activity of HOE 901 and human insulin.

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Figure 2Median exogenous serum insulin concentrations.


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Figure 3Median blood glucose concentrations.

A euglycemic clamp study was performed to address the characteristics of the action profile over 24 h after subcutaneous injection at the abdominal site (27). In a randomized, double-blind, crossover design, HOE 901 with either 15 or 80 µg/ml zinc was injected at a dose of 0.2 U/kg body weight in 12 healthy male subjects. To suppress exogenous insulin secretion, a concomitant somatostatin infusion was given. As a result, C-peptide secretion was suppressed during the entire clamp period. The median glucose infusion rate is depicted in Fig. 4. NPH shows its typical pattern, reaching a maximum effect after 5 h and from 10 h onward, gradually decreasing in activity. HOE 901 has a lag time of about 5 h and thereafter provides a constant peakless profile over the whole duration of the 24-h clamp.

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Figure 4Median glucose infusion rates.

Using 125I-labeled HOE 901 with 15 or 80 µg/ml zinc in 12 healthy male subjects, the disappearance rate from the injection site was compared with that of NPH insulin. Insulins were administered at a dose of 0.15 IU/kg, and external -counting was performed for 24 h (28). As can be seen from Fig. 5, the residual radioactivity of NPH insulin was significantly less after 24 h than that of the HOE 901 insulin formulations.

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Figure 5Median residual radioactivity.

STUDIES WITH HOE 901 IN DIABETIC PATIENTS— In a 4-day study, 12 type 1 diabetic patients using four injections of NPH insulin per day together with premeal regular insulins were switched over to a once-daily injection of HOE 901 at the same overall unit basis (29). The blood glucose profiles were similar those for four NPH injections per day, and no accumulation at the injection site occurred.

Phase II clinical trials with a treatment duration of 4 weeks have been completed in ~1,000 type 1 and 2 diabetic patients. The primary endpoint of these short-duration studies was fasting plasma glucose.

In type 1 diabetic subjects, basal insulin was supplied with either one injection of HOE 901 in the evening or with NPH insulin given once or twice daily. Regular insulin was injected before meals. Two studies revealed statistically significant lower fasting plasma glucose levels with HOE 901 treatment (30,31), which, in one study, translated into a significant HbA1 reduction versus NPH (–0.14%; P = 0.0299) (31). Interestingly, this was accompanied with comparable rates of hypoglycemia, even with a significantly lower rate of nocturnal hypoglycemia in one study (36 vs. 55%; P = 0.0037) (31). In type 2 diabetic subjects, HOE 901 was again given only once daily, whereas NPH was given either once or twice daily. One study in patients suboptimally managed (HbA1>7%) on a maximal dose of oral agents showed a significant improvement in fasting plasma glucose for either HOE 901 or NPH with no difference in hypoglycemic events (32). In another study in type 2 diabetic patients, both HOE 901 and NPH as an adjunct to oral therapy improved fasting plasma glucose and HbA1c to the same level, but there were significantly fewer hypoglycemic episodes on HOE 901 (7.3 vs. 19.1%; P< 0.037) (33). Considering that all type 2 diabetic patients in these two studies were new to insulin treatment, it is not surprising that overriding treatment effects within the first 4 weeks did not reveal differences between treatments, as was the case in type 1 diabetic patients already pretreated with insulin. In all studies, adverse events, including injection-site reactions, were not different between HOE 901 and NPH insulin treatment. Measurement of IgG insulin antibodies showed no difference between treatments, either.

SAFETY OF INSULIN ANALOGS— Modification of the insulin molecule may alter its binding kinetics at the insulin receptor level and at the structurally homologous IGF-I receptor (34). This may lead to an enhanced mistogenic effect, as has been demonstrated with ASP (B10) human insulin. As a result, suprapharmacological doses of ASP (B10) revealed a dose-dependent increase in the incidence of adenocarcinomas and fibroepithelial mammary tumors in female Sprague-Dawley rats in the course of a 12-month safety study (34). Before starting the Phase II trial program, a similar study was performed with HOE 901, and no carcinogenic effect was observed (D. Mayer, personal communication). This is in agreement with different in vitro characteristics of HOE 901 and ASP (B10): competition binding of HOE 901 for the IGF-I receptor shows a slightly higher affinity when compared with human insulin, but clearly less than that with ASP (B10) insulin. More important, the IGF-I receptor-mediated growth-promoting activity of HOE 901 in muscle cells is not different from that of native human insulin (35).

General safety concerns with insulin analogs include local and systemic reactions and immunogenicity (36). Preliminary results of 4-week treatment trials do not show any significant difference here between HOE 901 and human insulin.

SOLUBLE FATTY-ACID ACYLATED INSULINS— Albumin is a constituent of the subcutaneous tissue fluid and has a slow disappearance rate, making binding of insulin to albumin an attractive option to prolong insulin action. Albumin has binding sites for nonesterified fatty acids, which therefore became the preferred targets of coupling to insulin. Acylation with an aliphatic carbonic acid chain is usually performed in the side chain of B29Lys. Fatty-acid insulins in clinical development are WW99-S32 from Eli Lilly and NN304 from Novo Nordisk.

In pigs, a soluble solution of 125I-labeled NN304 showed a significantly prolonged disappearance compared with NPH insulin, and the respective glucose disposal rate was more steady (37). Under euglycemic clamp conditions, NN 304 showed a slightly longer duration of action than did NPH, without a peak (38).

In healthy volunteers, the acylated N-palmitoyl B29Lys human insulin showed a highly reproducible and linear pharmacokinetic profile consistent with an intermediate-acting insulin. However, only a 20–25% potency compared to NPH was observed, with a highly variable time-action profile (39). Appropriate human data with NN304 are not available in the literature at present.

With new long-acting insulin analogs on the horizon, the future treatment options for diabetic patients will be broadened. Clearly the combination of designer-tailored short-acting and long-acting insulin analogs could provide the tools for a more physiological insulin replacement that achieves near-normal metabolic control.

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From the Department of Metabolism and Endocrinology, HMR Global Clinical Research and Development, Hoechst Marion Roussel, Bridgewater, New Jersey.

Address correspondence and reprint requests to Ralf Rosskamp, MD, Vice President, Clinical Research, Metabolism/Endocrinology, HMR Global Clinical Research/Development, Hoechst Marion Roussel, Inc., Route 202-206, P.O. Box 6800, Bridgewater, NJ 08807-0800.

Received for publication 27 May 1998 and accepted in revised form 30 October 1998.

This article is based on a presentation at a satellite symposium of the 16th International Diabetes Federation Congress. The symposium and the publication of this article were made possible by educational grants from Hoechst Marion Roussel AG.

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