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WO1992001476A1 - Nouveaux systemes de liberation de medicaments pour proteines et peptides utilisant de l'albumine comme molecule porteuse - Google Patents

Nouveaux systemes de liberation de medicaments pour proteines et peptides utilisant de l'albumine comme molecule porteuse Download PDF

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Publication number
WO1992001476A1
WO1992001476A1 PCT/US1991/003889 US9103889W WO9201476A1 WO 1992001476 A1 WO1992001476 A1 WO 1992001476A1 US 9103889 W US9103889 W US 9103889W WO 9201476 A1 WO9201476 A1 WO 9201476A1
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WIPO (PCT)
Prior art keywords
protein
drug
albumin
bridging
fatty acid
Prior art date
Application number
PCT/US1991/003889
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English (en)
Inventor
Joseph A. Walder
John M. Dagle
Scott Fowler
Dee A. Casteel
Gerald D. Hurst
Original Assignee
University Of Iowa Research Foundation
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Publication of WO1992001476A1 publication Critical patent/WO1992001476A1/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/643—Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]

Definitions

  • This invention relates to a novel drug carrier system to enhance the efficacy of peptides and proteins used as therapeutic agents. Improvements in the chemical methods for peptide synthesis and the use of recombinant DNA techniques to produce larger polypeptides and proteins have greatly expanded the therapeutic uses of these drugs.
  • Representative applications include the use of superoxide dismutase (SOD) as an anti-inflammatory and anti-ischemic agent, the soluble CD4 protein for the treatment of AIDS, polypeptide hormones such as human insulin, gamma-interferon and other lympho ines for the treatment of infectious diseases and certain types of cancer, growth factors such as growth hormone and erythropoeitin which is useful in the treatment of anemia, and tissue plasminogen activator useful as a thrombolytic agent.
  • SOD superoxide dismutase
  • soluble CD4 protein for the treatment of AIDS
  • polypeptide hormones such as human insulin, gamma-interferon and other lympho ines for the treatment of infectious diseases and certain types of cancer
  • growth factors such as growth hormone and erythropoeitin which is useful in the treatment of anemia
  • tissue plasminogen activator useful as a thrombolytic agent.
  • proteins and peptides are rapidly metabolized or excreted in the urine.
  • the renal threshold for filtration of molecules dissolved in plasma is in the range of 50,000 to 60,000 daltons. Smaller molecules are very rapidly filtered from the circulation by the kidneys. For example, SOD with a molecular weight of only 32,000 daltons has a half-life in plasma of only about 6 minutes.
  • Hemoglobin normally a tetramer of two alpha and two beta polypeptide chains can dissociate into alpha-beta di ers which also have a molecular
  • the dimers are cleared from the circulation with a half-life on the order of minutes. As the concentration of dimers becomes depleted the equilibrium ⁇ , ⁇ 2 > 2 ⁇ (l) becomes progressively shifted to the right. The newly formed dimers are likewise excreted. This process continues until all of the hemoglobin is eliminated. The overall half-life of hemoglobin in the circulation is about 90 minutes. Not only does this compromise its function as a blood substitute, but the massive dumping of protein in the urine also poses the risk of renal injury.
  • hemoglobin and other proteins of therapeutic interest have been covalently cross-linked to a macromolecular carrier such as dextran or albumin, again using random cross-linking reagents like glutaraldehyde (Wong, J.T. (1988) Bio aterials, Artificial Cells, and Artificial Organs JL6, 237- 245; Poznansky, M.J. (1988) Methods in Enzymology 137, Academic Press, New York, pp. 566-574).
  • a third approach which has been taken to increase the effective size of polypeptide and protein drugs, and thereby prevent their rapid renal excretion, is to decorate the molecule with multiple copies of a lower molecular weight substance.
  • the extensive modification of the protein that results using all three of these methods invariably leads to a very heterogenous mixture of products.
  • the composition can be characterized only by certain average properties making batch to batch uniformity difficult to insure. Extensive modification of the protein or peptide can also lead to a decrease or loss of activity; and especially in the case of cross-linking agents, problems of antigenicity can arise.
  • this approach nearly 15 years ago.
  • the primary objective of the present invention is to fulfill that need.
  • a yet further objective of the present invention is to provide a drug carrier system for proteins and peptides in which such molecules are modified at a unique or a limited number of sites with an apolar (hydrophobic) substituent to serve as a bridging group to mediate binding of the drug noncovalently to albumin.
  • a yet further objective of the invention is to provide reagents which can be used to selectively modify peptides and proteins with fatty acid derivatives.
  • a yet still further objective of the present invention is to provide a means of regulating the activity of peptides and proteins used thera- Probeically by competition with free fatty acids for binding sites on albumin, resulting in displacement of the drug.
  • the protein is modified at a single or limited number of sites with a long chain fatty acid derivative. Novel protein modifying agents to attach such groups are provided.
  • a method for regulating the activity of such drugs based on competition for binding sites on albumin with free fatty acids is also presented.
  • Albumin is the most abundant plasma protein and has multiple binding sites for apolar molecules.
  • albumin is used as a carrier molecule for the transport of peptide and protein drugs within the circulation.
  • the invention is especially useful for polypeptides below a molecular weight of about 60,000 which normally would be rapidly excreted in the urine. Biding of the protein to albumin is mediated by an attached derivative. Long chain fatty acid derivatives are the preferred bridging groups to provide high binding affinity.
  • the molecular weight of the albumin is 66,000 daltons. Normally very little of the protein (less than 100 mg/day) is excreted in the urine. Albumin plays an important role physiologically in the transport of free fatty acids within the circula ⁇ tion. There are at least 10 sites on the protein at which fatty acids can bind. The two highest affinity binding sites are selective for fatty acids. The remaining binding sites can accommodate a variety of apolar molecules, particularly organic anions.
  • the association constant for the binding of long chain fatty acids such as palmitate (C, g ) and stearate (C lg ) at the high affinity sites is on the order of 10 M ⁇ . There are 3 to 5 secondary sites for which the binding constant is about 10 M ⁇ , still relatively large.
  • the binding energy is due primarily to hydrophobic interactions. A smaller, but significant contribution is due to electrostatic interactions between the carboxylate group and a surface lysine residue of the protein adjacent to the apolar binding pocket.
  • the carboxylate group can be replaced with other negatively charged substituents, including but not limited to, phosphates, phosphonates, sulfates and sulfonates without appreciably affecting the binding affinity.
  • the molar ratio of free fatty acids to albumin in the blood is typically about 0.6. At the highest plasma concentration of free fatty acids observed physiologically, which occurs during strenuous exercise, the molar ratio is 4. Thus under all conditions there are vacant binding sites on the protein with affinities for free fatty acids of at least 10 M ⁇ .
  • a variety of apolar molecules can be used as the bridging group to promote the binding of peptides and proteins to albumin.
  • Organic anions are preferred.
  • the most preferred bridging groups are long chain fatty acid derivatives.
  • the length of the hydrocarbon chain can vary from about 10 to about 24 carbon atoms, preferably 16 to 20 carbon atoms.
  • Carbon-carbon double bonds, such as in derivatives of oleic acid, carbon-carbon triple bonds, and heteroatoms uncharged at physiological pH, including but not limited to oxygen and sulfur, can also be incorporated into the chain.
  • a number of different negatively charged groups may be introduced into the molecule to enhance the binding affinity to albumin, including carboxylates, phosphates, phosphonates, sulfates and sulfonates.
  • Functional groups which may be incorporated into the reagent to provide a means of attachment to the protein include: (1) activated carboxylic acid derivatives such as anhydrides, N-hydroxy- succinimide esters, or phenylesters; (2) alkylating agents such as alkyl halides, alpha-halo carboxylic acids and amides or N-substituted maleimides; and (3) carbonyl groups, aldehydes and ketones, which may be used to link the compound to amino groups of the protein by reductive alkylation.
  • activated carboxylic acid derivatives such as anhydrides, N-hydroxy- succinimide esters, or phenylesters
  • alkylating agents such as alkyl halides, alpha-halo carboxylic acids and amides or N-substituted maleimides
  • carbonyl groups, aldehydes and ketones which may be used to link the compound to amino groups of the protein by reductive alkylation.
  • the attachment of the apolar group is restricted to a unique site or a limited number of positions on the protein or peptide. This makes it possible to produce a specific derivative having well defined and desirable properties, in contrast to the random mixture of reaction products which results from cross-linking proteins and peptides to macromolecu ⁇ lar carriers.
  • the reagent may be directed to a specific region of the molecule on the basis of noncovalent binding interactions at the site.
  • Example 2 illustrates the use of such an affinity reagent to modify hemoglobin with a long chain fatty acid derivative selectively at the 2,3- diphosphoglycerate binding pocket located at the interface between the beta chains.
  • the sites of modification may be restricted by using reagents that react at the SH group of cysteine residues.
  • Cysteine is uniquely reactive among the amino acid residues naturally found in proteins and can be readily targeted with a number of different alkylating agents such as alpha-halo carboxylic acids and amides and maleimide derivatives (see Lundblad and Noyes, 1984).
  • most proteins have only a limited number of free cysteine residues.
  • beta 93, beta 112 and alpha 104 (six in total per tetramer) .
  • Beta 112 and alpha 104 are buried inside the molecule; only cysteine beta 93 is reactive. In SOD there are four cysteinss per 32,000 molecular weight dimer; only the two cysteine 111 residues are reactive. In proteins in which a cysteine residue is absent, one may be readily introduced using recombinant DNA techniques. For shorter polypeptides, synthetic methods can be used to replace one of the native amino acids within the sequence with cysteine, or an additional cysteine residue can be added to the molecule. There are, of course, certain amino acid residues that are critical for the activity of any given protein which cannot be modified, but generally there are a number of positions at which amino acid substitutions can be made with relatively little effect on the properties of the molecule.
  • the negatively charged substituent is provided by a carboxylate group, in (II) by a phosphate group.
  • the long chain apolar group (X) which will insert into the hydrophobic binding pocket of albumin can vary in length from about 10 to about 24 atoms, preferably 16 to 20 atoms. Heteroatoms such as oxygen and sulfur, as well as other apolar functionalities may be incorporated into the chain.
  • the spacer arm (Y) through which the fatty acid derivative is attached to the protein or peptide can be a simple aliphatic chain, or it may incorporate polar or charged groups to enhance water solubility. Aromatic substituents may also be incorporated into the spacer arm.
  • the functional group (Z) provides the means of attachment of the reagent to the protein or peptide. It may be any of the reactive groups used to modify proteins described above. Syntheses of representative examples of these two classes of compounds is described in Example 1. Example 2 demonstrates the reaction of these reagents with hemoglobin.
  • an association constant of only 10 5 M-1 will ensure that over 99% of a fatty acid derivatized protein or peptide will be bound to albumin. Even the secondary binding sites have association constants of 10 M ⁇ for free fatty acids. Because the concentration of albumin will generally be in vast excess over the therapeutic level of the protein or peptide drug, a 1:1 complex between albumin and the drug molecule will be formed; i.e. there will be a negligible fraction of species in which two or more molecules of the drug are bound to the same molecule of albumin. One important exception to this is in the use of hemoglobin as a blood substitute.
  • the plasma concentration of hemoglobin is on the order of 1 to 2 mM.
  • the level of albumin is depleted due to the loss of blood. Consequently, higher molecular weight species in which there are two or more molecules of hemoglobin attached to a single molecule of albumin can be formed.
  • Protein and peptide drugs used in accordance with the present invention are generally administered parenterally. Co-administration of the drug with albumin may be employed but is usually not required. Binding of the modified protein or peptide occurs rapidly to the endogenous albumin within the circulation, or to albumin within the interstitial fluid if the drug is given subcuta- neously or intramuscularly. Replacement of albumin may become necessary in the treatment of severe hemorrhage when using fatty acid modified hemoglobin derivatives as blood substitutes.
  • Suitable pharmaceutical carriers for the modified protein and polypeptide drugs of the present invention include but are not limited to physiological saline, a mixture of glucose and saline or Ringer's lactate.
  • Proteins or peptides modified with apolar substituents may remain fully active while associated with albumin. Such is the case for SOD and hemoglobin derivatives, for example.
  • the free polypeptide may retain activity, in which case the complex with albumin would provide an inactive depot form of the drug. Since the vast majority of the protein is bound to albumin, this provides an effective reservoir to buffer the free concentration of the drug. As the free form of the drug becomes metabolized or excreted, the equilibrium in equation (2) shifts progressively to the right, i.e. the protein becomes released from albumin. This maintains the concentration of the free drug, and hence the level of activity, in a relatively narrow range between doses.
  • the present invention also affords a novel class of long acting insulin derivatives which bind to albumin by means of an attached apolar group. Competition with endogenous free fatty acids gives rise to particularly useful pharmacokinetic properties for these derivatives. Normally there is an excess of available binding sites on albumin so that the modified insulin is almost 100% in the bound form. In this state, the insulin is inactive. Regular insulin is given intermittently to maintain normal metabolic control. If the levels of insulin administered are inadequate, the concentration of free fatty acids in the blood increases. This displaces some of the insulin bound to albumin and ameliorates the insulin deficiency.
  • Such apolar modified insulin derivatives are particularly useful in preventing the complications of diabetic keto- acidosis, in which case the free fatty acid concentration can be as high as 3 to 4 mM.
  • This new compound was synthesized by the following 5 step sequence.
  • hexadecanol (2.42 g, Aldrich) was condensed with 2- cyanoethyl-N,N-diisopropylamino chlorophosphine (2.84 g, American Bionetics) in dry methylene chloride in the presence of 2.58 g of diiso- propylethylamine. The reaction was complete within 10 minutes. The mixture was poured into ethyl acetate and was washed first with saturated sodium bicarbonate, then with brine. The solution was then dried with sodium sulfate and the solvent was evaporated.
  • This new fatty acid derivative was synthesized according to the following reaction sequence.
  • First 8-thiooctanoic acid (6J was prepared by refluxing 8- bro ooctanoic acid (10.65 g) with thiourea (3.65 g) in ethanol (25 ml) for 7 hours. Ten ml of a 0.1 N NaOH solution was then added and the reaction mix ⁇ ture was further refluxed for 3.5 hours and then allowed to cool to room temperature and stand overnight. After addition of water, the solution was extracted with ethyl acetate then acidified with 10% sulfuric acid. The organic layer was then washed with brine, evaporated to give an oily residue which was then redissolved in hexanes.
  • Methyl 8-thiooctanoate (2) was prepared from the acid, , by esterification with excess ethereal diazomethane.
  • t-Butyl 2-bromohexadecanoate (j3) was synthesized from 2-bromohexadecanoic acid (11.4 g) in dry t-butanol (250 ml) in the presence of dicyclohexylcarbodiimide (7.5 g, added as a solution in 50 ml of tetrahydrofuran) and 4-pyrrolidinopyri- dine (200 mg) . The reaction mixture was allowed to stir for 2 hours at room temperature and then evaporated to dryness.
  • the title compound JL2_ was prepared by treating 11 with trifluoroacetic acid for 20 hours at room temperature to remove the t-butyl protecting group. After removing the trifluoroacetic acid under reduced pressure, the product was recrystallized from ethyl acetate. M.p. 101-102 ° C. 2- ( [8-(Carbo(3,5-dicarboxy)phenoxy)octyl]thio) hexadecanoic acid (15)
  • the title compound was prepared by the reaction of palmitoyl chloride (1.7 ml) with 5-hydroxyiso- phthalic acid (1 g) in dry tetrahydrofuran. Both starting materials were purchased from Aldrich. The reaction was allowed to proceed for 5 hours at room temperature after which the solvent was evaporated. The residue was acidified with an aqueous HCL solution containing 2.1 equivalents of acid at 0 C. The product precipatated as a white solid and was collected by filtration. After washing with cold water, it was dried under vacuum and recrystallized from benzene. M.p. 115-118"C.
  • the compounds were first dissolved in dimethylsulfoxide at a concentration of 100 mM and diluted 100-fold into the reaction mixture to give a final concentration of 1 mM.
  • the hemoglobin concentration during the reaction was 0.5 mM.
  • the products of the reaction were identified and quantitated by analytical isoelectric focusing (Chatterjee, et al., 1986). In all cases the isoelectric point of the modified hemoglobin was decreased. For 5_, V2. an ⁇ j ⁇ i this is due to the attached negatively charged group. With JL6 (and also lj>) the decrease in isoelectric point is due to the loss of the positive charge of the modified amino group.
  • IHP binds very tightly to deoxhemoglobin at the 2,3-diphospho- glycerate binding site, indicating that these compounds react with an amino group of the protein in this region, most probably Val 1-beta or lysine 82- beta.
  • the two negatively charged carboxylate groups are sufficient to direct these reagents to react with deoxyhemoglobin at the beta cleft.
  • the yield of the reaction can be further improved by incorporating additional negatively charged substituents into the aromatic ring.
  • the reactions of deoacyHbA with 5_ and deojcyHbXL99 with 35 were performed on a larger scale to isolate the fatty acid modified derivatives for plasma half-life studies. The reactions were carried out under the same conditions as described above with 2 to 4 g of hemoglobin.
  • the derivative modified with 35 was purified by chromatography over DEAE-Sepharose (Pharmacia). Using 0.2 M glycine pH 8.0 as the column buffer (see Chatterjee et al., 1986), the desired product was eluted with 0.1 M NaCl.
  • the product of the reaction of deoxyHbA with 5_ was isolated by preparative isoelectric focusing in a pH 6.7 to 7.7 gradient. Ampholines were obtained from Pharmacia.
  • HbXL99 and HbXL99 further modified with the fatty acid derivative 16 were compared.
  • the plasma half-life of HbXL99 was found to be 3.2 hours as reported earlier (Snyder, S.R., et al., 1987). Renal excretion of HbXL99 is almost completely blocked due to the intramolecular cross-link between the alpha chains.
  • the plasma half-life of the fatty acid modified derivative was increased about 2-fold to 6 hours.

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Abstract

L'invention se rapporte à un système de libération de médicaments pour protéines et peptides qui utilise de l'albumine comme molécule porteuse. Le médicament est lié par liaison non covalente à l'albumine au moyen d'un substituant apolaire attaché tel que de préférence un dérivé d'acides gras à chaÎne longue.
PCT/US1991/003889 1990-07-26 1991-06-03 Nouveaux systemes de liberation de medicaments pour proteines et peptides utilisant de l'albumine comme molecule porteuse WO1992001476A1 (fr)

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Cited By (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5631347A (en) * 1995-06-07 1997-05-20 Eli Lilly And Company Reducing gelation of a fatty acid-acylated protein
US5646242A (en) * 1994-11-17 1997-07-08 Eli Lilly And Company Selective acylation of epsilon-amino groups
US5693609A (en) * 1994-11-17 1997-12-02 Eli Lilly And Company Acylated insulin analogs
US5700904A (en) * 1995-06-07 1997-12-23 Eli Lilly And Company Preparation of an acylated protein powder
US5750497A (en) * 1993-09-17 1998-05-12 Novo Nordisk A/S Acylated insulin
US6251856B1 (en) 1995-03-17 2001-06-26 Novo Nordisk A/S Insulin derivatives
US6444641B1 (en) 1997-10-24 2002-09-03 Eli Lilly Company Fatty acid-acylated insulin analogs
WO2002066067A3 (fr) * 2001-02-16 2003-02-13 King S College London Nouveau systeme d'administration de medicaments
US6869930B1 (en) 1993-09-17 2005-03-22 Novo Nordisk A/S Acylated insulin
US7601691B2 (en) 1999-05-17 2009-10-13 Conjuchem Biotechnologies Inc. Anti-obesity agents
US7982018B2 (en) 2006-10-16 2011-07-19 Conjuchem, Llc Modified corticotropin releasing factor peptides and uses thereof
US8710001B2 (en) 2006-07-31 2014-04-29 Novo Nordisk A/S PEGylated, extended insulins
WO2014138253A1 (fr) * 2013-03-05 2014-09-12 Arizona Board Of Regents, Acting For And On Behalf Of Arizona State University Translocation d'un polymère à travers un nanopore
US9018161B2 (en) 2006-09-22 2015-04-28 Novo Nordisk A/S Protease resistant insulin analogues
US9061067B2 (en) 2004-07-08 2015-06-23 Novo Nordisk A/S Polypeptide protracting tags
US9260502B2 (en) 2008-03-14 2016-02-16 Novo Nordisk A/S Protease-stabilized insulin analogues
US9387176B2 (en) 2007-04-30 2016-07-12 Novo Nordisk A/S Method for drying a protein composition, a dried protein composition and a pharmaceutical composition comprising the dried protein
US9395352B2 (en) 2007-04-06 2016-07-19 Arizona Board Of Regents On Behalf Of Arizona State University Devices and methods for target molecule characterization
US9481721B2 (en) 2012-04-11 2016-11-01 Novo Nordisk A/S Insulin formulations
US9593372B2 (en) 2008-10-06 2017-03-14 Arizona Board Of Regents On Behalf Of Arizona State University Nanopore based sequencer
US9688737B2 (en) 2008-03-18 2017-06-27 Novo Nordisk A/S Protease stabilized acylated insulin analogues
US9896496B2 (en) 2013-10-07 2018-02-20 Novo Nordisk A/S Derivative of an insulin analogue
US10265385B2 (en) 2016-12-16 2019-04-23 Novo Nordisk A/S Insulin containing pharmaceutical compositions
US10288599B2 (en) 2012-10-10 2019-05-14 Arizona Board Of Regents On Behalf Of Arizona State University Systems and devices for molecule sensing and method of manufacturing thereof
US10336713B2 (en) 2014-02-27 2019-07-02 Arizona Board Of Regents, Acting For And On Behalf Of, Arizona State University Triazole-based reader molecules and methods for synthesizing and use thereof
US10589324B2 (en) 2010-12-14 2020-03-17 Sasol Technology (Pty) Limited Cleaning of process equipment

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Cited By (38)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5750497A (en) * 1993-09-17 1998-05-12 Novo Nordisk A/S Acylated insulin
US6869930B1 (en) 1993-09-17 2005-03-22 Novo Nordisk A/S Acylated insulin
USRE37971E1 (en) 1994-11-17 2003-01-28 Eli Lilly And Company Selective acylation of epsilon-amino groups
US5646242A (en) * 1994-11-17 1997-07-08 Eli Lilly And Company Selective acylation of epsilon-amino groups
US5693609A (en) * 1994-11-17 1997-12-02 Eli Lilly And Company Acylated insulin analogs
US5922675A (en) * 1994-11-17 1999-07-13 Eli Lilly And Company Acylated Insulin Analogs
US7229964B2 (en) 1995-03-17 2007-06-12 Novo Nordisk A/S Insulin derivatives
US6620780B2 (en) 1995-03-17 2003-09-16 Novo Nordisk A/S Insulin derivatives
US6251856B1 (en) 1995-03-17 2001-06-26 Novo Nordisk A/S Insulin derivatives
US5631347A (en) * 1995-06-07 1997-05-20 Eli Lilly And Company Reducing gelation of a fatty acid-acylated protein
US5700904A (en) * 1995-06-07 1997-12-23 Eli Lilly And Company Preparation of an acylated protein powder
US6444641B1 (en) 1997-10-24 2002-09-03 Eli Lilly Company Fatty acid-acylated insulin analogs
US7601691B2 (en) 1999-05-17 2009-10-13 Conjuchem Biotechnologies Inc. Anti-obesity agents
US7906482B2 (en) 1999-05-17 2011-03-15 Advanced Diagnostics And Discovery Anti-obesity agents
WO2002066067A3 (fr) * 2001-02-16 2003-02-13 King S College London Nouveau systeme d'administration de medicaments
US9061067B2 (en) 2004-07-08 2015-06-23 Novo Nordisk A/S Polypeptide protracting tags
US8710001B2 (en) 2006-07-31 2014-04-29 Novo Nordisk A/S PEGylated, extended insulins
US9018161B2 (en) 2006-09-22 2015-04-28 Novo Nordisk A/S Protease resistant insulin analogues
US7982018B2 (en) 2006-10-16 2011-07-19 Conjuchem, Llc Modified corticotropin releasing factor peptides and uses thereof
US9395352B2 (en) 2007-04-06 2016-07-19 Arizona Board Of Regents On Behalf Of Arizona State University Devices and methods for target molecule characterization
US10330632B2 (en) 2007-04-06 2019-06-25 Arizona Board Of Regents On Behalf Of Arizona State University Devices and methods for target molecule characterization
US9387176B2 (en) 2007-04-30 2016-07-12 Novo Nordisk A/S Method for drying a protein composition, a dried protein composition and a pharmaceutical composition comprising the dried protein
US9260502B2 (en) 2008-03-14 2016-02-16 Novo Nordisk A/S Protease-stabilized insulin analogues
US10259856B2 (en) 2008-03-18 2019-04-16 Novo Nordisk A/S Protease stabilized acylated insulin analogues
US9688737B2 (en) 2008-03-18 2017-06-27 Novo Nordisk A/S Protease stabilized acylated insulin analogues
US9593372B2 (en) 2008-10-06 2017-03-14 Arizona Board Of Regents On Behalf Of Arizona State University Nanopore based sequencer
US10442771B2 (en) 2008-10-06 2019-10-15 Arizona Board Of Regents On Behalf Of Arizona State University Trans-base tunnel reader for sequencing
US10589324B2 (en) 2010-12-14 2020-03-17 Sasol Technology (Pty) Limited Cleaning of process equipment
US9481721B2 (en) 2012-04-11 2016-11-01 Novo Nordisk A/S Insulin formulations
US10288599B2 (en) 2012-10-10 2019-05-14 Arizona Board Of Regents On Behalf Of Arizona State University Systems and devices for molecule sensing and method of manufacturing thereof
US11137386B2 (en) 2012-10-10 2021-10-05 Arizona Board Of Regents, Acting For And On Behalf Of Arizona State University Systems and devices for molecule sensing and method of manufacturing thereof
US10267785B2 (en) 2013-03-05 2019-04-23 Arizona Board Of Regents On Behalf Of Arizona State University Translocation of a polymer through a nanopore
US9952198B2 (en) 2013-03-05 2018-04-24 Arizona Board Of Regents On Behalf Of Arizona State University Translocation of a polymer through a nanopore
WO2014138253A1 (fr) * 2013-03-05 2014-09-12 Arizona Board Of Regents, Acting For And On Behalf Of Arizona State University Translocation d'un polymère à travers un nanopore
US9896496B2 (en) 2013-10-07 2018-02-20 Novo Nordisk A/S Derivative of an insulin analogue
US10336713B2 (en) 2014-02-27 2019-07-02 Arizona Board Of Regents, Acting For And On Behalf Of, Arizona State University Triazole-based reader molecules and methods for synthesizing and use thereof
US10265385B2 (en) 2016-12-16 2019-04-23 Novo Nordisk A/S Insulin containing pharmaceutical compositions
US10596231B2 (en) 2016-12-16 2020-03-24 Novo Nordisk A/S Insulin containing pharmaceutical compositions

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