Status: approved; marketed

Organizations involved:

Berlex Laboratories, Inc. – USA mark.

Bayer Schering Pharma AG – Parent

Pharmion GmbH – Intl. mark.

Sanofi Aventis S.A. – Manuf.; R&D; Tech.

Roussel UCLAF – Former

Behringwerke AG – Former

Hoechst Marion Roussel Deutschland GmbH – Former

University of Washington – Tech

Genentech, Inc. – Tech

Washington Research Foundation (WRF) – Tech.

Cross ref.: See the entries for Hirudin Products (#180) and Hirudin, rDNA/Sanofi (desirudin; #182). See also the entry for bivalirudin (Angiomax), a fully synthetic hirudin peptide.

Description: Lepirudin (rDNA) or Refludan is a lyophilized (freeze-dried) formulation of a recombinant mutein (modified sequence) of hirudin protein produced by a transformed Saccharomyces cerevisiae (yeast) cell line. Lepirudin is identical to natural leech-derived hirudin, except for substitution of a leucine for isoleucine at the N-terminal end of the molecule, and the removal of a sulfate group on the tyrosine at position 63. Lepirudin or [Leu1-Thr2]-63-desulfohirudin is a polypeptide of 65 amino acids with a molecular weight of 6,979.5 Daltons (~7 kDa) and molecular formula of C287-H440-N80-O111-S6. Both leech-derived hirudin and lepirudin have potent coagulation/clotting inhibition activity based on their ability to bind thrombin and neutralize its activity.

The activity of lepirudin is measured in a chromogenic assay. One antithrombin unit (ATU) of lepirudin is required to neutralize one unit of World Health Organization (WHO) preparation 89/588 of thrombin. Approximately 16,000 ATU equals 1 mg of lepirudin (i.e., the specific activity of lepirudin is ~16,000 ATU/mg of protein).

Refludan is packaged in vials as a sterile powder for reconstitution and administration by intravenous infusion. Each vial contains 50 mg lepirudin, 40 mg mannitol, and sodium hydroxide to adjust the pH (7) of the solution. Lepirudin is freely soluble and readily reconstituted in Water for Injection USP or 0.9% Sodium Chloride Injection USP. The product is supplied in boxes containing 10 vials. Unopened vials are stored at 2-25˚C (36-77˚F; room temperature), with a shelf life of 24 months.

Nomenclature: Hirudin, rDNA/Berlex [BIO]; Refludan [TR]; lepirudin (rDNA) [FDA]; lepirudin [INN; USAN]; Revasc [TR foreign]; hirudin (Hirudo medicinalis isoform HV1), 1-L-leucine-2-L-threonine-63-desulfo- [CAS]; 138068-37-8 [CAS RN]; HBW 023 [SY]; Hoe-023 [SY]; [Leu1-Thr2]-63-desulfohirudin [SY]; NDC 0088-2150-57 [NDC]

Note, some sources have mixed up nomenclature terms for lepirudin (Refludan) and desirudin (see entry above).

Companies.: Refludan was originally developed by Roussel UCLAF, Hoechst Marion Roussel S.A. (HMR), which merged in late 1999 with Rhone-Poulenc S.A. to form Aventis Pharma, which merged into Sanofi Aventis S.A. in late 2004. Lepirudin was originally manufactured jointly by Roussel UCLAF (part of Rhone-Poulenc) and Hoechst Marion Roussel Deutchland GmbH (Marburg, Germany). Both of these companies merged into Aventis Pharm, now Sanofi Aventis S.A., which continues to manufacture Refludan. Refludan was originally marketed in the U.S. by Hoechst Marion Roussel (Kansas City, MO) and internationally by HMR

As a condition of the Hoechst/Rhone-Poulenc merger to form Aventis Pharma, the companies entered into a “Consent Agreement” with the Federal Trade Commission (FTC) in which it was agreed that Aventis would divest lepirudin. This action was taken to prevent Aventis from controlling marketing of both lepirudin (which Sanofi Aventis continues to manufacture) and desirudin (which Sanofi Aventis currently markets worldwide).

In Oct. 2001, Schering AG (now Bayer Schering Pharma AG) acquired exclusive development and marketing rights to lepirudin (Refludan), which is now marketed through by its subsidiary, Berlex Laboratories, Inc. in the U.S. In June 2002, Schering AG (sub)licensed exclusive marketing and distribution rights for Refludan outside the U.S. and Canada to Pharmion GmbH, the German subsidiary of Pharmion Corp. Pharmion paid Schering $10.0 million and pays a royalty of 14% of net sales of Refludan and up to $7.5 million of contingent payments. Sanofi Aventis S.A. continues to manufacture Refludan for marketing by Berlex and Pharmion.

In Spring 2006, Bayer AG acquired Schering AG, as was renamed Bayer Schering Pharma AG.

Manufacture: Lepirudin is expressed in cultured yeast (Saccharomyces cerevisiae) cells using conventional recombinant technology. The hirudin polypeptide gene was originally derived from the leech, Hirudin medicinalis. The amino acid sequence was determined and used as a template for design of the lepirudin gene sequence, which was cloned into a plasmid. This lepirudin expression vector was transfected into a S. cerevisiae strain. The integrity of the expression vector has been confirmed by restriction enzyme mapping and nucleotide sequencing of the master and working seed lots. Transfected yeast cells share the same phenotype as the host strain and are genetically stable with no significant change in cell viability, plasmid retention, or lepirudin yield after five years of storage in liquid nitrogen.

Yeast culture (fermentation) is performed by Roussel UCLAF (Romainville, France), now merged into Sanofi Aventis S.A. Each fermentation run starts with one cryopreserved vial of yeast cells from the working cell bank. Fermentation is carried out in a closed system and includes four stages: preparation of the seed culture; pre-fermentation; fermentation; and harvesting of the yeast cells. Purification involves separation of yeast cells by filtration; inactivation of proteolytic enzymes; and multiple chromatography steps. The eluate is concentrated, acidified, and desalted using serial dia-/ultrafiltration steps. Bulk product is shipped to Sanofi Aventis Pharma Deutschland GmbH (Marburg, Germany), originally Behringwerke AG, for final purification by several chromatography steps, ultra-/diafiltration, and lot release.

Each production batch is tested for identity by reverse phase high pressure liquid chromatography (RP-HPLC), amino acid composition and peptide mapping; purity by clarity of solution, coloration and pH; amount of lepirudin-related proteins; yeast proteins, microbial contamination and bacterial endotoxins; and evaluation of potency by content of lepirudin, activity and specific activity. Other tests include dissolution time and content of excipients.

One example from U.S. patent 5,095,092 (see “Tech. transfer” section below) describes starting with 754 liters of S. cerevisiae culture filtrate containing a total of 27.2 g of protein, adsorption onto 75 kg of DIAION HP 20 chromatography matrix, elution with ten consecutive washes (700 L of 20 mM tris/HCl, pH 8.5, and 250 L of 0.1 M acetic acid), resulting in four fractions containing 22.6 g of protein, with an 83% purification yield.

FDA class: Drug NDA

Approvals: Date = 19980309; first approval, NDA; orphan designation (granted 2/13/1997; expires 3/2005)

Indications: [full text of "INDICATIONS AND USAGE” section from product insert/labeling]:

REFLUDAN is indicated for anticoagulation in patients with heparin-induced thrombocytopenia (HIT) and associated thromboembolic disease in order to prevent further thromboembolic complications.

Status: The NDA was completed on Dec. 31, 1996, granted priority review, and was approved on March 6, 1998; review time of ~14.1 months (~1.18 year). Refludan was launched in the U.S. on July 1, 1998.

Refludan received marketing approval in the European Union on March 13, 1997 for “treatment of heparin-associated thrombocytopenia (HAT) and thromboembolic disease mandating parenteral antithrombotic therapy.”

FDA issued a letter to Aventis Pharma in May 2000 rejecting the company’s supplemental application for anticoagulation in adults with acute coronary syndrome. The company subsequently withdrew its European Union application for this indication, which it had filed in July 1999.

Tech. transfer: There are no entries in the Orange Book (no unexpired relevant patents).

U.S. 5,180,668, "Hirudin derivative,"was extended to March 6, 2012.

U.S. patent 5,095,092, “Process for the isolation and purification of hirudin,” assigned to Hoechst AG, now Sanofi Aventis S.A., describes yeast expression of hirudin (defined to include lepirudin) at concentrations up to a several hundred milligrams/liter of culture filtrate. This involves use of a complex medium including yeast extract, cornsteep, and peptone or meat paste, resulting in difficulties in the purification of the protein. To resolve this, purification includes hydrophobic chromatography on porous adsorber resin copolymers of styrene and divinylbenzene, e.g., Amberlite XAD and DIAION High Porous Polymers from Rohm and Hass Co. Purification involves batch chromatography and column chromatography. The patent includes description of aspects of large-scale purification of rDNA hirudin/lepirudin. See also U.S. 5,180,668 (expired on Jan 19, 2010, according to the Orange Book) and EP 324712, both assigned to Hoechst AG.

U.S. patent 5,902,735, “Functional DNA block and plasmid coding for hirudin, transformed yeast, method for hirudin, hirudin obtained, and its pharmaceutical use,” issued May 11, 1999, assigned to Hoechst AG, concerns vectors for yeast expression of hirudin/lepirudin using an expression block to obtain a single, mature, correct protein. These vectors result in expression of a hirudin/lepirudin precursor which possesses only a single cleavage site for yscF, so that its maturation to hirudin leads to the secretion of only the desired form.

U.S. patent 5,866,399, “Process for inactivating carboxypeptidase Y in hirudin-containing culture broths,” Feb. 2, 1999, assigned to Hoechst AG, now Sanofi Aventis S.A., concerns methods for deactivating a yeast enzyme that can degrade hirudin and analogs during manufacture. S. cerevisiae strains used form the enzyme carboxypeptidase Y (CPY), a thermally unstable enzyme which non-specifically cleaves off various amino acids from the C-terminus of proteins including hirudin/lepirudin, particularly during chromatographic purification. Lepirudin has a free C terminus and is unprotected from enzymatic attack by CPY. The enzyme is inactivated by heating the culture broth to 80˚-100˚C in one minute or less.

Yeast expression system technology used for manufacture of lepirudin was developed jointly by the University of Washington and Genentech, and has been nonexclusively licensed to Berlex Labs., the U.S. subsidiary of Bayer Schering Pharma AG, by the Washington Research Foundation (WRF), the licensing agent for this family of yeast expression patents. These patents, particularly the more recent ones, broadly claim processes and materials for expression of proteins in yeast. Patents include U.S. 5,854,018 (issued April 8, 1997) and 5,856,123 (continuations of 5,618,676) concerning recombinant production of proteins in yeast expression systems including Saccharomyces, Kluyveromyces, Pichia and Hansenula. All three U.S. patents will expire in 2014. The new patents are broader, as they claim processes and materials for expression of proteins in recombinant yeast systems general.

EP 0324712, "Hirudin derivative," assigned to Hoechst AG, expired in 2009.

Note, with both lepirudin and desirudin manufactured by expression in yeast by Sanofi Aventis, it is likely that some recombinant manufacture-related patents related to these products originally assigned to the original developing companies, e.g., Hoechst or Novartis, have now been (cross-) licensed to Sanofi Aventis.

Disease: Heparin-induced thrombocytopenia (HIT) is a rare, allergy-like, adverse reaction to heparin, an anticoagulant (anti-blood-clotting) commonly used in a variety of clinical settings. HIT is caused by a complex immune mechanism and is characterized by a rapid and serious decline in the patient’s blood platelet count, leading to an increased risk of severe thromboembolic (blood clotting) complications, frequently resulting in limb amputation or even death. Prior to Refludan, the only treatment for HIT was to discontinue heparin.

Trials: The pivotal Phase III trials supporting approval were reported in Circulation, 99:73-80 and 100:587-593 (1999), with a meta-analysis of trials reported in Blood, 2000;96:846-851.

Medical: Refludan provides a treatment for HIT (HAT), a potentially life-threatening condition, by providing an effective anticoagulant treatment that facilitates rapid recovery of platelet counts with reduced risk of blood clots. In clinical trials, formation of anti-hirudin antibodies was observed in about 40% of HIT patients treated with Refludan. This may actually increase the anticoagulant effect of Refludan, possibly due to delayed renal elimination of active lepirudin-anti-hirudin complexes. Monitoring of clotting time (aPTT) is required during prolonged therapy.

Market: Refludan total sales were EUR 21 million in 2001 (<$21 million at that time). The North American market accounted for more than 70% of Refludan sales. Refludan sales in 1999 were $14 million, and $9 million in 1998. Neither ofcurrent the marketing companies report Refludan annual sales.

The author’s rough/crude guess for 2006 worldwide sales is in the range of ~$60-$100 million.

The 2007 Average Wholesale Price (AWP) is $1,910.40/vial ($1,582.44 in 2004) (Red Book, 2007). For comparison, the 2005 AWP was $1,732.80/vial, and $1,582.44 in 2004.