Long lasting analgesic effect of anti-NGF and anti-TrkA antibodies in neuropathic pain model
Mara
D'Onofrio1,
Ivan
Arisi1,
Elena
Fiori1, 2, 3,
Rossella
Brandi1,
Francesca
Malerba1,
Federico
La Regina1,
Renata
Cerna1, 4,
Marzia
Giordano1,
Lorenzo
Di Battista1,
Sabrina
Turturro1,
Flaminia
Pavone3 and
Antonino
Cattaneo1, 4*
-
1
European Brain Research Institute, Italy
-
2
Sapienza University, Italy
-
3
CNR, Institute of Cell Biology and Neurobiology, Italy
-
4
Scuola Normale Superiore, Italy
Aim of Investigation:
So far, there are no effective treatments for neuropathic pain (NP) and current treatments suffer from unwanted side effects. The neurotrophin system has emerged as a promising target for NP, in particular the Nerve Growth Factor (NGF) and its high affinity receptor (TrkA). We have previously demonstrated that anti-NGF and anti-TrkA monoclonal antibodies (mAb, specifically synthesized in the same lab) induce a long lasting analgesic effect in NP models (Ugolini et al, PNAS 2007; Covaceuszach et al, Plos One 2012), up to 21 days after the last dosing. To understand the role of the neurotrophin system in NP, both as a crucial regulator and a potential therapeutic target, we evaluated in a mouse model of NP:
i) the time course of the analgesic effect and how long this effect was maintained following the end of the antibody treatment;
ii) the gene expression changes during and after the NGF and TrkA targeting analgesic treatments;
iii) structural and morphological changes in peripheral and central nervous system, following the antibody treatments.
The final aims of this project are:
I. identifying, developing and validating new specific therapeutic targets for NP;
II. developing a pipeline of second generation NGF-system-based compounds, for an improved control of NP.
Methods:
To induce NP, standard Chronic Constriction Injury (CCI) of the right sciatic nerve was performed in C57BL/6J adult male mice. Mice were intraperitoneally administered with mAb anti-TrkA or mAb anti-NGF (2 different doses: 70 or 100 microg/mouse/day) from day 3 until day 10 post CCI. The following experimental groups were analyzed with different methodologies: untreated non-operated naive mice, saline-injected CCI mice, CCI mice treated with anti-NGF mAb (two doses), CCI mice treated with anti-TrkAmAb (two doses). Measurement of serum antibody levels in blood was performed by ELISA. Analgesic effects were tested with a Dynamic Plantar Aesthesiometer. The behavioural test was performed both on ipsi-lateral and contra-lateral paws of all CCI mice. For these tests, at least n=10 animals were used for each experimental group.
The transcriptomic analysis was performed by the Agilent one-color microarray platform, using 8x60K whole mouse genome chips (grid-ID 028005). Three tissues were isolated: L4 Dorsal Root Ganglia (DRG), lumbar spinal cord and prefrontal cortex were collected for microarray profile at days D3, D11, D24, D90 post CCI. n=4 animals were used for each experimental point (treatment, time point, tissue), with the final dataset composed by n= 164 samples. Transcriptomic data analysis was performed using R-Bioconductor and online tools. After discarding features too close to background noise, microarray data were normalized (aligned) to the 75th percentile. The R package Limma was used to compile differentially expressed gene (DEG) lists. Principal Component Analysis, Multi-dimensional Scaling and hierarchical clustering were used to classify samples. Functional analysis of gene lists (Gene Ontology-like classification) was performed by R scripts and online tools.
All animal handling and experimentations followed standard national and international guidelines on animal use. All animal experimentations are authorized by a specific protocol approved by the Italian Ministry of Health (n. 21/2014, according to D.Lgs 26/2014).
This research was funded by: European Commission FP7 Collaborative grant n.603191, acronym PAINCAGE, title "The NGF system and its interplay with endocannabinoid signalling, from peripheral sensory terminals to the brain: new targets for the development of next generation drugs for neuropathic pain".
Results:
Treatments with anti-NGF mAb or anti-TrkA mAb induce significant dose- and time-dependent analgesic effects, which show a different behaviour, with a specific time course: the anti-NGF is active since the earliest post-CCI phase, while the anti-TrkA seems to become effectively analgesic some days later. The higher doses of both antibodies have long-lasting antiallodynic effect, from D10 to D90 post-CCI. Antibody levels measured in blood serum by ELISA are consistent with a progressive degradation of the drugs.
As to the transcriptomic study in the CCI model, both antibodies modulate a large set of genes in the peripheral nervous system (DRG) compared to saline-treated group. In the Central Nervous System the anti-NGF induces a large transcriptional modulation also in the spinal cord and cortex, while the anti-TrkA affects a very small amount of genes in the same central tissues.
Moreover, the DEGs modulated by the two antibodies in DRG show only a partial overlap. Gene categories affected by treatments include: synaptic components, immune system, inflammation, ionic channels, synaptic signalling.
Conclusions:
The behavioural and transcriptomic data prove that anti-NGF and anti-TrkA antibodies counteract NP in CCI model, showing a very long lasting analgesic effect and induction of regenerative processes. The anti-NGF and anti-TrkA antibodies are both analgesic, but with different dose and time-dependent effect: some differences are evident shortly after the CCI induction, while the analgesic phenotype is similarly effective on a longer time scale. This suggests that they follow partially different pharmacological mechanisms.
This idea is supported even more clearly by the transcriptomic profile: the two antibodies modulate different genes peripherally in DRGs, though acting on the same neurotrophin system, while in spinal cord and cortex only the anti-NGF seems to impact on the expression profile.
The DEGs induced by the anti-TrkA and anti-NGF mAbs in DRGs reveal a large number of potential new targets linked to long lasting analgesia. This is the first direct transcriptomic comparison of the analgesic effects of anti-NGF and anti-TrkA antibodies in a well characterized neuropathic pain model. The results strongly support the importance of NGF and TrkA for the pharmacological control of NP and characterization of new and more specific molecular targets.
Keywords:
neuropathic pain,
dorsal root ganglia,
NGF targeting,
Analgesic drugs,
CCI mouse model
Conference:
The Cerebellum inside out: cells, circuits and functions
, ERICE (Trapani), Italy, 1 Dec - 5 Dec, 2016.
Presentation Type:
poster
Topic:
Cellular & Molecular Neuroscience
Citation:
D'Onofrio
M,
Arisi
I,
Fiori
E,
Brandi
R,
Malerba
F,
La Regina
F,
Cerna
R,
Giordano
M,
Di Battista
L,
Turturro
S,
Pavone
F and
Cattaneo
A
(2019). Long lasting analgesic effect of anti-NGF and anti-TrkA antibodies in neuropathic pain model.
Conference Abstract:
The Cerebellum inside out: cells, circuits and functions
.
doi: 10.3389/conf.fncel.2017.37.00005
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Received:
28 Nov 2016;
Published Online:
25 Jan 2019.
*
Correspondence:
Prof. Antonino Cattaneo, Scuola Normale Superiore, Pisa, Italy, antonino.cattaneo@sns.it