Drug Targeting Organ-Specific Strategies

provide rapid synthesis of conjugates, which is particularly suitable for animal experiments
and ‘proof of concept’ studies, fusion proteins have the potential for bulk production of a defined
molecular entity for future clinical development. With regard to chemical conjugation,
the avidin–biotin technology as a linker strategy was introduced [84] as a highly versatile alternative
to direct linkage of vector and drug moiety [85]. It exploits the broad availability of
biotinylating reagents for a range of compounds and functional groups, and a single vector to
be used for the delivery of different drugs. Moreover, the avidin–biotin bond is extremely stable.
It proved advantageous for pharmacokinetic reasons to substitute the basic avidin with a
chemically neutralized form of avidin, designated NLA [86] or with streptavidin (SA) [87].
Recently, a fusion protein between OX26 and avidin was engineered, in which the variable
regions of an IgG3-antibody were substituted with those of OX26, and the C H3 region was
fused to the avidin monomer (anti-TfR IgG3-C H3-Av). The fusion protein thus forms an
avidin dimer, which displays high affinity biotin binding.The pharmacokinetics and brain uptake
of the fusion protein were favourable compared to the chemical conjugate of
OX26–avidin, as is evident from the values in Table 2.1 [88].
The biotin–avidin linker strategy is particularly suitable for synthetic peptide drugs. These
can be designed to facilitate monobiotinylation at a site that does not interfere with bioactivity
[89]. Monobiotinylation is recommended due to the multivalency of avidin. A 1 : 1 molar
conjugate of vector and (strept)avidin can bind up to four biotin residues, and the genetically
engineered fusion protein still has two biotin binding sites.Therefore, higher degrees of
biotinylation of the drug moiety would result in the formation of high molecular weight aggregates,
which are cleared rapidly from the circulation [90].
Chimeric peptides need to be stable in the circulation before brain uptake occurs, and either
amide bonds, thioether or disulfide linkers fulfil that requirement in the plasma compartment.
In addition, they must be stable during transcytosis through BBB endothelial cells.
Finally they also need to retain binding affinity in their drug moiety. If binding of a peptide
drug to the vector reduces binding affinity to the drug receptor on brain cells, then the release
of free drug in the brain would be required. Disulfide reducing enzymes such as protein disulfide-isomerase
are present in tissues intracellularly and on the plasma membrane [91]. Cleavage
of (-S-S-) linked chimeric peptides in brain in vivo is possible.A biotinylated opioid peptide
analogue ([Lys7]dermorphin-amide) with a disulfide biotin linker, N-hydroxysuccinimide
dithiopropionate, was cleaved from the vector OX26-SA in brain but not in plasma
[92].
An alternative coupling strategy that avoids potential steric hindrance of drug action and
eliminates the need for cleavability utilizes long, flexible spacer arms, e.g. biotin-derivatized
polyethylene glycol (PEG) linkers with molecular weights of 2000 or 5000 Da [93,94].
2.4.2.6 Pharmacological Effects of Chimeric Peptides
2.4 Drug Delivery Strategies 43
The cargo that is suitable for transport by chimeric peptides encompasses a wide array of
substances. Table 2.2 gives examples of studies, which measured CNS effects after peptide
drug delivery.
Among the peptide-based payloads that have been delivered is an analogue of the 28amino
acid peptide Vasoactive Intestinal Polypeptide (VIP) [89,95]. VIP is suitable for the