Patent Application
20220409602
The disclosure relates to dopamine D3 receptor ligand compositions and methods of use of these compositions for treatment of dyskinesia. These compositions are also used specifically in levodopa-induced dyskinesia experienced in Parkinson's disease (PD).
PD is a complex multisystem, chronic and so far incurable disease with significant unmet medical needs. In the United States, over a million people suffer from PD and 60,000 new cases are diagnosed each year with an estimated cost of USD 27 billion per year. As the incidence of PD increases with aging, with an additional trend of increased incidence in men over the age of 70, the expected burden will continue to escalate with the aging population. PD pathology is associated primarily with the death of dopamine neurons in the substantia nigra and manifests with motor and non-motor dysfunctions including tremor, bradykinesia, rigidity, cognitive deficits, and sleep disturbances.
These symptoms are managed by dopamine-replacement pharmacological treatments aiming at enhancing dopamine in the striatum with the dopamine precursor L-3,4-dihydroxy-phenylalanine (levodopa). Since its discovery in the 1961, levodopa remains the gold standard pharmacotherapy as it produces effective relief of the motor symptoms. However, the progressive nature of PD associated with the degenerative process within and beyond the nigrostriatal system causes a multitude of side effects within 5 years including the uncontrolled involuntary movements or levodopa-induced dyskinesia, the “wearing OFF” and the “ON/OFF” motor fluctuations. Over 80% of Parkinsonian patients treated with levodopa develop levodopa-induced dyskinesia after five years. These debilitating side effects spurred the discovery of alternative dopamine-replacement pharmacological treatments. Examples are general dopamine receptor agonists, inhibitors of enzymes that degrade dopamine, and the anti-dyskinetic drug in current use, amantadine. These alternative treatments helped mitigate some of the side effects. Unfortunately, each drug elicits a new range of additional side effects making it challenging to manage the disease. Surgical treatments including deep brain stimulation (DBS) are alternatives that improve PD symptoms and dyskinesia. However, DBS has potential adverse events associated with the surgical procedure, the device, and stimulation and its loss of benefit parallels the progressive degenerative changes associated with PD.
Provided here are compositions and methods to address these shortcomings of the art and provide other additional or alternative advantages. Embodiments include methods of treating dyskinesia by administering an effective amount of a dopamine receptor 3 ligand containing an arylpiperazine pharmacophore or a pharmaceutically acceptable salt, derivative, hydrate or solvate thereof. In certain embodiments, the dyskinesia is levodopa-induced dyskinesia. In certain embodiments, the dopamine receptor 3 ligand is a N-(4-(4-phenyl piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamide D3 ligand. In certain embodiments, the dopamine receptor 3 ligand is PD13R (as shown in Formula 1 below). In certain embodiment, the dopamine receptor 3 ligand is administered as a thiophenyl benzoate salt of PD13R.
In certain embodiments, the dopamine receptor 3 ligand containing an arylpiperazine pharmacophore or a pharmaceutically acceptable salt, derivative, hydrate or solvate thereof, also contains a phenyl thiophene moiety. In certain embodiments, the arylpiperazine pharmacophore and the phenyl thiophene moiety are separated by a spacer. In certain embodiments, the dopamine receptor 3 ligand containing an arylpiperazine pharmacophore or a pharmaceutically acceptable salt, derivative, hydrate or solvate thereof, also contains one or more of a heterocyclic ring, such as triazole, imidazole, pyrimidine, pyrrazole, and pyridine. In certain embodiments, the dopamine receptor 3 ligand contains one or more of a piperazine bioisostere and a bicyclic bioisostere, or a pharmaceutically acceptable salt, derivative, hydrate or solvate thereof. In certain embodiments, the dopamine receptor 3 ligand is orally administered at a dosage ranging from about 1 mg/kg to about 20 mg/kg.
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Embodiments will be readily understood by the following detailed description in conjunction with the accompanying drawings. These drawings illustrate the principles of the disclosure and no limitation of the scope of the disclosure is thereby intended.
Dopamine actions are mediated through five-member family of G-protein-coupled receptors (GPCRs) classified into two subtypes, D1-like and D2-like receptors based on sequence homology, G-protein coupling and pharmacology. D1R and D5R are coupled to the stimulatory D1-like G-protein family members (Gs/olf), while D2R, D3R, and D4R are coupled to the inhibitory D2-like G-protein family members (Gi/o). Although dopamine D3 receptor (D3R) has been implicated in PD and levodopa-induced dyskinesia, the high sequence homology between the D3R and D2R, including in the transmembrane region and the orthosteric binding site that binds dopamine, has made it challenging to translate promising leads to the clinic.
D3R plays critical role in the central nervous system health and disease. Although it is well known for its involvement in drug abuse and addiction, reward, cognition, schizophrenia, impulse control disorders and Parkinson's disease, D3R has been implicated in a variety of other functions including neuroinflammation, neurogenesis, protein aggregation and neurotrophic factor secretion. The expression of D3R in Parkinson's disease animal models and patients mirrors the dynamic state of the disease impacted by the timing of the PD-like symptoms development, the evolution of the neurodegenerative process and the therapeutic intervention such as the levodopa treatment. Previous reports have also suggested that D3R expression is species specific, in rat models the D3R expression is limited to dopaminergic areas known to be associated with cognitive and emotional functions. In the NHP models, the D3R expression closely resemble human pattern with a widespread expression in motor and associative structures of the basal ganglia and in limbic system. In all these species however, D3R is down regulated in the basal ganglia after the stabilization of PD-like symptoms and D3R increases in expression and binding in caudate, putamen and globus pallidus after levodopa treatment and development of levodopa-induced dyskinesia. D3R was investigated for its role in the genesis and expression of levodopa-induced dyskinesia and as a viable therapeutic target for treatment of such effects.
In both rodent and nonhuman primate models of PD with levodopa-induced dyskinesia, D3R expression is decreased during the development of PD-like symptoms and is abnormally up-regulated in the caudate and putamen after prolonged levodopa treatment and levodopa-induced dyskinesia expression. These data suggest the involvement of D3R in the pathogenesis of motor complications and levodopa-induced dyskinesia following levodopa pharmacotherapy.
Two novel D3 receptor ligands were investigated, PD13R and SWR-3-73, which belong to two separate classes of pharmacophores: the arylpiperazine and the diazaspiro, respectively in treating levodopa-induced dyskinesia in a nonhuman primate model of PD. Both compounds had high affinity and selectivity for D3R and they acted as partial agonists via G-protein by inhibiting the adenyl cyclase signaling pathway and decreasing cAMP production. PD13R inhibited the expression of levodopa-induced dyskinesia while improving motor functions and sleep efficiency in dyskinetic animal models. SWR-3-73 did not inhibit expression of levodopa-induced dyskinesia nor the Parkinson's-like symptoms.
Embodiments include methods of treating dyskinesia by administering an effective amount of a dopamine receptor 3 ligand containing an arylpiperazine pharmacophore or a pharmaceutically acceptable salt, derivative, hydrate or solvate thereof. In certain embodiments, the dyskinesia is levodopa-induced dyskinesia. In certain embodiments, the dopamine receptor 3 ligand is a N-(4-(4-phenyl piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamide D3 ligand. In certain embodiments, the dopamine receptor 3 ligand is PD13R. In certain embodiment, the dopamine receptor 3 ligand is administered as a thiophenyl benzoate salt of PD13R.
In certain embodiments, the dopamine receptor 3 ligand containing an arylpiperazine pharmacophore or a pharmaceutically acceptable salt, derivative, hydrate or solvate thereof, also contains a phenyl thiophene moiety. In certain embodiments, the arylpiperazine pharmacophore and the phenyl thiophene moiety are separated by a spacer. In certain embodiments, the dopamine receptor 3 ligand containing an arylpiperazine pharmacophore or a pharmaceutically acceptable salt, derivative, hydrate or solvate thereof, also contains one or more of a heterocyclic ring, such as triazole, imidazole, pyrimidine, pyrrazole, and pyridine. In certain embodiments, the dopamine receptor 3 ligand contains one or more of a piperazine bioisostere and a bicyclic bioisostere, or a pharmaceutically acceptable salt, derivative, hydrate or solvate thereof. In certain embodiments, the dopamine receptor 3 ligand is orally administered at a dosage ranging from about 1 mg/kg to about 20 mg/kg. In certain embodiments, the dopamine receptor 3 ligand is administered at a dosage ranging from about 5 mg/kg to about 20 mg/kg. In certain embodiments, the dopamine receptor 3 ligand is administered at a dosage ranging from about 5 mg/kg to about 15 mg/kg.
Embodiments include methods of treating levodopa-induced dyskinesia in a subject in need thereof by administering to the subject an effective amount of a dopamine receptor 3 ligand containing N-(4-(4-phenyl piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamide or a pharmaceutically acceptable salt, derivative, hydrate or solvate thereof. In certain embodiments, the dopamine receptor 3 ligand containing N-(4-(4-phenyl piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamide or a pharmaceutically acceptable salt, derivative, hydrate or solvate thereof is administered orally. In certain embodiments, the dopamine receptor 3 ligand containing N-(4-(4-phenyl piperazin-1-yl)butyl)-4-(thiophen-3-yl)benzamide or a pharmaceutically acceptable salt, derivative, hydrate or solvate thereof is administered as a thiophenyl benzoate salt. In certain embodiments, the dopamine receptor 3 ligand is administered at a dosage ranging from about 1 mg/kg to about 20 mg/kg. In certain embodiments, the dopamine receptor 3 ligand is administered at a dosage ranging from about 5 mg/kg to about 20 mg/kg. In certain embodiments, the dopamine receptor 3 ligand is administered at a dosage ranging from about 5 mg/kg to about 15 mg/kg.
Embodiments include pharmaceutical compositions containing an effective amount of the D3 receptor partial agonist, PD13R (arylpiperazine-based pharmacophore). PD13R significantly suppresses the expression of levodopa-induced dyskinesia in a nonhuman primate model of PD. SWR-3-73 (diazaspiro-based pharmacophore) did not inhibit expression of levodopa-induced dyskinesia. The anti-dyskinetic effects of PD13R didn't affect the anti-Parkinsonian benefits of levodopa while improving sleep efficiency compared to amantadine. In certain animal models, PD13R demonstrated desirable CNS drug-like features with high affinity for the D3R (Ki=0.50 nM), high selectivity for the D3R over the D2R (1486-fold), >20,000-fold selectivity for D3R over D1R or D5R and >1600-fold selectivity for D3R over D4R. PD13R exhibited low efficacy in the forskolin-dependent adenylyl cyclase inhibition assay (19.4% maximum activity) and a prolonged half-life (>60 m in human and rat liver microsome assays) of over 10 hrs in the in vivo PK study. Together, these data demonstrated that PD13R is efficacious in treating levodopa-induced dyskinesias, and is orally active with desirable drug-like properties, including potency, selectivity, and bioavailability.
Levodopa-induced dyskinesia develops in up 95% of patients after 15 years of treatment. Yet, it remains the gold standard pharmacotherapy for PD. Attenuating dyskinesia has been a significant challenge in the clinical management of Parkinson's disease. The attempt to attenuate dyskinesia by reducing or adjusting the dose of levodopa and implementing other adjunctive dopamine-replacement drugs produced inconsistent outcome. PD13R eliminates the expression of levodopa-induced dyskinesia without affecting the anti-Parkinsonian effects of Levodopa. This lead compound PD13R is a D3R partial agonist demonstrated in the forskolin-dependent adenylyl cyclase inhibition assay producing 19.4% of the activity of the maximum response observed with quinpirole.
The anti-dyskinetic effects of PD13R were similar to those observed with amantadine treatment, the currently prescribed drug for dyskinesia that was used as positive control. Based on several clinical studies, amantadine, which is an NMDA glutamate receptor inhibitor, was designated as efficacious for the treatment of dyskinesia by the Movement Disorder Society Evidence Based Medical Review. Interestingly, different effects on sleep patterns were detected between amantadine and the two D3R partial agonists. Both PD13R and SWR-3-73 did not affect sleep efficiency, while animals treated with amantadine exhibited a significant increase in nocturnal activity and decrease in sleep efficiency. Previous reports have highlighted the potential side effects of amantadine with the most common including cardiovascular dysfunction, myoclonus, orthostatic hypotension, peripheral edema, urinary tract infection, nervousness, insomnia, anxiety, hallucinations, delirium, confusion, nausea and constipation. Thus, better anti-dyskinetic agents remain a critical medical need. This novel D3R partial agonist, PD13R has demonstrated a high sub-nanomolar affinity for the D3R (Ki=0.50 nM), 1486-fold higher selectivity for the D3R over the D2R, 20,000-fold higher selectivity for D3R over D1R or D5R and over 1600-fold selectivity for D3R over D4R. PD13R has a prolonged half-life of over 10 hours in vivo, and a partial agonistic activity (19.4% maximum activity) in the forskolin-dependent adenylyl cyclase inhibition assay.
In the CNS disorders, improving the D3 versus D2 compound selectivity has been the most challenging aspect in developing selective D3R ligands. The sequence similarity between D3R and D2R is 90%, while sequence identities in relevant interactions sites with ligands such as the transmembrane region are highest for D2 versus D3 at 79%. The docking simulation analysis for PD13R and SWR-3-73 reveals high selectivity for D3R versus D2R. The overlay of D3R and D2R crystal structures docking pose of the PD13R revealed a loss of the polar contact at the key residues TYR 365 and SER 182, the TYR 365 became TYR 408 and reoriented completely away from the pocket leading to loss of the polar contact with the ligand. Furthermore, the bottom part of the binding pocket is unoccupied in the D2R. This un-filled portion of the pocket is important because cavity-filling connects selectivity to affinity, and the unfilled void would decrease selectivity and binding affinity to D2R. The optimal cavity filling of PD13R for D3R vs D2R could be mediated by the spacer linking the arylpiperazine to the arylamid, enabling a full cavity filling and improved selectivity and affinity to D3R. These finding are consistent with previous SAR studies demonstrated that the length of the spacer impact the affinity and selectivity to the D3R. Decreasing the spacer length from butylene to propylene or ethylene markedly reduces affinity to D3R; similar results were obtained when the spacer is increased from butylene to pentamethylene. The evidence show that even though both compounds PD13R and SWR-3-73 acted as partial D3R agonists, only the arylpiperazine-based scaffold was efficacious in treating dyskinesias. Substitution in the 4-position of the phenyl ring of the arylpiperazine lead to losses in binding to D2R and a 1486-fold high selectivity for D3R over D2R. Taken together, these data suggest that arylpiperazine, along with the spacer and the phenyl thiophene, drives the optimal activity and cavity filling of D3R. These findings are consistent with the structure activity relationships for D3R antagonists in substance abuse concluding that in order to obtain high affinity for D3R (<10 nm), high selectivity over D2R (>100 fold) the arylpiperazine-based scaffold are the most promising. Specifically, the structural features for potent and selective D3 antagonists include 1) an extended aryl amide, 2) piperazine containing an aromatic or heteroaromatic moiety in position 4 and 3) 4-atom (n-butyl) linking chain between the two functional groups. Although the arylpiperazine element is the primary pharmacophore, the selectivity for D3R also depends on the length of the linker. The arylpiperazine pharmacophore was mostly described as antagonist, which has created confusion in the Parkinson's disease field, as it is also a common structure of partial D3R agonist.
The lead compound PD13R, a partial D3R agonist, exerts approximately 19% adenylyl cyclase inhibition of a full agonist. Importantly, PD13R did not affect the therapeutic benefits of levodopa, manifested by the improvement in the Parkinson's disease rating score. The PD-like symptoms evaluated included tremors, bradykinesia, and posture and general activity measured by an accelerometer device, the Actiwatch mini. In addition, motor and cognitive functions were measured using the object retrieval task with barrier detour. A clear improvement in all PD-like symptoms and movement coordination were observed in animals treated with PD13R compared to control or those treated SWR-3-73.
As used herein, the terms “treating” or “treatment” of a subject includes the administration of an arylpiperazine pharmacophore-containing composition to a subject with the purpose of preventing, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting dyskinesia, or a symptom of Parkinson's disease. The terms “treating” and “treatment” can also refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage.
By the term “effective amount” of a therapeutic agent is meant a nontoxic but sufficient amount of such agent to provide the desired effect. The amount of a therapeutic agent that is “effective” will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents, and the like. Thus, it is not always possible to specify an exact “effective amount.” However, an appropriate “effective” amount in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an “effective amount” of a beneficial can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts. An “effective amount” of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, a dosage may be expressed as a number of milligrams of a compound described herein per kilogram of the subject's body weight (mg/kg). Dosages of between about 0.1 and 150 mg/kg may be appropriate. In some embodiments, about 0.1 and 100 mg/kg may be appropriate. In other embodiments, a dosage of between 1 and 50 mg/kg may be appropriate. In other embodiments, a dosage of between 1 and 20 mg/kg may be appropriate. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the drug and/or drug formulation to be administered (e.g., the potency of the therapeutic agent (drug), the concentration of drug in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.
As used herein, the term “pharmaceutically acceptable” component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When the term “pharmaceutically acceptable” is used to refer to an excipient, it is generally implied that the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
Embodiments include methods of treating dyskinesia by an oral administration of an effective amount of a dopamine receptor 3 ligand containing an arylpiperazine pharmacophore or a pharmaceutically acceptable salt, derivative, hydrate or solvate thereof. The terms “administered orally” or “oral administration” herein include any form of delivery of a therapeutic agent or a composition thereof to a subject wherein the agent or composition is placed in the mouth of the subject, whether or not the agent or composition is swallowed. Thus “oral administration” includes buccal and sublingual as well as esophageal administration.
All the animals (n=3) have been previously treated with MPTP as described in detail. Briefly, MPTP (Sigma Aldrich) (dissolved in physiological saline) at a concentration of 2 mg/ml was subcutaneously injected (2 mg/kg B.wt) for 5 consecutive days. After a wash out period of 72 hours from last MPTP injection, the marmosets were returned to their home cages and monitored twice daily for rest of the study period.
For induction of dyskinesias, Levodopa (Sigma Aldrich) and carbidopa (Sigma Aldrich) at 1:1 were administered orally once daily for 5 days of a week. The drugs were mixed in either Ensure® pudding, cottage cheese, or marshmallows. The marmosets were started on lower dose (5 mg/kg B.wt) during the first week and gradually increased to 10 and 15 mg/kg B.wt over the course of next 2 weeks. Once the animals were acclimated to the drugs, the dose was increased to 20 mg/kg B.wt and continued for next 6 weeks. To test the anti-dyskinetic effects of the drugs, amantadine (Sigma Aldrich; 10 mg/kg B.wt) or the compounds PD13R and SWR-3-73 (both at 10 mg/kg B.wt) synthesized as previously described were orally administered along with Levodopa and carbidopa (1:1, 20 mg/kg B.wt) and the changes in dyskinesia symptoms and diurnal and nocturnal activities were analyzed using the dyskinesia disability score and the Actiwatch mini, respectively.
The severity of symptoms in the marmosets was categorized using a validated Parkinsonian rating scale for NHP. The PDRS correlates highly with striatal dopamine concentrations detected by postmortem immunohistopathology and it is modeled on the Unified Parkinson's Disease Rating Scale (UPDRS) and the Hoehn & Yahr scale used clinically to categorize PD patients. The PDRS was performed in daylight by video recording animals for 30 minutes. The evaluation was carried out biweekly before and after MPTP injections. The videos were scored by a blinded operator using PDRS, with a maximal disability score of 57 in the following manner: 0=normal, 1=mild, 2=moderate, 3=severe; rest tremor, action tremor, tremor of the head, tremor right arm, tremor left arm, freezing, locomotion, fine motor skills right hand, fine motor skills left hand, bradykinesia right arm, bradykinesia left arm, posture, hypokinesia, balance, posture, startle response, gross motor skills right hand and gross motor skills left hand, apathy (defined as a state of indifference), vocalization, drooling or frothing, tongue/face/lips. Independently of the PDRS, rigidity was assessed by evaluating the resistance to passive joint movements and the range of motion during reaching for food. Prior to MPTP lesion, we trained animals using their favorite food (i.e. marshmallows) as reward. The marmosets were trained and conditioned to perform the rewarding visually guided task of reaching and grabbing a marshmallow. The evaluation was performed before and after MPTP.
The severity of dyskinesia in the marmosets was categorized using a validated dyskinesia rating scale for NHPs. Dyskinesia was analyzed from 5-minute videos of the monkeys recorded at every 30 minutes on Tuesday, Wednesday and Thursday of each week. The videos were recorded between 9:30 AM-4:30 PM. Each day the vehicle or drugs were administered at 11:30 AM. The severity of dyskinesias was scored for different segments of the animal's body on a scale from 0 to 3, with 0=absent, 1=mild, 2=moderate, 3=severe. The body segments scored were: 1. Right upper limb dyskinesias (0-3); 2. Left upper limb dyskinesias (0-3); 3. Right lower limb dyskinesias (0-3); 4. left lower limb dyskinesias (0-3); 5. Trunk dyskinesias (0-3); 6, Head/facial dyskinesias (0-3). The severity of the rating was based on the frequency and amplitude of the abnormal movements. The total score is deducted from the six sub-scores to yield a dyskinesia score of 0 to 3: with 0=absent, 1=mild, manifesting by transient abnormal involuntary movements with choreic form, flicking of arms and limbs, increased running and hopping in the cages. 2=moderate, characterized by frequent (over 50%) and intermittent uncontrolled irregular movements of limbs interfering with normal activity, while the animals are able to reach for treats. 3=severe, characterized by a sustained dyskinesia manifesting with chorea and athetosis with sinuous writhing movements of the limbs, and dystonia with sustained extension of hind limbs and knees. The animals are unable to reach and grasp for treats (Video 1).
Object Retrieval Task with Barrier Detour (ORTBD)
The object retrieval task with a barrier detour is a reward based behavioral testing system that was previously described to evaluate motor and cognitive functions of NHP. Briefly, the task requires the test subject to retrieve a reward (marshmallow) fastened to a tray from the open side (bypassing the barrier) of a transparent box. For the current study, the testing apparatus was modified to fit the marmoset's home cage and the animals were acclimatized to the apparatus prior to testing. Behavioral analysis was done for 3 consecutive days with 20 trials per day before and 1 hr after administration of the drugs. All parameters measured were previously described in detail. During each trial the orientation of the open side of the box was randomly changed to either left or right of the animal or towards the opening of the cage. The entire process was recorded using a video camera and the recordings were then analyzed and scored by a blinded experimenter. During each trial, the following responses were scored (1) ability of the animal to reach the front, left, or right side of the box, scored under the term “reach act”; (2) hand of choice for the reach, either left or right, scored under the term “hand used”; (3) the outcome of the reach, either success or failure, scored under result section.
Using the above parameters, additional variables were analyzed: 1) Motor problem: Reaching into the open side of the box but without retrieving the reward. 2) Initiation time: Latency from the screen being raised to the subject touching the box or reward. 3) Execution: Retrieving the reward from the box on the first reach of the trial (indicates competence on the task). 4) Correct: Eventually retrieving the reward from the box on the trial (>1 reach on the trial to retrieve the reward because unlimited reaches per trial were allowed). 5) Reach number: Number of times the animal made an attempt and touched the box. 6) Hand preference: Hand (left or right) subject used for the first reach of the trial. 7) Hand bias: Total number of left and right hand reaches on each trial. 8) Awkward reach: Reaching with the hand farthest away from the box opening (either the left or right side). 9) Perseverative response: Repeating a reach to the side of the box that was previously open but then closed. 10) Barrier reach: Reaching and touching the closed side of the test box. The results from the data analysis were plotted using Graph pad prism statistical software.
The diurnal and nocturnal behavior of the marmosets was monitored using the actiwatch mini (Camntech, UK) as previously described. The device is an accelerometer that measures the intensity of the test subject's omnidirectional movements in units or counts that are directly proportional to the animal's activity. The device (2 cm in diameter) was placed on a collar around the neck of the marmoset. The animals were acclimatized to the collar in short sessions of 15 min followed by gradual increments of 30 min, 1 hr, 3 hrs, 6 hrs and 12 hrs. Once acclimatized, the actiwatch mini was attached to the collar and the activity-rest data was recorded for a period of 24 hours on 3 separate days of each treatment. The actiwatches were placed on animals at 8:30 AM and the devices were preset to start recording activity data from 9:00 AM. At 11:30 AM either the vehicle or the drugs were orally administered to the animals. The following day, the actiwatch was removed after 9:00 AM and the data was transferred to a computer through an actiwatch reader using the actiwatch activity and sleep analysis-7 software (Camntech, UK). For sleep analysis, the period of sustained quiescence (marmosets sleep cycle) starting at 7:00 PM in the evening to early morning 6:30 AM (approximately 11½ hrs) was analyzed using the sleep analysis-7 software to quantify the sleep quality and wakeful periods. The duration of sleep time was corrected for individual variations in the animal's behavior to fall asleep at different time of the evening, thereby keeping the period of sleep time analyzed same for all the animals. The analyzed data was then exported to Excel® and plotted using the Graph pad prism statistical software. To determine the change in activity after the administration of drugs, actiwatch data in 10-minute bins were exported using the actiwatch activity and sleep analysis-7 software and plotted in Graph Pad Prism.
Statistical analysis was done with Graph Pad Prism statistical software. Significance in differences between 2 groups was performed by applying Student's t-test where appropriate. For comparison of multiple groups Two-Way ANOVA with Bonferroni post-hoc analysis was performed to identify the significant differences. A P-value of less than 0.05 was considered to be statistically significant.
A potent D3R ligand, PD13R, was identified that is highly selective for the D3R over other dopamine receptors. Molecular modeling studies were performed to determine the binding region of PD13R and the D3R binding pocket. The Rhodium protein docking simulation program was used to implement a fully automated search over the agonist-bound D3R cryo-electron microscopy (cryo-EM) structure (PDB ID 7CMV) and predict the binding site. Rhodium's unique docking pose analysis is based on changes in free energy during ligand-protein interaction, which is utilized in affinity optimization in ligand design. The model focuses on cavity-filling and on matching hydrogen-bond donors and acceptors at the ligand-protein interface, rather on optimizing their counts. The docking site selection is based on the long-range potential rules and do not take in consideration analyst-chosen pockets. The optimal poses of PD13R revealed the structure of D3R bound to PD13R and localization of the amide group between TYR 365 and ASP 110 with polar contacts (
The synthesis of PD13R involved the conversion of 4-(thiophen-3-yl)benzoic acid to an amide by a reaction with 4-aminobutan-1-ol using the coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMT-MM). Carbon tetrabromide and triphenylphosphine were used to convert the DMT-MM to a primary bromide. The final reaction involved a nucleophilic displacement of the bromide with the arylpiperazine under basic conditions (K2CO3, CH3CN).
PD13R was tested in the in vitro competitive D3R and D2R radioligand binding assays and rat liver microsome (RLM) stability assay. The D3R tolerated better the electron donating and electron withdrawing substituents on the phenyl ring of the arylpiperazine as indicated by the potent and high sub-nanomolar affinity for D3R with Ki=0.50 nM. The D3R over D2R data was as follows: D3R Ki=19.8 nM, D2R Ki>17,000 nM, D2R/hyperD3R>858.
Structure activity relationship (SAR) comparison between D3R versus D2R demonstrated that substitution in the 4-position of the phenyl ring of the arylpiperazine lead to substantial losses in D2 receptor binding irrespective of the electron character of the substituent and a 1486-fold high selectivity for D3R over D2R. This data is consistent with the docking results demonstrating that the phenylpiperazine region is critical to the binding, while the high degree of selectivity for D3R observed was driven by the optimal cavity filling influenced by the length of the spacer and the phenyl thiophene region.
To determine the selectivity of PD13R toward other DA receptors, an in vitro screen was performed for D1R, D4R, and D5R binding. The results showed a 1486-fold high selectivity for D3R over D2R, >20,000-fold selectivity for D3R over D1R or D5R and >1600-fold selectivity over D4R.
The ability of the compound PD13R to couple G-proteins in the forskolin-dependent adenylyl cyclase inhibition assay and the β-arrestin binding assay were then investigated. Quinpirole and haloperidol were used as a full agonist and antagonist, respectively. Compounds were tested for efficacy in these assay at a dose equal to 10 Ř their D3R Ki values in order to ensure >90% receptor occupancy and the results were compared with the impact of the full agonist. There was no activity in the β-arrestin binding assay and partial agonism in the forskolin-dependent adenylyl cyclase inhibition assay producing 19.4%±5.4 S.E.M., n=3) of the activity of the maximum response observed with quinpirole.
Table 1 presents the physicochemical and in vitro properties of PD13R. RLM and HLM are the half-lives for stability (minutes) using rat and human liver microsomes, respectively. Compounds were tested for activity at the D3 dopamine receptor at a dose 10× the Ki value of PD13R to ensure 90% of the sites were occupied. Compounds were evaluated for efficacy using adenylyl cyclase inhibition and β-arrestin binding assays (n=3). Values represent the percent of the maximum response where quinpirole was used as a full agonist reference compound and haloperidol was used as an antagonist.
The motor and non-motor Parkinson's-like symptoms were previously characterized in the MPTP marmoset model including postural and action tremors, altered range of motion during reaching, bradykinesia and problem solving during the skilled action of retrieving a reward. These symptoms were improved with levodopa treatment. In this study, levodopa-induced dyskinesia was modelled, including debilitating side effects that PD patients develop after long-term levodopa pharmacotherapy. Previous studies have demonstrated that the marmosets readily develop levodopa-induced dyskinesia after extended period of levodopa treatment. To induce dyskinesia, the Parkinsonian marmosets were started on a low dose of levodopa/Carbidopa (1:1, 5 mg/kg. B. Wt.) followed by medium dose (10 & 15 mg/kg. B. Wt.). Once the animals acclimatized to the drugs, they were switched to high dose (20 mg/kg. B. Wt.) and maintained for 6 weeks. To measure dyskinesia, Dyskinesia disability rating scale was used that is scored for different segments of the animal's body including face, trunk, arms and legs (see prior section) and a wearable device, the actiwatch-mini to monitor the animals' activities while they are dyskinetic. During the low and medium dose of levodopa therapy, the Parkinsonian marmosets displayed a generalized increase in motility, absence of action tremors, and increase in vocalization with no apparent symptoms of dyskinesias. In the first week of high dose levodopa treatment, however, the Parkinsonian marmosets started displaying mild dyskinesia (
Marmosets were treated with dyskinesia-inducing high dose of levodopa (20 mg/kg) in combination with either vehicle or increasing doses of PD13R. Animal treated with levodopa combined with vehicle reached the peak-dose period of dyskinesias in 30 min after dosing with highest dyskinesia disability score of 3 and lasted for 2 hours. The marmoset model showed that the peak-dose dyskinesia and the ON response coincide following Levodopa treatment. This outcome of levodopa treatment is consistent with the outcome observed in patients with levodopa-induced dyskinesias. When the Parkinsonian marmosets were treated with levodopa (20 mg/kg) and PD13R at dose of 0.1 mg/kg no anti-dyskinetic effects were observed. While treatment with levodopa+PD13R at 1 mg/kg dose reduced the duration of the peak-dose period to 1.5 hours with a progressive decrease during the end-of-dose period in dyskinesia disability score (
PD13R Improved the PDRS and Normalized Daily Activity of the Parkinsonian Marmosets with Levodopa-Induced Dyskinesia.
The anti-dyskinetic effects of PD13R that could lead to the improvement of the Parkinson's disease-like symptoms were investigated. The PDRS were first used to evaluate the Parkinsonian syndrome during levodopa-induced dyskinesia. The Parkinsonian marmosets displayed resting tremors, bradykinesia, hypokinesia and apathy with an average PDRS of 25 (
Amantadine is currently the main drug used in clinical practice under the name “GOCOVRI” or “ADS-5102” for treating levodopa-induced dyskinesias. The anti-dyskinetic beneficial effects of PD13R were compared to amantadine as a positive control and to a second D3R receptor partial agonist SWR-3-73. Parkinsonian marmosets were treated with dyskinesia-inducing high dose of levodopa (20 mg/kg) in combination with either amantadine (10 mg/kg), SWR373 (10 mg/kg) or with PD13R (10 mg/kg). The data showed that both amantadine and PD13R significantly inhibited the expression of dyskinesias throughout the onset, peak-dose and end-of-dose periods of levodopa treatment (
The anti-dyskinetic effects of PD13R, SWR-3-73 and amantadine were all compared at 10 mg/kg on the peak-dose levodopa-induced dykinesias using the actiwatch-mini (
The actiwatch enable us to non-invasively and empirically analyze the activity and the quality of sleep during nighttime in dyskinetic marmosets treated with levodopa in combination with increasing doses of PD13R (0.1 to 10 mg/kg), with SWR-3-73, or with amantadine. Quantitative analysis of the actograms demonstrated significant increase in nocturnal activity, in time spent moving and wakefulness at nighttime in dyskinetic animals treated with amantadine compared to vehicle, to SWR373- or to PD13R-treated animals (
In summary, numerous benefits have been described which result from employing the concepts described herein. The foregoing description of the one or more forms has been presented for purposes of illustration and description. Modifications or variations are possible in light of the above disclosure.
This application claims the benefit of and priority to U.S. Provisional Application No. 63/202,489, filed Jun. 14, 2021, which is incorporated by reference herein in its entirety.
This invention was made with government support under grant numbers: R56 AG059284 and OD P51 OD011133 awarded by National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63202489 | Jun 2021 | US |
