Levemir; NN304; B29Lys-myristoyl des-B30 human insulin; B29Lys(epsilon-tetradecanoyl), des-B30 human insulin, recombinant
Status: approved and marketed
Organizations involved:
Novo Nordisk A/S: Manuf.; R&D; Tech.; Intl. mark.
Novo Nordisk Pharm. Industries, Inc. – USA mark.
Washington Research Foundation (WRF) – Tech.
Genentech, Inc. – Tech.
University of Washington – Tech.
Cross ref.: See the Insulin Products entry (#630).
Description: Levemir is a formulation of insulin detemir, a long-acting, fatty acid acylated, neutral, soluble, recombinant insulin analog manufactured from a Saccharomyces cerevisiae (yeast) expressed human insulin precursor with further chemical enzymatic modification. The lysine at position 29 of the insulin B-chain, which is not required for activity, is covalently bound to a 14-carbon (myristoyl) fatty acid chain, and the threonine (Thr) at position 30 (B30) of the insulin B-chain is removed. The epsilon-amino group of the side chain of B29 Lys is linked by a peptide bond to the myristoyl fatty acid. The fatty acid on residue B29 Lys binds irreversibly to circulating human albumin, with this complex resulting in slower metabolism of the molecule (desired). Removal of B30 Thr results in the negative charge region of the C-terminal being closer to the side chain, allowing the molecule to better mimic a nonesterified fatty acid. Insulin detemir has a molecular mass 5916.9 Da, appears as a white to whitish crystalline powder, and is soluble in water.
Levemir is packaged in 3 mL penfill Type I glass cartridges with a plunger (bromobutyl) and a stopper (bromobutyl/polyisoprene) for subcutaneous injection. Cartridges are packed in cartons of 1, 5 and 10 cartridges (although not all carton sizes may be marketed). Cartridges contain 100 U/ml solution for injection, with each 1 mL containing 100 U insulin detemir (300 U/3 mL). One unit of insulin detemir contains 0.142 mg salt-free anhydrous insulin detemir. One unit (U) of insulin detemir corresponds to one IU of human insulin. Excipients in Levemir are mannitol, phenol, metacresol, zinc acetate dihydrate, disodium phosphate dihydrate, sodium chloride, hydrochloric acid 2N (pH adjustment), sodium hydroxide 2N (pH adjustment), and Water for Injection. Mannitol is used to modify tonicity, sodium chloride as a chemical stabilizer, disodium phosphate dihydrate as a pH buffering agent, zinc (as zinc acetate) as a physical stabilizer and phenol and metacresol as antimicrobial preservatives. The shelf life is 2 years stored at 2-8°C (refrigerated). Levemir Penfill is designed to be used with Novo Nordisk delivery systems and NovoFine needles.
In March 2009, Novo Nordisk launched a redesigned FlexPen autoinjector in the U.S. This allows diabetes patients to use less force when pushing the button to inject their insulin and easily identify their insulin with prominent color branding. FlexPen is only available with Novo Nordisk insulin products, Levemir (insulin detemir [rDNA origin] injection), NovoLog (insulin aspart [rDNA origin] injection) and NovoLog Mix 70/30 (70% insulin aspart protamine suspension and 30% insulin aspart injection, [rDNA origin]). Similar to the original FlexPen, the improved FlexPen allows users to set an appropriate dose, and does not allow a dose larger than the amount of insulin remaining in the pen. It also permits dose corrections without loss of insulin, and provides doses in one-unit increments up to 60 units per injection (each pen contains 300 units in total). The dose scale of the improved FlexPen automatically returns to zero after each injection to allow visual confirmation of dose delivery. Like the original FlexPen, once in use, FlexPen does not require refrigeration (below 86˚F) for up to 42 days if used with Levemir.
Insulin detemir was the first protein pharmaceutical with an engineered fatty acid linkage. It was designed to provide an improved basal insulin profile which is smoother, longer-acting, and with less day-to-day variation than conventional NPH insulin preparations. Insulin detemir’s metabolic action is prolonged by binding of its myristoyl fatty acid residue to albumin in the blood and peripheral tissues. The lipid side chain at the end of the B-chain neither disrupts the aggregation properties (important to stabilize the molecule, since insulins are generally more stable as hexamers/crystals), nor the molecule’s insulin activity (which is identical to that of human insulin). The binding capacity of human albumin for insulin detemir exceeds five monomers. Unlike other forms of insulin, insulin detemir is not solid by nature and does not stimulate macrophage attack at the site of injection. Currently available basal insulins produce a variable response in controlling blood glucose. For example, NPH insulins must be re-suspended before use, resulting in variations in the amount injected. Insulin detemir has a unique mode of prolonged action (protraction), resulting in more consistent insulin levels and more predictable day-to-day control of blood glucose levels.
Biological.: The purpose of the molecular changes in insulin detemir is to achieve a high affinity for human albumin as a way of protracting insulin’s biological effect, providing a long acting soluble insulin product.
Nomenclature: insulin detemir [BIO USAN INN]; Levimir [TR in Europe]; 29B-(N6-myristoyl-L-lysine)-30B-de-L-threonineinsulin (human) [CAS]; LysB29 (N--tetradecanoyl)des(B30) insulin human [CAS]; 169148-63-4 [CAS RN]; B29Lys-myristoyl des-B30 human insulin [SY]; B29Lys(epsilon-tetradecanoyl), desB30 human insulin [SY]; NN304 [SY]
Companies.: Novo Nordisk A/S developed and manufactures NovoLog products. Novo Nordisk Pharmaceuticals, Inc. markets NovoLog products in the U.S.
Manufacture: As described in “Synthesis of the Soluble, Long-Acting Insulin NN304,” J. Markussen, et al., Abstract 805, Amer. Diabetes Assoc. 56th Annual Meeting, June 1996, LysB29-tetra-decanoyl, des-B30 human insulin is manufactured in three steps, starting with a modified recombinant insulin precursor expressed by Saccharomyces cerevisiae (yeast). Note, this information was provided by Novo Nordisk after asking for public information about the manufacturing process, but this process differs from that officially reported as the final commercial process by European Union regulators (see below). This precursor has an extension (Ext-Lys-Ala-Pro-Arg) in the N-terminal of the insulin B-chain with a bridge (Ala-Ala-Ala) connecting LsyB29 to GlyA1. The first step is specific hydrolysis using immobilized lysyl endopeptidase, with Ext-Lys cleaved off the N-terminal and the bridge opened between LysB29 and AlaA-3. The 2nd step is non-aqueous acylation using three equivalents of tetradecanoyl-N-hydroxysuccinimide ester, which reacts with the N-terminal amino acids groups of AlaB-3, AlaA-3, and the epsilon-amino group of LysB39. Finally, the N-terminals, PheB1 and GlyA1, of the two chains, are deprotected (removed) by hydrolysis using immobilized trypsin.
The European Product Assessment Report (EPAR) reports that insulin detemir is produced using Saccharomyces cerevisiae. transformed with a plasmid based on the yeast 2µ plasmid. Plasmid stability is monitored as an in-process control during fermentation. Segregational stability is also routinely monitored by the in-proces controls for insulin precursor positive phenotype in samples from production fermentations. The cell bank system used consists of Original Mother Culture (OMC), New Mother Culture (NMC), Master Cell Bank (MCB) and Working Cell Bank (WCB). The initial stages of production, i.e. the fermentation and recovery steps, are common to Novo Nordisk’s currently licensed (unmodified) human insulins until the purification step. The insulin precursor is produced and drawn continuously. In the recovery stage, the continuous process changes into a batch process. Agar medium is inoculated with yeast cells from a WCB vial. Subsequently, the yeast is transferred to the seed fermenter. When vigorous growth has developed, this culture is transferred aseptically to the main fermenter. The insulin precursor is concentrated by cation exchange chromatography followed by two crystallisation steps. The crystallised product is stored at -18°C until further processing. The insulin human precursor (insulin B1-B29-Ala-Ala-Lys-insulin A1-A21) is passed through a reactor with immobilized enzyme [apparently, Achromobacter lyticus protease (ALP)]. This is followed by chromatographic steps to reduce product related impurities. The myristoyl group (CH3(CH2)12CO-) is introduced by acylation onto the epsilon-amino group on the side chain of lysine in position B29 of the DesB30-insulin, forming insulin detemir. This is followed by chromatographic steps and precipitation. The active substance is crystallised and dried. Insulin detemir 100 U/ml is manufactured by mixing a solution with neutral pH containing all excipients except zinc and a weak alkaline solution containing insulin detemir and zinc acetate. This bulk solution is then filtered (0.22 µm) into a stainless steel filling tank. The formulated bulk is filled into the final containers in a class A environment.
Three major degradation products have been identified as product related substances. Of these, only B3-desamido insulin detemir is formed in significant amounts during storage at recommended conditions (0.9% after 18 months at 2-8°C). Three components in the storage medium for the WCB are from bovine origin, i.e. peptone, beef extract and pepticase. Peptone is also used in the propagation of the MCB. In the production of pepticase, two secondary raw materials of animal origin are used; these are bovine derived lactose and a material of porcine origin. Two additional raw materials of bovine origin, amicase and lactose are used in the manufacture of the enzyme Achromobacter lyticus protease (ALP). Both are produced from bovine milk from health animals. Validation studies on virus removal/inactivation were not performed on the basis that viruses are unable to replicate in the yeast cell line, and that the production processes for all materials include steps that are considered virus inactivating – high temperature treatment (all materials) and high pH treatment (peptone).
During early development (as described in the EPAR), insulin detemir was produced from a dedicated insulin detemir precursor that was expressed and secreted from yeast during the fermentation process as a single chained structure with disulfide bridges. This process was used for the production of the active substance for early toxicity studies and early Phase I and II trials. As drug development progressed, a new and more specific acylation method was developed and a production strategy was adopted in which the insulin human precursor, insulin B1-B29-Ala-Ala-Lys-insulin A1-A21, is used as the starting material. This process was used for the production of the active substance for pre-clinical (long-term toxicity and reproduction studies), Phase I, Phase II and Phase III clinical trials and is the commercial production process. Two product-related substances have been identified in the insulin detemir active substance: the A21-desamido form and the B3-desamido form. Comparisons have confirmed that the structural and physico-chemical properties of active substance from the two processes are identical. Note, no pharmacokinetic/bioequivalence studies were conducted to show the comparability of the products of the two processes.
Three formulations of finished product were used in clinical trials (as decribed in the EPAR). Early clinical trials - Phase I and Phase II- were carried out with formulation A. Formulation B was used in later Phase I and Phase II trials, as well as early Phase III trials. Formulation C was used in late Phase I and Phase III trials and is the marketed formulation. The concentration of active substance was increased from 600 nmol/ml (formulation A) to 1,200 nmol/ml (formulation B) and finally 2,400 nmol/ml (formulation C) as clinical trials in human subjects with diabetes revealed a higher molar requirement of insulin detemir compared to human insulin to obtain the same glucose lowering effect. The facilities for production/packaging and quality control of the finished product are all located in Denmark.
Bench-scale synthesis of LysB29-tetra-decanoyl, des-B30 human insulin is reported in Biochem. J., vol. 312, p. 725-31, 1995. See 1XDA, Brookhaven Protein Data Bank, for crystal structure coordinates.
FDA class: Drug NDA
Approvals: Date = 20050616; original NDA (no. 021536); Indication = for the treatment of adults with diabetes mellitus (type 1 and type 2)
Date = 20051020; NDA supplement; Indication = approval for use in childrens
Date = 20120512; NDA supplement; Indication = for use in children ages two to five years with type 1 diabetes
Indications: [Full text of the "INDICATIONS AND USAGE" section of the product insert/labeling, 6/2012]:
Status: On Dec. 9, 2002, Novo Nordisk A/S filed an NDA for approval of insulin detemir for the treatment of diabetes mellitus. On Oct. 8, 2003, FDA issued an approvable letter that cited certain clinical issues and requested additional information. Novo Nordisk had hoped to launch the product in the first half of 2004. FDA approval has not yet been granted, with the application apparently on hold, e.g., awaiting trial results, rejected or abandoned.
A Marketing Authorization Application (MAA) for European Union (EU) approval was filed on Nov. 5, 2002 for use in patients with both type 1 and type 2 diabetes. The EU approved Levimir on June 1, 2004. On April 4, 2005, the EU granted supplemental approval to extend the approved indications to include treatment of diabetes in children and adolescents 6 to 17 years of age. With this, Novo Nordisk became the first company in the European Union to offer both a long-acting insulin analog (Levemir; see related entry) and a rapid-acting insulin analog (NovoRapid) for children with diabetes.
Levemir was launched in the U.S. in March 2006.
In March 2007, the European Union granted supplemental approval of Levemir for once-daily use in combination with oral anti-diabetes drugs. This provided a new treatment option for people with Type 2 diabetes having difficulties in maintaining acceptable blood glucose levels using oral diabetic drugs alone. This approval was supported by the PREDICTIVE trial.
In Oct. 2007, Levemir received approval in Japan for type 1 and 2 diabetes. As of Oct. 2007, it was marketed in 58 countries worldwide.
With its May 2012 supplemental approval, Levemir became available for type 1 diabetes patients from age two through adulthood and adult patients with type 2 diabetes.
Tech. transfer: The U.S. product insert cites, "6,582,404; 6,004,297; 6,235,004, and other patents pending." U.S. 6,582,404, "Dose setting limiter," concerns cartridge delivery devices. U.S. 6,004,297 and 6,235,004, "Injection syringe," concern dose setting mechanisms for syringes. These patents are assigned to Novo Nordisk.
The Orange Book cites 5,750,497, "Acylated insulin," expiring May 16, 2019; 5866538, including claims for insulin detemir; 5,866,538, "Insulin preparations containing NaCl," expiring Jun 20, 2017, claiming formulations; 6,011,007 and 6869930, "Acylated insulin, expiring Feb 2, 2014, both expiring Feb 2, 2014. "Exclusivity Expiration" is cited as Apr 6, 2015.
Trials: In its pivotal trials, the safety and efficacy of Levemir given once or twice daily was compared to NPH human insulin or insulin glargine in controlled clinical studies involving a total of 6,004 patients with diabetes (3,724 with type 1, and 2,280 with type 2). In one study, a treat-to-target efficacy study of 475 patients using Levemir or NPH insulins, 70% of those treated with Levemir achieved an A1C of approximately 6.6%, meeting the target level recommended by the American Diabetes Association. There was no significant difference between the two treatment arms. In other studies, Levemir achieved a level of glycemic control similar to that provided by other basal insulins, as measured by A1C.(2)
Clinical studies have shown that insulin detemir provides more predictable and consistent control of blood glucose levels, lower fasting plasma glucose levels, and a lower risk of nocturnal hypoglycemia in insulin-requiring diabetics, compared to NPH insulin, l. Hemoglobin A1C levels were similar for patients receiving insulin detemir and NPH insulin, and insulin detemir was associated with less weight gain.
Levemir has been shown to reduce fasting blood glucose and the risk of hypoglycaemia, especially at night-time. In addition, studies have shown that people using Levemir do not experience the undesirable weight gain often associated with conventional insulin preparations.
In June 2006, results were reported from the German cohort (subset) of the PREDICTIVE (Predictable Results and Experience in Diabetes through Intensification and Control to Target: An International Variability Evaluation) trial in 10,276 diabetic patients showing that Levemir improved blood sugar control and reduced episodes of major hypoglycemia with no weight gain in actual clinical practice. The primary endpoint was safety. Levemir is the first insulin to show less weight gain versus other basal insulins (in 12 of 12 controlled clinical trials). An analysis of a sub-group of type 2 diabetes patients (n=511) focusing on patients who switched from insulin NPH or glargine to Levemir showed that these patients experienced significant improvement in A1C, and reduced episodes of major hypoglycemia, with both patient groups also experienced weight reduction.
In Sept. 2007, new data from the PREDICTIVE trial that the reduced weight gain seen with Levemir can be sustained over a period of two years. A total of 497 men and women were randomised to Levemir (331) or NPH (166). The weight advantages observed in a succession of clinical trials with Levemir were further validated by a sub-group analysis of the PREDICTIVE 303 study, which found that diabetes patients switched to Levemir from standard NPH insulin or insulin glargine (Lantus) actually lost weight. Levemir’s ability to achieve blood glucose targets without the degree of weight gain experienced by diabetics taking – and especially starting on – other basal insulins has been a key differentiator for the Novo Nordisk brand, most of all in relation to its more established long-acting rival Lantus. While 13 out of 13 published clinical trials have shown a weight benefit with Levemir versus other basal insulins, this 24-month study in Type 1 diabetes was the first long-term comparison of a modern basal insulin with the commonly used NPH insulin.
Medical: Levemir is a long-acting insulin analogue used as basal insulin, in combination with meal-related short- or rapid acting insulin. Dosage of Levemir should be adjusted individually. Levemir should be administered once or twice daily depending on patients’ needs. For patients who require twice daily dosing to optimise blood glucose control, the evening dose can be administered in the evening or at bedtime. Levemir is administered subcutaneously by injection in the thigh, abdominal wall, or the upper arm. The time action profile of insulin detemir is statistically significantly less variable than for NPH insulin.
Market: Novo Nordisk reported in Jan. 2005 that Levemir holds 9% of the market for long-acting insulin analogs, less than a year after its introduction in its first market. Total 2006 worldwide sales for NovoLog and Levemir products combined were ~$1.908 billion. The author’s rough/crude guess for total 2006 sales of Levemir is $400 million.
In June 2005, with Levemir’s approval, Novo Nordisk reported, “Levemir has already been approved for use in 37 countries worldwide, including countries throughout Europe. As of January 2005, approximately 88,075 patients have been treated with Levemir.”
The 2007 Average Wholesale Price (AWP) is $167.48/five 3 mL cartridges (Red Book, 2007).
The U.K. National Health Service (NHS) cost for five pre-filled Levimir FlexPen devices (5 x 300 units) is £39 (as of 11/2004; U.S. $68.34 in 4/2006). The price in Australia for five pre-filled Levimir FlexPen devices (5 x 300 units) was reported to be about $159 (in 2004) (U.S. ~$116 in 4/2006).
In Jan. 2013, In Denmark, in a reassessment of reimbursement grant to diabetes drugs, the Reimbursement Committee moved the analog insulins Lantus and Levemir from general to limited reimbursement. In addition, the Board made a distinction between products, and recommended that patients must first be treated with Sanofi's Lantus, and only if that does not work, they can be treated with Novo Nordisk's Levemir. Overall, this does not bode well for the next generation insulins from Novo Nordisk , Lilly and Sanofi which are expected to reach the market over the next few years.
This new differentiation was made solely on the basis of the price differential between the two products. Both Lantus and Levemir are considered to have the same efficacy and safety profile, but there is a significant price difference.
Companies involvement:
Full monograph
196 Insulin detemir, rDNA
LEVEMIR is indicated to improve glycemic control in adults and
children with diabetes mellitus.
Important Limitations of Use:
• LEVEMIR® is not recommended for the treatment of diabetic
ketoacidosis. Intravenous rapid-acting or short-acting insulin is
the preferred treatment for this condition.
Nomenclature:
insulin detemir, rDNA [BIO USAN INN]
Levemir [TR]
29B-(N6-Myristoyl-L-lysine)-30B-de-L-threonineinsulin
(human) [CAS]
LysB29 (N.epsilon-tetradecanoyl)des(B30) insulin human [CAS]
B29Lys-myristoyl.des-B30 human insulin [SY]
B29Lys(epsilon-tetradecanoyl), desB30 human insulin [SY]
insulin, B29Lys-myristoyl.des-B30, recombinant [SY]
NN304 [SY]
molecular weight (kDa) = 6 [50 a.a. polypeptide]
FDA Class: NDA Drug
Year of approval (FDA) = 2005
Date of 1st FDA approval = 20050617
(in format YYYYMMDD)
Biosimilars/biobetters-related U.S. Patents: | 2016, based on extension of 5,750,497) |
U.S. Patent Expiration Year: | 2016 |
U.S. Biosimilars Data Exclusivity Expiration: | 2017 |
U.S. Biosimilars Orphan Exclusivity Expiration: | 2012 |
U.S. Biosimilars Launchability Year: | 2017 |
U.S. Biobetters Launchability Year: | 2016 |
Biosimilars/biobetters-related EU Patents: | 2014, based on EP 0792290 |
EU Patent Expiration Year: | 2014 |
EU Biosimilars Data Exclusivity Expiration: | 2014 |
EU Biosimilars Orphan Exclusivity Expiration: | 2014 |
EU Biosimilars Launchability Year: | 2014 |
EU Biobetters Launchability Year: | 2014 |
Index Terms:
biopharmaceutical products
bovine materials used<!-- bovinesource -->
exempt from CBER lot release requirements
hormones
insulin, recombinant human
porcine plasma
recombinant DNA
yeast source materials
Bean strain, Clostridium botulinum
pepsin digestion
peptides, synthetic
Saccharomyces cerevisiae (yeast)
XV2181, Saccharomyces cerevisiae (yeast) strain
acetylglucosamidase
autologous cells, human
cresol, meta-
disodium phosphate
hydrochloric acid (HCl)
inosine
Lysine-Biogel chromatography matrix
mannitol
phenol
protamine
sodium chloride
sodium hydroxide
tetracycline
trypsin digestion
Water for Injection
zinc acetate
approval dates uncertain (FDA reports erroneous, conflicting, or simply has lost the original approval dates) (FDAapproved)
EU200 Currently Approved in EU
UM001 Marketed Product in US
US200 Currently Approved in US
EM001 Marketed Product in EU
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