Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
1999-02-05
2001-07-17
Geist, Gary (Department: 1623)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S026400, C536S026440, C530S350000, C514S052000
Reexamination Certificate
active
06262253
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the oral delivery of the therapeutic substances granulocyte-colony stimulating factor (GCSF) and erythropoletin (EPO) by administration of a complex comprising these substances linked to vitamin B
12
(VB
12
) or an analogue thereof. More particularly, the invention relates to methods for the synthesis of these complexes and to methods for the amplification of the amount of GCSF or EPO delivered per VB
12
carrier molecule.
An oral delivery system is known, because of recent work undertaken by one of the current inventors, which is described in PCT Application WO87/02251 (PCT/AU86/0299), whereby an active substance linked to at least one carrier molecule, which is VB
12
or an analogue thereof, can use the natural VB
12
uptake system mediated by the binding of VB
12
to intrinsic factor (IF) to transport the resultant complex from the intestinal lumen into the circulation. Once delivered into serum or the lymphatic drainage system the complex substantially retains the bioactivity of the native active substance.
In common with virtually all proteins, peptides and other large bioactive molecules there is currently no method for the oral delivery of either GCSF or EPO. The oral route of administration is the most preferable means of delivering a pharmaceutically active agent, and as such there is a large and valuable market for any process which permits the oral delivery of either of these proteins to humans. Such a process would be available by the formation of a complex between VB
12
and GCSF or EPO.
SUMMARY OF THE INVENTION
It is thus the object of the invention to describe complexes between VB
12
analogues and either GCSF or EPO that retain both significant affinity for intrinsic factor (IF) in the VB
12
portion of the complex and significant bioactivity of the GCSF or EPO portion of the complex. The invention also concerns a process for the synthesis of these complexes. This may be achieved at least in part, by using a spacer compound, which is linked covalantly between the VB
12
portion and the GCSF or EPO.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
One aspect of the invention provides a complex which comprises at least one active substance linked to at least one carrier molecule, which is VB
12
or an analogue of VB
12
wherein the ability of the carrier to undergo the binding reactions necessary for uptake and transport of VB
12
in a vertebrate host and the activity of the active substance are substantially maintained. This occurs by providing a complex between GCSF and a carrier selected from vitamin B
12
or a vitamin B
12
analogue wherein the GCSF and the carrier are covalently linked through a diradical spacer, the complex being capable of binding to intrinsic factor with high affinity with maintenance of GCSF bioactivity.
The complex preferably has the general form V—X—A—Y—Z, where V is the carrier molecule VB
12
or an analogue (including derivatives) thereof that retains IF affinity, Z is the active substance selected from EPO or GCSF, A is a spacer arm of variable composition and length, X is the functional group through which V is attached to A, and Y is the functional group through which Z is attached to A. The nature of functional group X, its site of attachment to V and the nature of spacer arm A are chosen to maximise the IF affinity of the complex. The nature of functional group Y, its site of attachment to Z and the nature of spacer arm A are chosen to maintain substantially the bioactivity of Z.
Preferably X is selected from: —NHNH—, —NH—, —O—, S—, —SS—or —CH
2
—. A is preferably an optionally substituted, saturated or unsaturated, branched or linear, C
1-50
so alkylene, cycloalkylene or aromatic group, optionally with one or more carbons within the linear chain being replaced with N, O or S, and wherein the optional substituents are selected from carbonyl, carboxy, hydroxy, amino and other groups. Y is preferably a covalent linkage between spacer chain A and protein Z, where Y is selected from —NHCO—, —CONH—, —CONHNHCO—, —N═N, —N═CH—, NHCH
2
, —NHN═CH—, —NHNHCH
2
, —SS—, —SCH
2
—, —CH
2
S—, —NHCRNH— [where R=O, S or NH
2
], —COO—, —OCO—, and Z is GCSF or EPO.
In the context of the present invention, the term “active substance” (ie Z) includes all, part, an analogue, homologue, derivative or combination thereof of either granulocyte colony stimulating factor (GCSF) or erythropoietin (EPO).
The carrier is VB
12
or VB
12
analogue. The VB
12
analogues include any variant or derivative of VB
12
(cyanocobalamin) which possesses binding activity to intrinsic factor. Preferred analogues of VB
12
also include aquocobalamin, adenosylcobalamin, methylcobalamin, hydroxycobalamnin, cyanocobalamin, carbanalide, and 5-methoxybenzylcyanocobalamin ([(5-MeO)CN—Cbl] as well as the desdimethyl, monoethylamide and the methylacide analogues of all of the above. Other analogues include all alkyl cobalamins in which the alkyl chain is linked to the corrin nucleus by a direct CoC covalent bond. Other analogues include chlorocobalamin, sulfitocobalamin, nitrocobalamin, thiocyanatocobalamin, benzimidazolecyanocobalamin derivatives such as the: 5,6-dichlorobenzimidazole, 5-hydroxybenzimidazole, trimethylbenzimidazole, as well as adenosylcyanocobalamin [(Ade)CN—Cbl], cobalamin lactone, cobalamin lactam and the anilide, ethylamide, monocarboxylic and dicarboxylic acid derivatives of VB
12
or its analogues.
Preferred derivatives of VB
12
also include the mono-, di-and tricarboxylic acid derivatives or the propionamide derivatives of VB
12
. Carriers may also include analogues of VB
12
in which the cobalt is replaced by zinc or nickel. The corrin ring of VB
12
or its analogues may also be substituted with any substituent which does not effect its binding to IF, and such derivatives of VB
12
or its analogues are part of this invention. Other derivatives of VB
12
or its analogues which have a functional group which is able to react with the spacer compound are also part of the invention.
It is preferred that the complex may comprise GCSF linked through a disulphide bond to a (dithiopyridyl propionamido) dodecylamino [DTP-dodecylamino] derivative of VB
12
.
Another preferred embodiment of the invention provides a process for the production of a complex comprising GCSF linked through a disulphide bond to a (dithiopyridyl propionamido) dodecylsuberylhexylamino derivative of VB
12
, (a long-chain analogue of the DTP-dodecylamino VB
12
) which displays higher affinity for intrinsic factor.
Another preferred embodiment of the invention provides a process for the production of a complex comprising GCSF linked through a disulphide bond to an (dithiopyridyl propionamido) dodecylcarboxamidomethyl derivative of VB
12
, in which the spacer is linked to the VB
12
through an axial CoC bond.
Another embodiment of the invention provides a process for the production of a complex comprising at least one active substance linked to at least one carrier molecule through a spacer, the carrier molecule being VB
12
or an analogue thereof, wherein the ability of the carrier to undergo the binding reactions necessary for uptake and transport of VB
12
in a vertebrate host and the activity of the active substance are substantially maintained, the process comprising one or more of the following steps:
a) reacting together the active substance and the carrier and the spacer compound to form the complex;
b) reacting the active substance with the spacer, and then reacting the product with the carrier to form the complex;
c) reacting the carrier with the spacer, and then reacting the product with the active substance to form the complex;
d) following method of (a), (b) or (c) but with the additional step of having chemically modified the carrier and/or the active substance in a previous step to provide a functional group on the carrier and/or active substance which will react with the spacer compound; or
e) following the method of (a), (b), (c) or (d) but with the additional step
Russell-Jones Gregory John
Westwood Steven William
Biotech Australia Pty Limited
Crane L. E.
Foley & Lardner
Geist Gary
LandOfFree
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