CA2150956A1 - Glycolipid enzyme-polymer conjugates - Google Patents

Glycolipid enzyme-polymer conjugates

Info

Publication number
CA2150956A1
CA2150956A1 CA002150956A CA2150956A CA2150956A1 CA 2150956 A1 CA2150956 A1 CA 2150956A1 CA 002150956 A CA002150956 A CA 002150956A CA 2150956 A CA2150956 A CA 2150956A CA 2150956 A1 CA2150956 A1 CA 2150956A1
Authority
CA
Canada
Prior art keywords
conjugate
substance
polymer
glucocerebrosidase
poly
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
CA002150956A
Other languages
French (fr)
Inventor
Robert G. L. Shorr
Myung-Ok Cho
Carl W. Gilbert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Enzon Pharmaceuticals Inc
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of CA2150956A1 publication Critical patent/CA2150956A1/en
Abandoned legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/24Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal 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
    • A61K47/51Medicinal 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
    • A61K47/56Medicinal 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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59Medicinal 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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60Medicinal 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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00Preparations for testing in vivo
    • A61K49/001Preparation for luminescence or biological staining
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y302/00Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01045Glucosylceramidase (3.2.1.45), i.e. beta-glucocerebrosidase
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Abstract

Conjugates having activity against gylcolipids such as glucocerebroside and non-antigenic polymers such as polyethylene glycol are disclosed. The conjugates circulate for extended times and have prolonged activity in vivo when compared to unmodified enzymes. The conjugates are useful in the treatment of Gaucher's disease.

Description

~ WO94tl3311 PCT~S93/11920 ~151~9~6 GLYCOLIPID ENZYME-POLYMER CONJUGATES
~ACRGROUND OF THE INVENTION

The present invention relates to conjugates having prolonged hydrolyzing activity in vivo against glycolipids such as glucocerebroside.

Gaucher's disease is an autosomal recessive genetic disorder which effects about 20,000 people in the Unites States. The disease is the most common lysosomal storage disorder and describes a defect in the afflicted's naturally-occurring glucocerebrosidase (GCS). This defect causes pathological storage of the complex lipid, glucocerebroside, primarily in organs and tissues of the reticuloendothelial system. The disease is systemic and patients may experience enlargement of the liver and spleen as well as replacement of the bone marrow with lipid-filled cells known as Gaucher cells.
Unfortunately, there is currently no cure for patients suffering from this disease. Treatment for the disease is largely symptomatic. For example, analgesics are used for relief of pain, blood and platelet transfusions are often indicated. In cases where the disease is severe, a splenectomy is indicated to remove the enlarged spleen.

Gaucher's disease is considered to be a good candidate for enzyme-replacement therapy. For example, U.S. Patent No. 3,910,822 discloses the use of GCS
isolated from human placental tissue as a treatment of Gaucher's disease. In addition, PCT Publication Nos. WO
90/07573 and WO 89/05850 describe preparing GCS using recombinant DNA ~chniques. While these advancements are significant, effective treatment and management of the WO94/13311 PCT~S93/11920 21~09~ ~

disease for many patients remains elusive. A chief drawback with current therapies is the relatively short period of time that the replacement enzyme is active in vivo. Accumulated glucocerebroside is not always fully metabolized. Thus, the long term effects of the lingering glycoprotein has not been addressed.

U.S. Patent No. 4,935,465 describes protein conjugates including glucocerebrosidasereversibly linked to water soluble polymers. The linking groups described therein are based on maleic acid derivatives which are quickly hydrolyzed in vivo and thus release the unmodified protein from the polymer. The present inventors, however, have found that alternative linking lS groups having a much greater resistance to in vivo hydrolysis provide conjugates which are enzymatically-active over longer periods and thus are better suited to act on accumulated glycolipids and thus reduce the residual amounts of glucocerebroside in the plasma and normalize spleen, liver and skeletal abnormalities.

SUMNARY OF THE lNv~..lON

The present invention provides biologically active conjugates having prolonged activity against glycolipids such as glucocerebroside. The conjugates contain a substance such as an enzyme or enzyme fragment having the ability to hydrolyze glycolipids and a substantially non-antigenic polymeric substance. In one preferred aspect of the invention, the conjugates include glucocerebrosidase or a substance having glucocerebrosidase activity covalently attached to an activated form of a polyalkylene oxide such as polyethylene glycol. In this regard, the polymer will ~WO94/13311 PCT~S93/11920 21~Q956 The present invention also provides methods of preparing the conjugates. The methods include reacting a substance having activity against glycolipids with a substantially non-antigenic polymeric substance under conditions sufficient to effect conjugation of the substituents while main~;ning at least a portion of the anti-glycolipid activity. Such conditions include reacting the polymer with the enzyme-like substance in molar ratios ranging from about 5:l to 125:l. The resultant conjugates have from about l to 25 polymeric strands attached to each enzyme-like substance.

The invention also provides methods of treating Gaucher's disease. In this aspect of the invention, treatment includes administering an effective amount of the conjugates described herein to patients or mammals requiring such therapy.

As a result of the present invention, conjugates having substantially prolonged enzyme-like activity against glycolipids in vivo are provided. The conjugates are substantially resistant to in v vo hydrolysis and thus uniquely allow less frequent administration of the therapeutic conjugate when compared to unmodified enzyme preparations and prolonged activity against accumulated glycolipids.

For a better understanding of the present invention, reference is made to the following description and its scope will be pointed out in the appended claims.

WO94/13311 PCT~S93/11920 ~SO~

BRIEF DESCRIPTION OF ~E DRAWING

Figure l is a graph demonstrating the comparative results of PEG-Glucocerebrosidase conjugates and unmodified glucocerebrosidase in vivo.

DET~Tr~ DE8CRIPTION OF THE l~.v~.~lON

The conjugates include substances having the ability to hydrolyze glycolipids. Such substances preferably have a glucocerebrosidase or glucocerebrosidase enzyme-like activity. These substances can be prepared or obtained from a variety of sources, including recombinant or mammalian extracted GCS. It is preferred that the enzyme included in the conjugate be prepared using recombinant te~niques. In this regard, the recombinantly prepared glucocerebrosidase such as that disclosed in PCT WO 89/05850 may be used herein. The contents of this PCT publication are hereby incorporated by reference. Alternatively, glucocerebrosidase may be obtained from mammalian sources such as human placental tissue as disclosed in U.S. Patent No. 3,9l0,822. It is to be understood that other substAnc~s including pro-enzymes and fractions of enzymes or pro-enzymes can also be included in the conjugates of the present invention.
As used herein, the expression "the ability to hydrolyze glycolipids" means any substance which demonstrates in vivo activity against mammalian glycolipids especially glucocerebroside. These substances are prepared by using techniques known to those of ordinary skill in the art such as tissue culture, extraction from plant or animal sources or by recombinant DNA methodologies. Transgenic sources of enzymes, pro-enzymes and fractions thereof are also contemplated. Such materials are obtained from ~ WO94/13311 PCT~S93/11920 215~95~

transgenic animals, i.e. mice, pigs, cows, etc. wherein the enzyme is expressed in milk, blood, or tissues.
Catalytic antibodies specific for glycolipid catalysis are also contemplated. Such antibodies can be prepared using recombinant technologies where antibodies specific to a glycolipid binds to the glycolipid and cleaves the CHO-lipid bond. The method by which the enzymatic substance is prepared for the conjugates of the present invention is not limited to those described herein.
The substantially non-antigenic polymer substances included in the conjugates are preferably poly(alkylene oxides). Within this group of substances are alpha-substituted polyalkylene oxide derivatives such as methoxypolyethylene glycols or other suitable alkyl substituted derivatives such as Cl-C4 alkyl groups. It is preferred, however, that the non-antigenic material be a monomethyl-substituted PEG homopolymer. Alternative polymers such as other polyethylene glycol homopolymers, polypropylene glycol homopolymers, other alkyl-polyethylene oxides, bis-polyethylene oxides and co-polymers or block co-polymers of poly(alkylene oxides) are also useful. In those aspects of the invention where PEG-based polymers are used, it is preferred that they have molecular weights of from about l,OO0 to about lO,OOO. Molecular weights of about 2,000 to 7,500 are preferred and molecular weights of about 5,000 are especially preferred.

Alternative non-antigenic polymeric substances include materials such as dextran, polyvinyl pyrrolidones, polysaccharides, starches, polyvinyl alcohols, polyacryl amides or other similar non-immunogenic polymers. Those of ordinary skill in the art WO94113311 PCT~S93/11920 21~0956 will realize that the foregoing is merely illustrative and not intended to restrict the type of non-antigenic polymeric substances suitable for use herein.

As stated above, covalent modification of the enzyme-like material is preferred to provide the hydrolysis-resistant conjugate. The covalent modification reaction includes reacting a substance having the desired activity against glycolipids with a substantially non-antigenic polymeric substance under conditions sufficient to effect conjugations while maintaining at least a portion of the hydrolytic activity against glycolipids.

Oftentimes, the polymers are activated in order to effect the desired linkage with the enzymatically-acting substance. By activation, it is understood by those of ordinary skill in the art that the polymer is functionalized to include a desired reactive group. See, for example U.S. Patent Nos. 4,179,337 and 5,122,614 which are incorporated by reference herein. In these disclosures, the hydroxyl-end groups of polyalkylene glycols are converted and activated into reactive functional groups. One particularly preferred activated form of PEG for use in the present invention is poly(ethylene glycol)-N-succinimide carbonate. This activated polymer forms stable, hydrolysis-resistant carbamate (urethane) linkages with amino groups of the enzymatically active materials. Isocyanante-activated PEG's are also of use. While the references incorporated herein describe epsilon amino group modifications of lysine, other conjugation methods are also contemplated.
Carbohydrate and/or acid group or other amino acid modifications are also within the scope of the present ~ WO94/13311 PCT~S93/1~20 21509~6 invention. Covalent linkage by any atom between the enzyme and polymer is possible. Moreover, non-covalent conjugation such as lipophilic or hydrophilic interactions are also contemplated.

The process of the present invention includes preparing or providing the activated polymer and thereafter reacting it with a substance having the desired enzymatic or hydrolytic activity, in this case, the ability to hydrolyze glycolipids. The reaction is carried out in a buffer such as O.lM phosphate buffer at a pH of from about 6.0 to about 8Ø The conjugate substituents are reacted with an appropriate amount of the polymer, which is typically present in a several-fold molar excess over the enzymatic-like substance. The polymeric excess will range from about 5 to about 125 molar ratio excess and preferably from about 50 to about 120 molar excess of the polymer to the enzyme-~ike substance. The reaction is carried out at temperatures of from about 0 to 25 C over time periods ranging from a few minutes to as long as 12 hours. Temperatures of from about 20 to about 25 C are preferred and time periods of around 1 hour are sufficient to carry out the conjugation reaction.
Following the conjugation reaction, the desired product is recovered using known t~chniques and purified using column chromatography or similar apparatus if necessary. Depending upon the reaction conditions, the conjugates have from about 1 to about 25 polymeric strands attached to the enzyme-like substance. By controlling the molar excess of the polymer reacted with the enzyme, for example, the artisan can tailor the number of polymeric strands attached. Conjugates W094/13311 PCT~S93/11920 215095~

containing from about 5 to about 20 polymeric strands are preferred while conjugates containing from about lO to 18 polymeric strands are most preferred.

Another aspect of the present invention provides methods of treatment for Gaucher's disease. The method includes administering an effective amount of the compositions described herein to alleviate the Gaucher's disease symptoms. Those of ordinary skill in the art will realize that the amount of the conjugate used in the method of the present invention will vary somewhat from patient to patient, however, conjugates capable of delivering from about O.l IU/kg to about 200+ IU/kg per administration and preferably 25 IU/kg are contemplated.
The optimal dosing of the conjugate can be determined from clinical experience.

Further in this regard, the amount of the conjugate administered to treat Gaucher's disease is an amount that is sufficient to significantly reduce pathological glycolipid concentrations in vivo, and, in particular, the glycolipid glucocerebroside. The maximal dosage for humans is the highest dosage that does not cause clinically important side effects.
An important feature however is that by covalently combining the polymeric substance and the glycolipid hydrolyzing substance as described herein, the conjugates are substantially resistant to hydrolysis in vivo. The conjugates thus act on accumulated glucocerebroside in vivo to a greater extent than prior art compositions.

~ WO94/13311 PCT~S93/11920 2~5~9~

EXAMPLBS

The following examples serve to provide further appreciation of the invention but are not meant in any way to restrict the effect of the scope of the invention.

EXAMPLE I
Modification of Glucocerebrosidase with SC-PEG

In this example, recombinant glucocerebrosidase was conjugated with the activated poly(ethylene glycol)-N-succinimide carbonate (SC-PBG) described in U.S. Patent number 5,122,614. The polymer had a molecular weight of about 5000. The recombinant glucocerebrosidase (rGCS) was prepared in accordance with the method described in PCT Publication No. WO 89/05850.

Procedures: 1.8 mg of recombinant glucocerebrosidase (rGCS) in 92 Mm NaOAc pH 5.8/10 %
glycerol/18 % EtOH was dialyzed with 0.1 M Sodium phosphate buffer solution, pH 7.0 using a Centricon-10 (a product of the Amicon Corporation of Beverly, MA).
The final concentration of the rGCS was ~0.5 mg/ml. 18 mg of SC-PEG (10-fold excess by weight = 120 molar excess) was added to the enzyme solution and the reaction mixture was stirred for 1 hr. at room temperature. The reaction was quenched by adding 0.1 M glycine. The unreacted PEG was removed by dialysis into a buffer solution having a pH of 6.5. The modification was checked by SDS-gel and the enzyme activity was measured WO94/13311 PCT~S93/~1920 21~09~

by Fluorimeter F-2000 Fluorescence Spectrophotometer, a product of Hitachi, Japan.

Results: The moler~ r veight of PEG-GCS was approximately estimate~ to be -120,000-160,000 by SDS-gel. This corresponds to conjugates having about -12-20 PEG
strands attached. The enzyme activity was measured by using the artifi~ial substrate 4-methyl- umbelliferyl-~-D-glucopyranoside (4-MUG) and 90 ~ 5 ~ of the GCS activity was retained.

~XAMPL~ II
Modification of GCS with SC-PEG

In this example, the procedure of example I is repeated using that a 20 fold molar excess of SC-PEG and same rGCS.

Procedures: 100mg of rGCS is dialyzed with 0.lM
phosphate buffer, pH 7.0 using a Minisette (available from Filtron of Northborough, MA) to yield a 1 mg/ml solution. To this solution 167 mg of SC-PEG (a 20 fold molar excess) is added and stirred for 1 hr. at room temperature (-20 C). The reaction stopped by adding a 20 fold molar excess of 0.lM glycine.
The unreacted reagents are removed by dialysis using the Minisette into a 20 millimolar NaOAC/phosphate 0.025~ Tween 80 buffer, pH 6.5.

~ 094113311 PCT~S93/11920 21509~

Results: PEG-GCS is estimated to have a molecular weight range of about 65,000-95,000 Daltons by SDS-PAGE. This suggests that between l and 7 PEG 5000 strands are attached to each enzyme molecule. It is estimated that greater than 80% activity of the GCS is retained.

As can be seen from the foregoing example, by varying the amounts of the reactants, the amount of PEG
attached to the GCS can be varied. This, in turn, will vary the circulating life of the conjugate. Lower modifications yield conjugates with circulating lives substantially longer than native GCS but less than the higher modified PEG-rGCS made in Example I.

EXANPLE III

In this example, the circulating half-lives of various glucocerebrosidase products was compared. The circulating half-life of both recombinant GCS and PEG-rGCS were determined in rats. Six rats about 300 + 25 gm were used for this experiment. Three rats were injected i.p. with rGCS and three were similarly injected with PEG-rGCS at a dose of 60 IU/kg. At various time points the rats were bled and plasma prepared. The plasma was stored at 4C until assayed. Glucocerebrosidase activity was determined using the 4-MUG fluorescent assay. The half-lives (T,h) are reported in the following table. The half-life for placental GCS was reported by Brady, et al, New England Journal of Medicine 291: 990-993 (1974); the half-life of Ceredase TM was reported by Whittington, et al, Drugs 44(l) 72-93 (1992). The results are reported below.

WO94/13311 PCT~S93/11920 21~956 CIRCULATING ~ALF-~IVES OF G~u~ ~R~O~IDA8E

Preparation Tl/2 r Placental GCS 25 minutes Chemically Modified Placental GCS
(Ceridase~ 3-ll minutes Recombinant GCS1 approx. l hr (alpha phase) PEG-rGlucocerebrosidase2 6-7 hours (alpha phase) 2~

CeridaseTM: a product of the Genzyme Corporation Prepared in accordance with the method of Ginns, et al in PCT WO 89/05850 2 As prepared in Example I

As can be seen from the table, PEG-GCS conjugates prepared in accordance with the present invention have a substantially prolonged increase in circulating life when compared to unmodified enzymes.

EXANPLE IV

In this example, the activity of the PEG-rGCS
prepared in accordance with example I was compared to that of unconjugated rGCS prepared according to the method of Ginns, et al., supra, in a 72 hour plasma circulating half-life study. As was the case in example III, 3 rats were injected i.p. with 60 IU/kg with PEG-rGCS and 3 rats were injected with 60 IU/kg of rGCS.

_ WO94/~311 PCT~S93/11920 Referring now to FIG. l, it can be seen that not only does the conjugate provide the rapid activity associated with unmodified glucocerebrosidase at the outset, but the inventive conjugates provide higher levels of activity throughout a three day period. While applicants are not bound by theory, it is believed that such higher levels of enzyme activity in vivo over time serve to provide persistent activity against glucocerebroside. Such activity represents a major advance toward reducing residual glycolipid concentrations in Gaucher's Disease patients. This in turn will help alleviate some of the sequelae associated with later stage Gaucher's Disease such as enlarged spleen, liver and skeletal abnormalities.
While there have been described what are presently believed to be the preferred embodiments of the present invention, those skilled in the art will realize that changes and modifications may be made thereto without departing from the spirit of the invention. It is intended to claim all such changes and modifications as fall within the true scope of the invention.

Claims (31)

WHAT IS CLAIMED IS:
1. A biologically active conjugate comprising a substance having the ability to hydrolyze glycolipids and a substantially non-antigenic polymeric substance.
2. The conjugate of claim 1, wherein said glycolipid is glucocerebroside.
3. The conjugate of claim 1, wherein said polymer is a poly(alkylene oxide).
4. The conjugate of claim 3, wherein said poly(alkylene oxide) is an alpha-substituted polyalkylene oxide derivative.
5. The conjugate of claim 3, wherein said poly (alkylene oxide) is selected from the group consisting of polyethylene glycol homopolymers, polypropylene glycol homopolymers, alkyl-capped polyethylene oxides, bis-polyethylene oxides and copolymers or block copolymers of poly(alkylene oxides).
6. The conjugate of claim 5, wherein said polymer has a molecular weight of from about 1,000 to about 10, 000.
7. The conjugate of claim 6, wherein said polymer has a molecular weight of from about 2,000 to about 7,500.
8. The conjugate of claim 7, wherein said polymer has a molecular weight of about 5,000.
9. The conjugate of claim 1, wherein said substance is covalently linked to said polymer.
10. The conjugate of claim 1, wherein said substance is linked to said polymer via a carbamate linkage.
11. The conjugate of claim 1, wherein said polymeric substance is selected from the group consisting of dextran, polyvinyl pyrrolidones and other non-immunogenic polymers.
12. The conjugate of claim 1, wherein said substance is glucocerebrosidase.
13. The conjugate of claim 12, wherein said glucocerebrosidase is of recombinant origin.
14. The conjugate of claim 12, wherein said glucocerebrosidase is of mammalian origin.
15. The conjugate of claim 14, wherein said mammalian glucocerebrosidase is placental glucocerebrosidase.
16. The conjugate of claim 12, wherein said glucocerebrosidase is of transgenic origin.
17. The conjugate of claim 1, wherein said substance is selected from the group consisting of enzyme fragments, proenzymes and catalytic antibodies.
18. The conjugate of claim 1, wherein said conjugate comprises from about 1 to about 25 polymeric strands attached to said substance.
19. The conjugate of claim 18, wherein said conjugate comprise from about 5 to about 20 polymeric strands attached to said substance.
20. The conjugate of claim 19, wherein said conjugate comprises from about 10 to about 18 polymeric strands attached to said substance.
21. A method of preparing a conjugate having the ability to hydrolyze glycolipids, comprising reacting a substance having activity against glycolipids with a substantially non-antigenic polymeric substance under conditions sufficient to effect conjugation of said substance and said polymeric substance while maintaining at least a portion of the substance's activity.
22. The method of claim 21, wherein said polymer is a poly (alkylene oxide).
23. The method of claim 22, wherein said polyalkylene oxide is a alpha-substituted polyalkylene oxide derivative.
24. The method of claim 22, wherein said poly (alkylene oxide) is a polyethylene glycol.
25. The method of claim 24, wherein said polyethylene glycol is a methoxypolyethylene glycol.
26. The method of claim 24, wherein said polyethylene glycol is activated to include a linking group for attaching said polymer to said substance.
27. The method of claim 26, wherein said activated polyethylene glycol is PEG-N-succinimidyl carbonate.
28. The method of claim 26, wherein said activated polyethylene glycol is PEG-isocyanate.
29. The method of claim 21, wherein said conditions include reacting said polymer with said substance in a molar ratio of from about 5:1 to 125:1.
30. The method of claim 29, wherein said condition include reacting said polymer with said substance in a molar ratio of from about 50:1 to 120:1.
31. A method of treating Gaucher's disease comprising administering an effective amount of the composition of claim 1.
CA002150956A 1992-12-10 1993-12-08 Glycolipid enzyme-polymer conjugates Abandoned CA2150956A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US98980292A 1992-12-10 1992-12-10
US07/989,802 1992-12-10

Publications (1)

Publication Number Publication Date
CA2150956A1 true CA2150956A1 (en) 1994-06-23

Family

ID=25535471

Family Applications (1)

Application Number Title Priority Date Filing Date
CA002150956A Abandoned CA2150956A1 (en) 1992-12-10 1993-12-08 Glycolipid enzyme-polymer conjugates

Country Status (8)

Country Link
US (2) US5620884A (en)
EP (1) EP0675727B1 (en)
JP (1) JPH08507677A (en)
AU (1) AU5743994A (en)
CA (1) CA2150956A1 (en)
DE (1) DE69331805T2 (en)
DK (1) DK0675727T3 (en)
WO (1) WO1994013311A1 (en)

Families Citing this family (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2175578A1 (en) * 1993-11-03 1995-05-11 Alec Sehon Cell control and suppression
EP0952841A4 (en) * 1994-08-05 2000-11-02 Molecular Structural Biotechno Site-specific biomolecular complexes
US6004579A (en) * 1995-09-14 1999-12-21 Lxr Biotechnology, Inc. Compositions which inhibit apoptosis, methods of making the compositions and uses thereof
SE9601817D0 (en) * 1996-05-13 1996-05-13 Karsten Buschard Novel glycolipid complexex and their uses
DE69735669T2 (en) 1996-07-15 2007-03-29 Macrozyme Dnm B.V. DEOXYNOJIRIMYCIN DERIVATIVES AND THEIR USE AS GLUCOSE SYLCERAMIDASE INHIBITORS
WO1998012274A1 (en) 1996-09-23 1998-03-26 Chandrashekar Pathak Methods and devices for preparing protein concentrates
US6008037A (en) * 1996-11-14 1999-12-28 Polymer Technology Corporation Use of water soluble enzyme-polymer conjugates for cleaning contact lenses
EP0973384A4 (en) 1997-02-13 2004-10-13 Sky High Llc Organ preservation solution
US6251866B1 (en) 1997-08-05 2001-06-26 Watson Laboratories, Inc. Conjugates targeted to the interleukin-2 receptor
ATE276358T1 (en) * 1998-06-23 2004-10-15 Novozymes As POLYPEPTIDE-POLYMER CONJUGATE
US6638526B1 (en) 1998-06-23 2003-10-28 Novozymes A/S Polypeptides conjugated to copolymers of ethylene oxide and propylene oxide to reduce allergenicity
AU770911B2 (en) * 1998-07-17 2004-03-04 Novozymes A/S A polypeptide-polymer conjugate with improved wash performance
EP1196190B1 (en) * 1999-07-26 2003-03-19 G.D. SEARLE & CO. Use of long-chain n-alkyl derivatives of deoxynojirimycin and a glucocerebrosidase enzyme for the manufacture of a medicament for the treatment of glycolipid storage diseases
WO2001049830A2 (en) * 1999-12-30 2001-07-12 Maxygen Aps Improved lysosomal enzymes and lysosomal enzyme activators
US7138262B1 (en) 2000-08-18 2006-11-21 Shire Human Genetic Therapies, Inc. High mannose proteins and methods of making high mannose proteins
WO2003017938A2 (en) * 2001-08-22 2003-03-06 Watson Pharmaceuticals, Inc. Conjugates targeted to target receptors
US6887952B1 (en) * 2004-02-12 2005-05-03 Biosite, Inc. N-aryl-carbamic acid ester-derived and valeric acid ester-derived cross-linkers and conjugates, and methods for their synthesis and use
US7740861B2 (en) * 2004-06-16 2010-06-22 University Of Massachusetts Drug delivery product and methods
EP1794588A2 (en) * 2004-09-09 2007-06-13 Biosite Incorporated Methods and compositions for measuring canine bnp and uses thereof
EP1802289B1 (en) 2004-09-17 2011-12-07 University of Massachusetts Compositions and their uses for lysosomal enzyme deficiencies
WO2006078813A2 (en) * 2005-01-21 2006-07-27 Biosite Incorporated Arginine analogs, and methods for their synthesis and use
AU2006213822B2 (en) 2005-02-09 2011-05-26 Covidien Lp Synthetic sealants
JP5489462B2 (en) * 2005-10-24 2014-05-14 ユニバーシティ オブ マサチューセッツ Compositions for gene therapy of bone pathology and methods of use thereof
US7833766B2 (en) * 2006-02-07 2010-11-16 Shire Human Genetic Therapies, Inc. Stabilized compositions of proteins having a free thiol moiety
WO2007097934A2 (en) * 2006-02-17 2007-08-30 Elusys Therapeutics, Inc. Methods and compositions for using erythrocytes as carriers for delivery of drugs
CA2704056A1 (en) * 2007-10-29 2009-05-07 University Of Massachusetts Encapsulated nanoparticles for nucleic acid delivery
WO2010080720A2 (en) * 2009-01-12 2010-07-15 Nektar Therapeutics Conjugates of a lysosomal enzyme moiety and a water soluble polymer
WO2010104883A1 (en) * 2009-03-09 2010-09-16 Molecular Express, Inc. Methods and compositions for liposomal formulation of antigens and uses thereof
MX344786B (en) * 2009-07-28 2017-01-06 Shire Human Genetic Therapies Compositions and methods for treating gaucher disease.
US9194011B2 (en) 2009-11-17 2015-11-24 Protalix Ltd. Stabilized alpha-galactosidase and uses thereof
WO2011107991A1 (en) * 2010-03-02 2011-09-09 Protalix Ltd. Glucocerebrosidase multimers and uses thereof
JP6230160B2 (en) 2012-03-02 2017-11-15 シャイア ヒューマン ジェネティック セラピーズ インコーポレイテッド Compositions and methods for treating type III Gaucher disease
ES2927758T3 (en) 2013-03-14 2022-11-10 Alere San Diego Inc 6-Acetylmorphine analogues and procedures for their synthesis and use
DK3307326T3 (en) 2015-06-15 2020-10-19 Angiochem Inc METHODS FOR THE TREATMENT OF LEPTOMENINGAL CARCINOMATOSIS
AU2017267728B2 (en) 2016-05-18 2021-11-04 Alere San Diego, Inc. 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine analogs and methods for their synthesis and use
CA3066915A1 (en) 2017-06-11 2018-12-20 Molecular Express, Inc. Methods and compositions for substance use disorder vaccine formulations and uses thereof
KR20230017207A (en) 2020-04-30 2023-02-03 사이로파 비.브이. Anti-CD103 antibody

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) * 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US3910822A (en) * 1974-03-14 1975-10-07 Us Health Isolation of glucocerebrosidase from human placental tissue
US4749570A (en) * 1981-12-31 1988-06-07 The Governors Of The University Of Alberta Targeting conjugates of albumin and therapeutic agents
GB8430252D0 (en) * 1984-11-30 1985-01-09 Beecham Group Plc Compounds
JPS61275978A (en) * 1985-05-31 1986-12-06 Toshiba Corp Arithmetic processing unit
EP0393143A4 (en) * 1987-12-23 1991-09-11 Us Commerce Cloned dna for synthesizing unique glucocerebrosidase
ATE138686T1 (en) * 1988-12-23 1996-06-15 Genzyme Corp CHO CELLS CAPABILITY TO PRODUCE ENZYMATICALLY ACTIVE RECOMBINANT GLUCOCEREBROSIDASE
US5122614A (en) * 1989-04-19 1992-06-16 Enzon, Inc. Active carbonates of polyalkylene oxides for modification of polypeptides
DE69213714T2 (en) * 1991-01-21 1997-01-23 Genzyme Corp PRODUCTION OF ENZYMATIC ACTIVE GLUCOCEREBROSIDASE FROM RECOMBINANT CELLS

Also Published As

Publication number Publication date
DK0675727T3 (en) 2002-08-05
WO1994013311A1 (en) 1994-06-23
DE69331805T2 (en) 2002-11-28
EP0675727B1 (en) 2002-04-10
US5705153A (en) 1998-01-06
EP0675727A4 (en) 1997-01-08
DE69331805D1 (en) 2002-05-16
AU5743994A (en) 1994-07-04
EP0675727A1 (en) 1995-10-11
US5620884A (en) 1997-04-15
JPH08507677A (en) 1996-08-20

Similar Documents

Publication Publication Date Title
EP0675727B1 (en) Glycolipid enzyme-polymer conjugates
US10907142B2 (en) Oligosaccharides comprising an aminooxy group and conjugates thereof
US9885024B2 (en) PEG-urate oxidase conjugates and use thereof
JP5341945B2 (en) Aggregate-free urate oxidase for the preparation of non-immunogenic polymer conjugates
Burnham Polymers for delivering peptides and proteins
CN102573920B (en) Glycopolysialylation of non-blood coagulation proteins
US5969040A (en) Factor IX -- polymeric conjugates
CN101194016B (en) A variant form of urate oxidase and use thereof
CN101002945B (en) Novel complex used for treating tumor
HU228916B1 (en) Polyethylen-glycol/carbamate oxydase conjugates and pharmaceutical compositions containing thereof
AU2001249975A1 (en) Aggregate-free urate oxidase for preparation of non-immunogenic polymer conjugates
CN102943067A (en) Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
AU595173B2 (en) Method and use for site-specific activation of substances
CZ137597A3 (en) Bdnf and nt-3 conjugates with water-soluble polymer
CN102580066A (en) Combination therapy for treating protein deficiency disorders
CN101405019A (en) Medicine for treating tumor and use thereof
Poznansky et al. Insulin: carrier potential for enzyme and drug therapy
CN104530182A (en) Glycopolysialylation of non-blood coagulation proteins
Poznansky Enzyme-protein conjugates: new possibilities for enzyme therapy
US5256413A (en) Method and use for site-specific activation of substances
CA2616187A1 (en) Human growth hormone conjugated with biocompatible polymer
Poznansky Enzyme—Albumin polymers: New approaches to the use of enzymes in medicine
Torchilin et al. Long acting thrombolytic immobilized enzymes
US20120197003A1 (en) Modified oligopeptides for the treatment of pancreatitis, stomach ulcers, and other hyperenzymemias based on enzyme peptide inhibitors and methods for obtaining them
WO2012070966A1 (en) Modified oligopeptides for treating pancreatitis, gastric ulcers and other hyperenzymemias based on a peptidic enzyme inhibitor and method for producing said modified oligopeptides

Legal Events

Date Code Title Description
EEER Examination request
FZDE Dead