Background and Purpose
Anaemia of chronic disease is characterized by impaired erythropoiesis due to functional iron deficiency, often caused by excessive hepcidin. Lexaptepid pegol, a pegylated ...structured l‐oligoribonucleotide, binds and inactivates hepcidin.
Experimental Approach
We conducted a placebo‐controlled study on the safety, pharmacokinetics and pharmacodynamics of lexaptepid after single and repeated i.v. and s.c. administration to 64 healthy subjects at doses from 0.3 to 4.8 mg·kg−1.
Key Results
After treatment with lexaptepid, serum iron concentration and transferrin increased dose‐dependently. Iron increased from approximately 20 μmol·L−1 at baseline by 67% at 8 h after i.v. infusion of 1.2 mg·kg−1 lexaptepid. The pharmacokinetics showed dose‐proportional increases in peak plasma concentrations and moderately over‐proportional increases in systemic exposure. Lexaptepid had no effect on hepcidin production or anti‐drug antibodies. Treatment with lexaptepid was generally safe and well tolerated, with mild and transient transaminase increases at doses ≥2.4 mg·kg−1 and with local injection site reactions after s.c. but not after i.v. administration.
Conclusions and Implications
Lexaptepid pegol inhibited hepcidin and dose‐dependently raised serum iron and transferrin saturation. The compound is being further developed to treat anaemia of chronic disease.
Background and Purpose
Calcitonin gene‐related peptide (CGRP) plays an important role in the pathology of migraine, and recent clinical trials suggest the inhibition of CGRP‐mediated processes as a ...new therapeutic option in migraine. In this study, we describe the generation of NOX‐L41, a CGRP‐neutralizing mirror‐image (l‐)aptamer (Spiegelmer) and investigate its in vitro and in vivo function.
Experimental Approach
A CGRP‐binding Spiegelmer was identified by in vitro selection. Binding studies were performed using surface plasmon resonance (SPR), and the inhibitory activity was determined in cell‐based assays. The pharmacokinetic profile comparing i.v. and s.c. dosing was analysed in rats. Intravital two‐photon microscopy was employed to follow extravasation from meningeal vessels. Finally, in vivo efficacy was tested in a model of electrically evoked meningeal plasma protein extravasation (PPE) in rats.
Key Results
We identified NOX‐L41, a novel CGRP‐neutralizing Spiegelmer. SPR studies showed that NOX‐L41 binds to human and rat/mouse CGRP with sub‐nanomolar affinities and is highly selective against related peptides such as amylin. In vitro, NOX‐L41 effectively inhibited CGRP‐induced cAMP formation in SK‐N‐MC cells. In rats, NOX‐L41 had a plasma half‐life of 8 h. Pharmacodynamic studies showed that NOX‐L41 extravasates from blood vessels in the dura mater and inhibits neurogenic meningeal PPE for at least 18 h after single dosing.
Conclusions and Implications
This is the first description of the CGRP‐neutralizing Spiegelmer NOX‐L41. Preclinical studies confirmed a role for CGRP in neurogenic PPE and provided proof‐of‐concept for the potential use of this new drug candidate for the treatment or prevention of migraine.
During the past decade, RNA has become a focus of investigation into new therapeutic schemes: antisense RNA, interfering RNA and trans-cleaving ribozymes are used to silence undesired gene ...expression. As an additional option with its own therapeutic potential, ribozymes may be employed to specifically alter the sequence of RNA. Among these RNA based strategies the mode of action varies: while antisense and interfering RNAs are capable of making specific contacts to other RNA molecules with the result of employing the cellular machinery for degradation of the RNA target, trans-cleaving ribozymes fold into specific three-dimensional structures to form catalytic centres and to specifically cleave a chosen RNA target. Beyond this, trans-splicing ribozymes have been engineered to first cleave a RNA target followed by ligation of a new RNA fragment delivered with the ribozyme. The latter strategy potentially extends the application of ribozymes from inhibition of gene expression to RNA repair, i. e. correction of genetic disorders at the level of RNA, and has already shown promising results in cell culture experiments. On the other side, advances in RNA synthesis, ribozyme engineering, delivery methods and expression systems have greatly enhanced the prospects of ribozymes, antisense and interfering RNAs in gene therapy. This review provides an overview of existing strategies for potential RNA based gene therapy. It is focussed on the engineering of ribozymes and functional RNAs to be used as drugs and on the basic molecular principles of their action.
Background and Purpose
Calcitonin gene‐related peptide (
CGRP
) plays an important role in the pathology of migraine, and recent clinical trials suggest the inhibition of
CGRP
‐mediated processes as ...a new therapeutic option in migraine. In this study, we describe the generation of
NOX
‐
L
41, a
CGRP
‐neutralizing mirror‐image (
l
‐)aptamer (
S
piegelmer) and investigate its
in vitro
and
in vivo
function.
Experimental Approach
A
CGRP
‐binding
S
piegelmer was identified by
in vitro
selection. Binding studies were performed using surface plasmon resonance (
SPR
), and the inhibitory activity was determined in cell‐based assays. The pharmacokinetic profile comparing i.v. and s.c. dosing was analysed in rats. Intravital two‐photon microscopy was employed to follow extravasation from meningeal vessels. Finally,
in vivo
efficacy was tested in a model of electrically evoked meningeal plasma protein extravasation (
PPE
) in rats.
Key Results
We identified
NOX
‐
L
41, a novel
CGRP
‐neutralizing
S
piegelmer.
SPR
studies showed that
NOX
‐
L
41 binds to human and rat/mouse
CGRP
with sub‐nanomolar affinities and is highly selective against related peptides such as amylin.
In vitro
,
NOX
‐
L
41 effectively inhibited
CGRP
‐induced
cAMP
formation in
SK
‐
N
‐
MC
cells. In rats,
NOX
‐
L
41 had a plasma half‐life of 8 h. Pharmacodynamic studies showed that
NOX
‐
L
41 extravasates from blood vessels in the dura mater and inhibits neurogenic meningeal
PPE
for at least 18 h after single dosing.
Conclusions and Implications
This is the first description of the
CGRP
‐neutralizing
S
piegelmer
NOX
‐
L
41. Preclinical studies confirmed a role for
CGRP
in neurogenic
PPE
and provided proof‐of‐concept for the potential use of this new drug candidate for the treatment or prevention of migraine.
Over the past two decades, the structure and mechanism of catalytic RNA have been extensively studied; now ribozymes are understood well enough to turn them into useful tools. After we have ...demonstrated the twin ribozyme mediated insertion of additional nucleotides into a predefined position of a suitable substrate RNA, we here show that a similar type of twin ribozyme is also capable of mediating the opposite reaction: the site-specific removal of nucleotides. In particular, we have designed a twin ribozyme that supports the deletion of four uridine residues from a given RNA substrate. This reaction is a kind of RNA recombination that in the specific context of gene therapy mimics, at the level of RNA, the correction of insertion mutations. As a result of the twin ribozyme driven reaction, 17% of substrate are converted into the four nucleotides shorter product RNA.
In recent years major progress has been made in elucidating the mechanism and structure of catalytic RNA molecules, and we are now beginning to understand ribozymes well enough to turn them into ...useful tools. Work in our laboratory has focused on the development of twin ribozymes for site‐specific RNA sequence alteration. To this end, we followed a strategy that relies on the combination of two ribozyme units into one molecule (hence dubbed twin ribozyme). Here, we present reverse‐joined hairpin ribozymes that are structurally optimized and which, in addition to cleavage, catalyse efficient RNA ligation. The most efficient variant ligated its appropriate RNA substrate with a single turnover rate constant of 1.1 min−1 and a final yield of 70%. We combined a reverse‐joined hairpin ribozyme with a conventional hairpin ribozyme to create a twin ribozyme that mediates the insertion of four additional nucleotides into a predetermined position of a substrate RNA, and thus mimics, at the RNA level, the repair of a short deletion mutation; 17% of the initial substrate was converted to the insertion product.
Site‐determined functionalization of large RNA molecules can be achieved by a strategy involving twin ribozymes and small synthetic oligonucleotides. The twin ribozyme mediates the exchange of a ...predefined patch of RNA sequence against a modified oligonucleotide to result in specific functionalization of the large RNA.
Outside influence: External regulation of hairpin ribozyme derivatives by oligonucleotide effectors offers the possibility of switching activity in a defined way and opens up attractive methods for ...structure and function analysis. An oligonucleotide effector was used to reactivate two inactive mutants of the hairpin ribozyme (see figure). The effector replaced the mutated sequence of the ribozyme and restored its catalytically competent conformation. The same technique could be used to interrupt and then reactivate the catalytic cycle at specific steps and thus to study the structure and function of other ribozymes.
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