Biomedical Engineering Reference
In-Depth Information
examples to encourage ATH development since it treats a disease state that can be predicted to
occur in the premature population and cannot be properly addressed by other known agents. In this
case, ATH may be an orphan drug.
18.5
DEVELOPMENT OF COVALENT ANTITHROMBIN-HEPARIN COMPLEXES
18.5.1 C
ONCEPTS
FOR
C
OVALENT
A
NTITHROMBIN
-H
EPARIN
S
YNTHESIS
Forms of ATH incorporating structures with the inherent characteristics discussed above may
represent the optimum heparinoid. To achieve this lofty goal, the type of conjugate structure devised
will be crucial and dependent on the synthetic algorithm. Three paradigms can be envisaged to per-
manently link ATH. One option is for heparin molecules to be activated so that they can react with
AT to covalently join the two macromolecules. This sequence of ATH preparation is one that may
be more commonly followed. It is possible that some heparin derivatives may already possess reac-
tive functional groups that can form a covalent bond with AT under appropriate conditions or with
assistant reagents. Another alternative is that groups on AT can be activated so as to allow covalent
bonding to heparin. Methodology of this type may defi ne points on the AT polypeptide from which
linkage to the heparin chain can be made. A third possibility is to allow AT and heparin to interact
for noncovalent-binding associations to occur, followed by stabilization of the complex by using a
bifunctional agent. Thus, one end of the bifunctional compound will covalently bond to AT, and the
opposite end of the bifunctional compound will react with the heparin. In all the three reaction types,
there is the theoretical possibility that, once activated, heparin molecules may become linked together
or AT molecules may become bonded to themselves. To avoid these undesirable side products, careful
designs must be employed using selective chemistries, particular stoichiometries, or special conditions
that prevent their formation.
Consideration of conjugation by heparin activation involves awareness of the groups on GAGs
that can be feasibly activated. Heparin functional groups that are candidates for activation (or are
activated) include sulfonyl, carboxyl, amino, hydroxyl, and acetal. Both amino and acetal groups can
potentially provide linkage through the heparin chain terminus, while sulfonyl and carboxyl groups
would give bonds within the heparin molecule. Preference may tend toward heparin-chain terminus
bonding to the AT polypeptide if structures similar to a core protein with glycosidically linked GAG
chains found in heparin proteoglycans
116
a re desirable. However, amino or acetal groups for end-point
attachment of heparin to AT may be scarce, leaving the binding through internal functional groups
on heparin potentially more practical. Some standard procedures can be applied for preactivation
of heparin leading to ATH preparation. Carboxyl groups on heparins can be reacted with carbodi-
imide to produce an active intermediate that can then react with amino or hydroxyl groups from AT
N-terminal, lysyl, seryl, or threonyl residues yielding amide or ester covalent linkages. ATH produc-
tion using this protocol would directly join AT to the GAG. However, steric issues and optimum ori-
entation of heparin with respect to the AT may preclude the development of useful complexes without
fi rst activating the internal heparin carboxyls with a spacer arm. Coupling through AT may have more
options for chemically activatable groups but protocols may be more complicated. Amino acid R-
groups include carboxyl, alkyl hydroxyl, phenyl hydroxyl, amino, imino, thiol, and thio ether, which
provide a wide range of potential reactions to be exploited. However, the diversity of functional groups
that could be activated for conjugation can, in themselves, interfere or overlap with each other leading
to nonselectivity of linkage type and number or intramolecular AT linkage. Additionally, AT tertiary
structure engenders a range of environments that alter reactivity of any one functional R-group type.
Therefore, chemistries for activation and linkage must be more refi ned to get a consistent ATH yielded
from initial activation of a small number of loci on AT. A potential example of ATH preparation by
AT activation would be to react AT with N-hydroxysuccinimidyl-4-azidobenzoate. This reaction
would give a photoactivatable benzoyl-amide on a lysyl amino. Incubation of this AT derivative
with heparin, followed by treatment with light, gives bonding between the benzoyl linker and
