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LRRK2 Inhibitors May Benefit Parkinson’s Patients With and Without Genetic Mutation, Study Finds

LRRK2

Inhibiting the activity of LRRK2 kinase — an enzyme whose mutated form is one of the most common genetic causes of Parkinson’s disease — may benefit patients both with and without this disease-related mutation, a study finds.

Molecules that block the activity of the LRRK2 kinase — such as DNL201 and DNL151, both being developed by Denali Therapeutics — are currently being tested in clinical trials.

The results of this study, “LRRK2 inhibition prevents endolysosomal deficits seen in human Parkinson’s disease,” were published in Neurobiology of Disease. The research was supported by the Michael J. Fox Foundation.

Mutations in the leucine rich repeat kinase 2 (LRRK2) gene are one of the most commonly known genetic causes of Parkinson’s disease. Evidence indicates that in people with idiopathic Parkinson’s, in which the disease has no known cause, the LRRK2 protein is overly active, regardless of the patient’s mutation status — whether or not they have a mutated LRRK2. That overly active protein leads to the malfunctioning of lysosomes, the special compartments within cells that digest and recycle different types of molecules. Lysosomal dysfunction is involved in the formation of  protein aggregates, or clumps, called Lewy bodies, which contribute to Parkinson’s and, therefore, neurodegeneration.

Therapies that can inhibit, or block LRRK2 are currently being tested in human clinical trials. However, it is still unclear whether blocking LRRK2 protein activity in people with idiopathic Parkinson’s can prevent lysosomal dysfunction and consequent neurodegenerative processes.

To learn more, investigators at the University of Pittsburgh now studied post-mortem brain samples, specifically from a motor brain region called the substantia nigra, which is severely damaged in Parkinson’s. The researchers characterized lysosomal abnormalities in the surviving dopaminergic neurons — the main source of dopamine, the loss of which is a hallmark of this disease — of idiopathic Parkinson’s patients.

When compared with healthy controls, Parkinson’s patients had more abnormal lysosomes. These changes occurred during the early stages of lysosomal development, the researchers found.

The team then investigated whether these post-mortem cellular findings could be replicated in an animal model of Parkinson’s. Rats were given two distinct dose regimens of rotenone, a pesticide that inhibits mitochondria, or the “powerhouses” of cells. Blocking mitochondria leads to cellular death and the onset of parkinsonian features.

Nine to 14 daily doses of rotenone reproduced many idiopathic Parkinson’s features, including lysosomal defects. This caused neurodegeneration in the striatum and substantia nigra, two brain areas involved in motor control.

Interestingly, five daily doses of the pesticide weren’t enough to cause cell death, but did increase the accumulation of Parkinson’s-related alpha-synuclein protein and produce changes in lysosomes.

“These data demonstrate that, in rotenone-treated rats, [alpha]-synuclein protein levels rise in the dopaminergic neurons prior to the onset of frank neurodegeneration,” the researchers said.

When overactive LRRK2 was blocked in rotenone-treated rats, the protein’s activity was reduced. That, in turn, improved the overall health of lysosomes and prevented the accumulation of alpha-synuclein. These effects were observed in animals without a genetic predisposition to develop Parkinson’s, suggesting that the LRRK2 kinase inhibitors may be effective beyond LRRK2-mutated patients.

“Our work suggests that drugs that block LRRK2, some of which have entered clinical trials, will be useful for people with typical Parkinson’s disease,” J. Timothy Greenamyre, MD, PhD, the study’s lead author, said in a press release.

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PF-360 Provides Some Benefits But Does Not Improve Dopaminergic Function, Mouse Study Shows

PF-360 mouse study

Treatment with PF-360, an investigational leucine-rich repeat kinase 2 (LRRK2) inhibitor, can efficiently decrease LRRK2’s phosphorylation levels, known to be elevated in Parkinson’s patients, in the brains of a mouse model of Parkinson’s disease, a preclinical study reports.

However, despite some observed dose-dependent therapeutic effects, including gait improvement, no robust changes in dopaminergic function were observed.

Results of the study were recently presented during the Society for Neuroscience’s 2018 conference in San Diego in a poster titled “Assessment of the Anti-parkinsonian Effects of the Potent and Selective LRRK2 Kinase Inhibitor PF-360 in the AAV-A53T Mouse Model of Parkinson’s Disease.”

The study was the result of a collaboration between several institutions including Charles River Discovery, Merck, Pfizer, Atuka Inc., and The Michael J. Fox Foundation for Parkinson’s Research.

The LRRK2 gene provides instructions for making a kinase, which is a protein that regulates the function of other molecules. Mutations in this gene put the protein into an overly activated state.

Mutations in the LRRK2 gene are one of the most commonly known genetic causes of Parkinson’s disease and usually result in the malfunctioning of lysosomes — special compartments within cells that digest and recycle different types of molecules. Lysosomal dysfunction is involved in the formation of Lewy body protein aggregates and, therefore, neurodegeneration.

Scientists believe that blocking LRRK2’s activity has the potential to slow disease progression.

Using a selective LRRK2 inhibitor called PF-360, researchers studied the dose-response efficacy of the potential therapy in two different mouse models (C57BL/6J and LRRK2-G2019S) that were injected with a “biological cocktail” of an adeno-associated virus combined with a human mutated A53T alpha-synuclein (AAV-A53T) — the major component of protein clumps called Lewy bodies, a hallmark of Parkinson’s.

They used 90 C57BL/6J mice 10-12 weeks old and 105 LRRK2-G2019S mice, 75 of which were 11-12 weeks old and 30 were 5-6 months old. In mouse “time,” 12 weeks is equal to adulthood.

This induced the degeneration of dopaminergic neurons in an area of the brain called the substantia nigra and decreased dopamine and tyrosine hydroxylase — the enzyme responsible for catalyzing levels of L-DOPA, the precursor to dopamine — in the striatum, mimicking Parkinson’s disease.

Mice were treated for 42 days with a diet containing PF-360 or a placebo (control), which was begun seven days prior to AAV-A53T injections.

PF-360 inhibited LRRK2 phosphorylation in the animals’ brain cortex and lungs at a specific site of the protein called serine 935 (serine is an amino acid, or the proteins’ building block). This protein region is required for interaction of LRRK2 with other molecules.

Phosphorylation (the adding of a phosphate group) alters a protein’s structure turning it, for instance, into an activated or deactivated state. As such, phosphorylation is the most common mechanism of regulating protein function and transmitting signals throughout the cell.

Pronounced therapeutic effects were observed with increasing doses (1 mg/kg, 3 mg/kg, 10 mg/kg, 30 mg/kg, and 60 mg/kg of PF-360) in both animal strains and age groups.

AAV-A53T injection led to motor impairments such as decreased speed (longer stride duration, shorter step length), slower swing speed, and reduced hind limb protraction (forward extension).

LRRK2-G2019S mice at 11-12 weeks old recovered their hind limb protraction and retraction with 10 mg/kg of PF-360, while older animals at 5-6 weeks of age had their overall speed (stride duration and swing speeds) improved with 30 mg/kg of the treatment.

No gait changes were observed after 42 days of PF-360 treatment in C57BL/6J mice. However, there was an insignificant treatment-related trend toward increased tyrosine hydroxylase-positive cells in the substantia nigra of C57BL/6J animals.

After treatment, a significantly higher number of tyrosine hydroxylase-positive cells were observed in older LRRK2-G2019S mice.

An increase in tyrosine hydroxylase-positive cells is indicative of an increase in the number of nerve cells that can produce either L-DOPA or dopamine.

Neurochemical analysis revealed that PF-360 delivery to younger animals did not improve striatum levels of dopamine or the intermediate end products of dopamine’s metabolism (3,4-dihydroxyphenylacetic acid and homovanillic acid).

However, treatment significantly increased homovanillic acid levels in older LRRK2-G2019S mice.

Given that most evidence suggests an LRRK2 contribution to Parkinson’s disease via abnormal phosphorylation, this study shows that although PF-360 can reduce LRRK2 phosphorylation levels, both in the brain and in the periphery, it failed to show robust improvements in dopaminergic function.

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CENTOGENE, Denali Partnering to Recruit LRRK2 Parkinson’s Patients for Clinical Trials

CENTOGENE, Denali collaboration

CENTOGENE and Denali Therapeutics are teaming up to identify and recruit Parkinson’s disease patients who carry mutations in the LRRK2 gene for future clinical trials, the companies announced.

CENTOGENE will carry out a targeted global recruitment campaign to identify and characterize Parkinson’s patients with LRRK2 mutations  and sequence the LRRK2 gene in this population, using its proprietary CentoCard, a dried blood spot collection kit.

CENTOGENE-recruited patients will participate in clinical trials supporting Denali’s LRRK2 inhibitor therapy program.

Mutations in the LRRK2 gene are one of the most commonly known genetic causes of Parkinson’s disease and usually result in the malfunctioning of lysosomes — special compartments within cells that digest and recycle different types of molecules.

Lysosomal dysfunction is involved in the formation of Lewy body protein aggregates and, therefore, neurodegeneration. LRKK2 regulates the formation and function of lysosomes, which are impaired in Parkinson’s disease and may eventually be restored by inhibiting LRRK2 activity, both in patients with a genetic LRRK2 mutation as well as in those with sporadic Parkinson’s disease.

“Our exclusive collaboration underscores CENTOGENE’s CEO and founder of CENTOGENE, said in a press release. “We believe we can contribute to Denali’s development of disease modifying medicines for patients with Parkinson’s disease. CENTOGENE will help Denali speed up the enrollment of patients in clinical studies for its LRRK2 program.”

CENTOGENE’s dried blood spot collection kit contains a validated procedure to extract high-quality DNA, enzymes, and biomarkers from patients’ blood samples. The technology has several advantages, including ease of handling — the samples are stable once they are dry and can be sent to CENTOGENE by regular mail — no sensitivity over time or to temperature, and cost-effectiveness.

This aids in the delivery of biological samples and makes genetic, enzymatic, and biomarker testing available worldwide.

After the identification of LRRK2 Parkinson’s patients, data will be sent to Denali to potentially recruit patients for its clinical trials testing LRRK2 inhibitor therapies.

Denali’s Parkinson’s pipeline includes two investigational LRRK2 inhibitors currently in the early phase of development, DNL201 and DNL151.

The company’s latest results revealed that DNL201 was safe and well-tolerated by healthy individuals in a Phase 1 clinical trial. The investigational therapy is currently being tested in a dose-escalation Phase 1 study in healthy volunteers in the Netherlands.

“Denali is the first company to conduct clinical trials with LRRK2 inhibitors for the treatment of Parkinson’s disease. This partnership with CENTOGENE is a central part of our global efforts to identify and recruit PD [Parkinson’s disease] patients with a mutation in the LRRK2 gene into our planned clinical studies,” said Carole Ho, MD, chief medical officer and head of development at Denali. “We are impressed with CENTOGENE’s approach and technology and believe that our joint efforts will accelerate the enrollment of PD patients and the completion of our clinical trials.”

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