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Title:  Method of targeting pharmaceuticals to motor neurons

United States Patent:  6,670,322

Issued:  December 30, 2003

Inventors:  Goodnough; Michael C. (Stoughton, WI); Johnson; Eric A. (Madison, WI); Tepp; William H. (Stoughton, WI); Malizio; Carl J. (Madison, WI)

Assignee:  Wisconsin Alumni Research Foundation (Madison, WI)

Appl. No.:  870262

Filed:  May 30, 2001

Abstract

A method of targeting therapeutic molecules to motor neurons is disclosed. In one embodiment, this method comprises the steps of (a) synthesizing a prodrug comprising a therapeutic molecule covalently bound to a polymeric delivery vehicle, and (b) conjugating the prodrug to a botulinum neurotoxin heavy chain.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, the present invention is a method of targeting therapeutic molecules to motor neurons. This method preferably comprises the step of (a) synthesizing a prodrug comprising a therapeutic molecule covalently bound to a polymeric delivery vehicle and (b) conjugating the prodrug to a botulinum neurotoxin heavy chain. In a preferred method of the present invention, the prodrug comprises at least two copies of the therapeutic molecule.

In another preferred method of the present invention, the botulinum neurotoxin heavy chain is type A botulinum neurotoxin and the therapeutic molecule is a metalloprotease inhibitor.

It is an object of the present invention to target therapeutic molecules to motor neurons.

Other objects, features and advantages of the present invention will become apparent after one has reviewed the specification, claims and drawings.

DETAILED DESCRIPTION OF THE INVENTION

Our solution to the above-stated problem is to use a modified, nontoxic form of the botulinum neurotoxin molecule to specifically target and deliver currently identified therapeutics as well as compounds developed in the future to the appropriate cholinergic motor neuron terminals. Once delivered to the presynaptic motor neuron membrane, the chimeric delivery vehicle preferably consisting of the heavy chain of the neurotoxin covalently linked with a polymeric molecule capable of delivering multiple copies of therapeutic compounds will bring the desired therapeutic across the membrane and into the neuron where the drug will be an effective therapeutic. In one embodiment, the drug may be an antagonist of botulinum neurotoxin. In another embodiment, the drug is an enzymatic inhibitor, including peptide inhibitors.

In one embodiment, the present invention is a method of targeting pharmaceuticals to motor neurons, comprising a first step of synthesizing prodrugs comprising at least one copy, and preferably multiple copies, of a therapeutic molecule (the Examples below describe the model metalloprotease inhibitor captopril as a therapeutic molecule) covalently bound to a polymeric delivery vehicle. The second step comprises conjugating the prodrug to the heavy chain of botulinum neurotoxin, preferably type A. This combination will yield a pharmaceutically active compound that specifically targets and internalizes desired substances into cholinergic neurons including active inhibitors of the active fragment of the neurotoxin itself.

We envision that the method of the present invention will be suitable for all heavy chains of botulinum neurotoxin. The Examples below describe a preparation using type A heavy chain. Other heavy chains (i.e., all seven serotypes of heavy chains, A-G) can be prepared in a similar manner. One may obtain the heavy chain in a variety of manners. Preferably, one will obtain heavy chains as described below in the Examples. In another embodiment of the present invention, one will obtain the heavy chain via recombinant DNA methods.

Polymeric delivery vehicle is an inert carrier molecule ranging in size from 10-40 kD that can be chemically modified such that therapeutic molecules may be covalently linked to it.

The polymeric delivery vehicle preferably comprises amine reactive dextran, described below as having a molecular weight of approximately 10 kD and 40 kD. Other suitable polymers include polyethylene glycol and polyimines.

Dextran amines were preferably functionalized for conjugation with model inhibitors by addition of the water soluble heterobifunctional linker sulfosuccinimidyl-6-[alpha-methyl-alpha(2-pyridylthio)-toluamido]hexanoate (sulfo-LC-SMPT, FIG. 5). Other suitable linkers include heterobifunctional linkers containing amine and sulfhydryl reactive elements. Also useful are heterobifunctional linkers that are carboxy reactive as well as sulfhydral reactive.

The conjugation of the prodrugs to the heavy chain of botulinum neurotoxin will typically take place as follows:

We envision two preferable approaches to the synthesis of prodrugs that contain high molar concentrations of model inhibitor and have a traceable label.

Approach #1. We currently use specific dextrans that are amine reactive and contain a flourescent marker such as fluorescein, tetramethylrhodamine, or Cy3.5. The availability of fluorescent dextran conjugates with different sizes and charges will permit us to synthesize a wide variety of prodrugs. These labels are commercially available from Molecular Probes, Eugene, Oreg. and from Pharmacia Amersham, Pistcataway, N.J.

The initial step in this approach will be to generate carbonyls on the dextran that are reactive to a hydrazide containing heterobifunctional linker. The preferred linker of choice is 3-(2-pyridylthio)propionyl hydrazide (PDPH). The generation of hydrazide reactive carbonyls on the dextran is accomplished by gentle oxidation using sodium periodate according to the method of Ranadive, et al. (G. Ranadive, et al., Nucl. Med. Biol. 20:719-726, 1993). Briefly, this reaction involves reacting the dextran with 5-10 mM sodium periodate in 100 mM sodium acetate, pH 5.5, for 20-30 minutes in the dark at room temperature. The oxidation reaction is quenched with addition of glycerol to 20 mM final concentration. The dextran is dialyzed against 100 mM sodium acetate, pH 5.5, to remove glycerol and periodate and linker is added to a final concentration of 5-10 mM. This reaction is allowed to proceed for 2 hours at room temperature at which time excess linker is removed from the dextran by applying the mixture to a desalting column such as the Pharmacia PD10.

The desalted, derivatized dextran is then reacted with captopril. The reaction can be monitored by appearance of the leaving group 2-thiopyridine having a characteristic absorbance at 343 nm. After 2 hours incubation at room temperature, excess captopril is removed by desalting on PD10 columns. The amine groups on the dextran are then reacted with the linker sulfo-LC-SMPT. This generates free thiol groups that can then be conjugated directly to botulinum heavy chain via disulfide exchange.

Approach #2. The second approach to conjugating prodrug to heavy chain involves synthesis of prodrug using PDPH-activated amino dextran. Amine-reactive pharmaceutical compounds are added to PDPH-modified amino dextran and allowed to cross-link with the free amine groups present on the dextran. Following removal of excess pharmaceutical by desalting on PD10 columns or extensive dialysis in 25 mM phosphate buffered saline, pH 7.4, the prodrug is added to botulinum heavy chain and the free thiol group(s) on the heavy chain allowed to form disulfide bonds with the thiol reactive portion of the linker PDPH. The reaction is allowed to proceed overnight at 4oC. Excess prodrug is readily separated from the heavy chain-prodrug conjugates by addition of ammonium sulfate to 60% saturation. Under these conditions heavy chain-prodrug and unreacted heavy chain precipitate while the carbohydrate-based prodrug remains in solution.

A second approach to separation of unreacted dextran from botulinum heavy chain can be accomplished by immobilized-metal affinity chromatography (IMAC) according to the method of Schiavo, et al. (G. Schiavo, et al., Infect. Immun. 58:4136-4141, 1990). Briefly, chelating Sepharose (Pharmacia) charged with Zn is used to bind the heavy chain of botulinum toxin and heavy chain/polymer constructs. Unreacted dextran is washed from the column with 5-8 column volumes of running buffer and the heavy chain/polymer constructs eluted with 25 mM imidazole.

Preferred therapeutic agents include protease inhibitors. Specific inhibitors include aprotinin, chymostatin, amastatin, bestatin, leupeptin, antipain, trypsin inhibitors, chelating agents such as EDTA, TPEN, o-phenanthroline, and peptide inhibitors of proteases. Peptide inhibitors such as caspase inhibitors (apopain series) which are primarily four and five amino acid inhibitors of caspases are also preferred. Metalloprotease inhibitors such as phosphoramidon, pepstatin A, phebestin, and the TAPI series of matrix metalloprotease inhibitors are also preferred.

The prodrug-heavy chain may be targeted to specific muscle groups by directly injecting the affected muscles. Alternatively, one could deliver the prodrug-heavy chain combination orally.

Claim 1 of 11 Claims

We claim:

1. A method of targeting therapeutic molecules to motor neurons, comprising the steps of:

(a) synthesizing a prodrug comprising at least one therapeutic molecule covalently bound to a polymeric delivery vehicle wherein the delivery vehicle comprises amine-reactive polymers, and

(b) conjugating the prodrug to a botulinum neurotoxin heavy chain wherein the conjugation is via a reducible heterobifunctional linker.



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