Yi Ai

Point source concentration of GDNF may explain failure of phase II clinical trial.

Significant differences have been reported in results from three clinical trials evaluating intraputamenal infusion of glial cell line-derived neurotrophic factor (GDNF) for the treatment of Parkinsons disease. To determine if problems in drug bioavailability could have contributed to the discrepancies between studies, we have analyzed the distribution of intraputamenally infused GDNF in the rhesus monkey brain using the delivery system and infusion protocol followed in a phase 2 clinical trial that failed to achieve its primary endpoint. I125-GDNF was unilaterally infused into the putamen of three adult rhesus monkeys for 7 days. Three age- and sex-matched animals received vehicle infusions following identical procedures. GDNF levels in the brain, peripheral organs, blood and CSF were quantified and mapped by GDNF immunocytochemistry, GDNF ELISAs and I125 measurements. Infused GDNF was found to be unevenly concentrated around the catheter, with tissue levels dropping exponentially with increasing distance from the point source of the single opening in the catheter tip. The volume of distribution of GDNF around the catheter, as determined by immunocytochemistry, varied over four-fold between animals ranging from 87 to 369 mm3. The concentration of GDNF around the catheter tip and limited diffusion into surrounding brain parenchyma support the hypothesis that drug bioavailability was limited to a small portion (2-9%) of the human putamen in the clinical trial using this catheter and infusion protocol.

Intraputamenal infusion of GDNF in aged rhesus monkeys: Distribution and dopaminergic effects

Site‐specific delivery of trophic factors in the brain may be important for achieving therapeutic efficacy without unwanted side effects. This study evaluated the site‐specific infusion of glial cell line–derived neurotrophic factor (GDNF) into the right putamen of aged rhesus monkeys. After 4 weeks of continuous infusion at a rate of 22.5 μg/day, GDNF had diffused up to 11 mm from the catheter openings in the putamen into the rostral putamen, internal capsule, external capsule, caudate nucleus, and globus pallidus. Anisotropic flow along the external capsule tracts carried GDNF into the anterior amygdaloid area. Backflow of GDNF along the catheter track from the frontal cortex infiltrated juxtaposed corpus callosal and cortical tissue. GDNF was carried by retrograde transport to dopamine neurons in the ipsilateral substantia nigra, stimulating an 18% increase in the number of tyrosine hydroxylase (TH)–positive dopamine neurons and a 28% increase in dopamine neuron perikaryal size. Also, TH‐positive fiber density was increased in the ipsilateral globus pallidus, caudate nucleus, and putamen. Anatomic effects from GDNF stimulation of the dopaminergic system were restricted to the ipsilateral hemisphere. Retrograde GDNF labeling was also present in a few TH‐positive neurons in the locus coeruleus and a large cluster of TH‐negative neurons in the ventral anterior thalamus. Anterograde transport of GDNF was evident in axons in the pyramidal tract from the cerebral peduncle to the caudal spinal cord. Tissue injury from the intraparenchymal catheter and continuous infusion was confined primarily to a narrow zone surrounding the track and was mild to moderate in severity. J. Comp. Neurol. 461:250–261, 2003.

Progenitor proliferation in the adult hippocampus and substantia nigra induced by glial cell line-derived neurotrophic factor.

Neurogenesis is an ongoing process in the hippocampus and olfactory bulb of adult mammals, regulated in part by trophic factors. While glial cell line-derived neurotrophic factor (GDNF) is being directly delivered into the nigrostriatal system of the brain for the treatment of Parkinsons disease in clinical trials, little is known about its effects on cell genesis in the brain. Here, we investigated the effects of GDNF on progenitor cell proliferation and differentiation in two GDNF-responsive areas, the hippocampus and substantia nigra. GDNF (18 microg/day) was infused in the striatum of 2-month-old Sprague-Dawley rats for 28 days. New cells were identified by the nuclear incorporation of 5-bromo-2-deoxyuridine (BrdU) and analyzed by light and electron microscopic immunostaining and quantitative morphometric techniques. GDNF significantly increased cell proliferation in the hippocampus by 78% and in the substantia nigra by 52%. There was no evidence of neurogenesis in the substantia nigra, with new cells displaying glial features and none of the 1549 BrdU-positive cells co-labeled for the dopamine neuronal marker tyrosine hydroxylase (TH). Rather, GDNF upregulated TH in existing neurons, consistent with the restorative actions of this tropic factor. The hippocampus is a site that supports adult neurogenesis and new cells generated here were closely associated with granule cells in the dentate gyrus. Some were double labeled for the neuronal marker NeuN; others had features of astrocytes, the principal source of new adult neurons in the hippocampus. The effects of GDNF on the hippocampus are potentially important in memory and learning processes.

Six-month partial suppression of Huntingtin is well tolerated in the adult rhesus striatum

Huntingtons disease is caused by expression of a mutant form of Huntingtin protein containing an expanded polyglutamine repeat. One possible treatment for Huntingtons disease may be to reduce expression of mutant Huntingtin in the brain via RNA interference. Unless the therapeutic molecule is designed to be allele-specific, both wild-type and mutant protein will be suppressed by an RNA interference treatment. A key question is whether suppression of wild-type as well as mutant Huntingtin in targeted brain regions can be tolerated and result in a net benefit to patients with Huntingtons disease. Whether Huntingtin performs essential functions in the adult brain is unclear. Here, we tested the hypothesis that the adult primate brain can tolerate moderately reduced levels of wild-type Huntingtin protein for an extended period of time. A serotype 2 adeno-associated viral vector encoding for a short hairpin RNA targeting rhesus huntingtin messenger RNA (active vector) was bilaterally injected into the striatum of four adult rhesus monkeys. Four additional animals received a comparable vector encoding a scrambled control short hairpin RNA (control vector). General health and motor behaviour were monitored for 6 months. Upon termination, brain tissues were sampled and assessed blindly for (i) huntingtin messenger RNA knockdown; (ii) Huntingtin protein expression; and (iii) neuropathological changes. Reduction in wild-type huntingtin messenger RNA levels averaging ∼30% was measured in the striatum of active vector recipients 6 months post-injection. A widespread reduction in Huntingtin protein levels was also observed by immunohistochemistry in these animals, with an average protein reduction of ∼45% relative to controls measured by western blot analysis in the putamen of active vector recipients. As with control vector recipients, no adverse effects were observed behaviourally, and no neurodegeneration was found on histological examination of active vector recipients. Our results suggest that long-term partial suppression of wild-type Huntingtin may be safe, and thus if a comparable level of suppression of mutant Huntingtin is beneficial, then partial suppression of both wild-type and mutant Huntingtin may result in a net benefit in patients with heterozygous Huntingtons disease.

Trophic factor distribution predicts functional recovery in parkinsonian monkeys.

Glial cell line–derived neurotrophic factor (GDNF) promotes the survival, growth, and regeneration of dopamine neurons in the midbrain that degenerate in Parkinsons disease. However, translating successful animal studies into effective clinical therapy for Parkinsons disease has proved difficult. In this article, using pulsed infusion for convection‐enhanced delivery of GDNF, we have analyzed two variables hypothesized to be important for achieving efficacy: dose and GDNF distribution in the target tissue. Motor functions were significantly improved in rhesus monkeys with unilateral 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine–induced parkinsonism that received midbrain infusion of GDNF for 10 weeks. The volume of distribution of GDNF in the five trophic factor recipients varied more than fivefold, from 59 to 325 mm3, and significantly correlated with motor function improvements. Significant increases were evident in the number of midbrain dopamine neurons immunopositive for tyrosine hydroxylase in both the substantia nigra and ventral tegmental area. Based on neurochemical and quantitative morphological measures, GDNF administration promoted recovery of both the nigrostriatal and ventral tegmental area–nucleus accumbens dopaminergic pathways without producing evident side effects. Increasing the dose threefold did not increase efficacy, suggesting that after achieving a critical threshold, GDNF tissue distribution is more important than dose for trophic stimulation of dopamine neurons. Ann Neurol 2005;58:224–233

Correlation of R2 with total iron concentration in the brains of rhesus monkeys

To estimate the relationship between R2 = 1/T2 as measured with a double echo spin echo sequence and total iron concentration in gray matter structures in the brains of aging rhesus monkeys.

Widespread suppression of huntingtin with convection-enhanced delivery of siRNA

Huntingtons disease is an autosomal dominant neurodegenerative disease caused by a toxic gain of function mutation in the huntingtin gene (Htt). Silencing of Htt with RNA interference using direct CNS delivery in rodent models of Huntingtons disease has been shown to reduce pathology and promote neuronal recovery. A key translational step for this approach is extension to the larger non-human primate brain, achieving sufficient distribution of small interfering RNA targeting Htt (siHtt) and levels of Htt suppression that may have therapeutic benefit. We evaluated the potential for convection enhanced delivery (CED) of siHtt to provide widespread and robust suppression of Htt in nonhuman primates. siHtt was infused continuously for 7 or 28 days into the nonhuman primate putamen to analyze effects of infusion rate and drug concentration on the volume of effective suppression. Distribution of radiolabeled siHtt and Htt suppression were quantified by autoradiography and PCR, respectively, in tissue punches. Histopathology was evaluated and Htt suppression was also visualized in animals treated for 28 days. Seven days of CED led to widespread distribution of siHtt and significant Htt silencing throughout the nonhuman primate striatum in an infusion rate and dose dependent manner. Htt suppression at therapeutic dose levels was well tolerated by the brain. A model developed from these results predicts that continuous CED of siHtt can achieve significant coverage of the striatum of Huntingtons disease patients. These findings suggest that this approach may provide an important therapeutic strategy for treating Huntingtons disease.

Intraputamenal Infusion of Exogenous Neurturin Protein Restores Motor and Dopaminergic Function in the Globus Pallidus of MPTP-Lesioned Rhesus Monkeys

The neurorestorative effects of exogenous neurturin (NTN) delivered directly into the putamen via multiport catheters were studied in 10 MPTP-lesioned rhesus monkeys expressing stable parkinsonism. The parkinsonian animals were blindly assigned to receive coded solutions containing either vehicle (n = 5) or NTN (n = 5, 30 μg/day). Both solutions were coinfused with heparin using convection-enhanced delivery for 3 months. The NTN recipients showed a significant and sustained behavioral improvement in their parkinsonian features during the treatment period, an effect not seen in the vehicle-treated animals. At study termination, locomotor activity levels were increased by 50% in the NTN versus vehicle recipients. Also, DOPAC levels were significantly increased by 150% ipsilateral (right) to NTN infusion in the globus pallidus, while HVA levels were elevated bilaterally in the NTN-treated animals by 10% on the left and 67% on the right hemisphere. No significant changes in DA function were seen in the putamen. Volumetric analysis of putamenal NTN labeling showed between-subject variation, with tissue distribution ranging from 214 to 744 mm3, approximately equivalent to 27–93% of area coverage. Our results support the concept that intraparenchymal delivery of NTN protein may be effective for the treatment of PD. More studies are needed to determine strategies that would enhance tissue distribution of exogenous NTN protein, which could contribute to optimize its trophic effects in the parkinsonian brain.

Intracranial delivery of proteins and peptides as a therapy for neurodegenerative diseases.

Parkinsons disease is characterized by a progressive degeneration of the substantia nigra pars compacta dopamine neurons that innervate the striatum. Unlike current treatments for PD, GDNF administration could potentially slow or halt the continued degeneration of nigral dopaminergic neurons. GDNF does not cross the blood-brain barrier and needs to be administered directly into the brain. Due to the progressive nature of PD, sustained delivery of trophic factors may be necessary for optimal, long-term neuronal effects. Novel methods for sustained delivery of GDNF into the nigrostriatal pathway are currently being studied in non-human primates, including computer-controlled infusion pumps. Using this approach, we have demonstrated that chronic infusions of nominally 7.5 or 22.5 microg/day GDNF into the lateral ventricle, the putamen or the substantia nigra, using programmable pumps, promotes restoration of the nigrostriatal dopaminergic system and significantly improves motor functions in MPTP-lesioned rhesus monkeys with neural deficits modeling the terminal stages of PD and in aged rhesus monkeys modeling the early stages of PD. Based on the promising studies of the chronic effects of GDNF in non-human primate models of PD, a study was recently conducted in England on five advanced PD patients. Chronic GDNF infusion into the dorsal putamen, via programmable pumps, resulted in improved motor function in all patients and limited side effects were observed. However, while the data from this intraparenchymal clinical trial in humans look encouraging, extensive blinded efficacy trials will need to be conducted before it can be determined if chronic treatment with GDNF or other trophic molecules will prove useful in treating patients with PD.

Dopamine Neuron Stimulating Actions of a GDNF Propeptide

Background Neurotrophic factors, such as glial cell line-derived neurotrophic factor (GDNF), have shown great promise for protection and restoration of damaged or dying dopamine neurons in animal models and in some Parkinsons disease (PD) clinical trials. However, the delivery of neurotrophic factors to the brain is difficult due to their large size and poor bio-distribution. In addition, developing more efficacious trophic factors is hampered by the difficulty of synthesis and structural modification. Small molecules with neurotrophic actions that are easy to synthesize and modify to improve bioavailability are needed. Methods and Findings Here we present the neurobiological actions of dopamine neuron stimulating peptide-11 (DNSP-11), an 11-mer peptide from the proGDNF domain. In vitro, DNSP-11 supports the survival of fetal mesencephalic neurons, increasing both the number of surviving cells and neuritic outgrowth. In MN9D cells, DNSP-11 protects against dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA)-induced cell death, significantly decreasing TUNEL-positive cells and levels of caspase-3 activity. In vivo, a single injection of DNSP-11 into the normal adult rat substantia nigra is taken up rapidly into neurons and increases resting levels of dopamine and its metabolites for up to 28 days. Of particular note, DNSP-11 significantly improves apomorphine-induced rotational behavior, and increases dopamine and dopamine metabolite tissue levels in the substantia nigra in a rat model of PD. Unlike GDNF, DNSP-11 was found to block staurosporine- and gramicidin-induced cytotoxicity in nutrient-deprived dopaminergic B65 cells, and its neuroprotective effects included preventing the release of cytochrome c from mitochondria. Conclusions Collectively, these data support that DNSP-11 exhibits potent neurotrophic actions analogous to GDNF, making it a viable candidate for a PD therapeutic. However, it likely signals through pathways that do not directly involve the GFRα1 receptor.