Patent Application
20210322343
The present invention is directed to pharmaceutical compositions containing (Z)-2-(3,5,5-trimethyl-2-cyclohexen-1-ylidene) acetic acid for nasal administration, methods of manufacture and their uses in neurological disorders especially epilepsy. According to the present invention, nasal pharmaceutical composition containing (Z)-2-(3,5,5-trimethyl-2-cyclohexen-1-ylidene) acetic acid and its analog can be used to treat CNS disorders, such as epilepsy, pain, anxiety, spasticity and migraine.
The nasal pharmaceutical composition containing (Z)-2-(3,5,5-trimethyl-2-cyclohexen-1-ylidene) acetic acid of the present invention are suspensions or viscous liquid pharmaceutical compositions, namely, creams, gels and emulsions, that are formulated with therapeutically effective amounts of (Z)-2-(3,5,5-trimethyl-2-cyclohexen-1-ylidene) acetic acid, and are nasally administered to treat epilepsy disorder.
Neurological diseases, such as epilepsy, Parkinson's disease, multiple sclerosis, Alzheimer's disease, chronic age-related neurodegenerative diseases, are associated with changes in neural functions and the burden of these diseases is increasing globally with high healthcare costs. Epilepsy is a major neurological disorder globally with high prevalence in developing world.
Currently available chemotherapeutic agents are not capable of curing the seizures completely and in majority cases, epileptic patients have to rely on medication to control seizures throughout their life, while, most drugs have severe side-effects. In view of the large percentage of uncontrolled epileptics and the side effects experienced by patients with the existing medications, there is an urgent need for more selective and less toxic anticonvulsant drugs.
Nasal administration has been recognized as an appropriate method for systemic delivery of drugs due to several advantages, such as large surface area of nasal mucosa, rapid initiation of action comparable to injection/oral administrations, lower chances of enzymatic degradation compared with GI tract and bypass the first pass metabolism in the liver. Important aspect of nasal administration is that, some amount of drug can directly deliver through olfactory neurons into the brain tissues or cerebrospinal fluid which provide better treatment for central nerves system diseases. For example, zolmitriptan (U.S. Pat. No. 5,466,699A) was developed to treat migraine; it was then commercially available as a nasal spray formulation (U.S. Pat. No. 6,750,237). Procedures for nasal administration of drugs are reported in literature, for example, an oil based vehicle for testosterone is described in U.S. patent application Ser. No. 13/194,928 and Application No. PCT/IB2012/001127. Drugs with poor solubility are difficult to be developed into a formulation for nasal administration and they require suitable solvent with surfactant to increase the absorption of drugs through nasal route. Therefore, the use of suitable vehicle for nasal administration of drugs with no toxicity to the nasal mucosae is highly demanded.
The current invention is directed towards nasal application of (Z)-2-(3,5,5-trimethyl-2-cyclohexen-1-ylidene) acetic acid in form of pharmaceutical composition containing corn/olive oil and surfactant. In previous U.S. application Ser. No. 14/609,211, we reported that (E/Z)-2,2′-(3,5,5-trimethyl-2-cyclohexen-1-ylidene) acetic acid] (acid analogue of E/Z isoxylitones) exhibited activity at 500 mg/kg through intraperitoneal administration.
The instant invention is a nasal formulation of (Z)-2-(3,5,5-trimethyl-2-cyclohexen-1-ylidene) acetic acid that showed a ten-fold improvement in the activity of the compound that was surprising when compared with intraperitoneal administration. After establishing the anticonvulsant and antiepileptic activities of (Z)-2-(3,5,5-trimethyl-2-cyclohexen-1-ylidene) acetic acid, we developed a nasal formulation of the test compound. This formulation was applied through nasal route in different doses. Interesting observation was made i.e., at the dose of 50 mg/kg (body weight) of active compound, the nasal delivery of formulation was active enough to block the PTZ induced epileptiform activity in rats (since the animals in our test group were in the range of 200-210 g). The sub-chronic toxicity testing was performed for intraperitoneal administration of Z-acid and nasal application of Z-acid formulation in rats for a period of 3 months. At the end of the study, samples were processed and gross anatomical observations were made after sacrificing the animals. We did not found any sign of toxicity in the treated animals.
The syntheses of an anticonvulsant isomeric mixture of (E/Z)-2,2′-(3,5,5-trimethyl-2-cyclohexen-1-ylidene) acetic acid was carried out through hydrolysis of an isomeric mixture of E/Z ester 2 (scheme-1). The E/Z isomeric mixture of acid analog of isoxylitones was poorly soluble in water, E/Z isomeric mixture was characterized by spectroscopic studies (U.S. patent application Ser. No. 14/609,211).
Pure isomeric (Z)-2-(3,5,5-trimethyl-2-cyclohexen-1-ylidene) acetic acid was obtained in pure form from recrystallization of E/Z-mixture of acid (Scheme-1) and it was characterized by spectroscopic studies.
In the present invention, detailed anticonvulsant activities of pure (Z)-2-(3,5,5-trimethyl-2-cyclohexen-1-ylidene) acetic acid (Z-acid) are described and nasal application of (Z)-2-(3,5,5-trimethyl-2-cyclohexen-1-ylidene) acetic acid in form of pharmaceutical composition was developed to inhibit PTZ-induced seizures. The purity of sample of Z-acid was checked under different chromatographic conditions and by 1HNMR from each batch of synthesis before performing any pharmacological testing. The pure Z-acid was found stable in different organic solvents at room temperature and even at high temperature. It was found stable under all conditions used in various experiments.
Serine, Glycine and Lycine and gamma amino butyric acid were conjugated with Z-acid to develop more new analogs (Scheme-2). Among these analogs, Z-acid-Lys-Lys-OH was water soluble and it was found active through nasal route.
The screening of compounds was carried out both in acute seizure model (anticonvulsant activity) and chronic seizure model (antiepileptogenesis activity). The kindling model of epilepsy is considered to be a chronic model of epilepsy, which is primarily used for evaluating the test drug for anti-epileptogenic activity. The PTZ test is most commonly used in the primary screening for new antiepileptic drugs (AEDs). In the current study, acute model of scPTZ-induced seizure and kindling model of epilepsy was initially used to evaluate the anticonvulsant and anti-epileptic activity of Z-acid. Once established, we next moved to evaluate the activity of the Z-acid and its analog on the EEG pattern of epileptiform activity in brain of live animals through i.p. and nasal routes. The toxicity profile of Z-acid was also studies for a period of 3 months both through i.p. administration and nasal application.
Z-acid and its various analogs were tested in acute PTZ-induced seizure model in mice. Each batch of the freshly synthesized Z-acid analog was evaluated for anticonvulsant activity in acute test in order to confirm the reproducibility.
All experimental procedures were performed in accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985) and it was further approved by the Advisory Committee on Animal Standards of International Center for Chemical and Biological Sciences (ICCBS), University of Karachi (Protocol #2019-013). Male NMRI or Balb/c albino mice weighing 19-25 g were housed in an environmentally regulated room on a 12:12 h light and dark cycle with 21±1° C. and had free access to food and water.
Anticonvulsant effects of the test compounds were evaluated using subcutaneous PTZ-induced seizure test (s. c. PTZ). PTZ was prepared in saline whereas Z-acid was dissolved in 0.1 N NaOH. The Z-acid and its analogs were administered intraperitoneally (i. p.) at least 30 min before subcutaneous (s.c.) administration of convulsive dose of PTZ (110 mg/kg). Following PTZ administration, animals were observed for at least 1 hour for the presence or absence of different types of seizure patterns i.e., onset of body twitches, threshold seizures, generalized seizures with loss of righting reflex, loss of righting reflex with tonic forelimb seizures, loss of righting reflex with tonic forelimb and hind limb seizures. Latency to PTZ-induced threshold seizures was also calculated. The latency to threshold seizure is defined as the interval between the time of the PTZ-injection and the occurrence of first episode of threshold seizure. Protection of testing material against PTZ-induced mortality within 24 hours was also evaluated. In all experiments, diazepam (7.5 mg/kg i. p) and valproic acid (100 mg/kg) were used as standard drug control.
The chemical kindling was induced according to the modified method of De Sarro i.e. by repeated treatment of mice with sub-convulsive dose of pentylenetetrazole (s.c., 50 mg/kg) on alternate days. Four doses of Z-acid (150 mg/kg/day, 200 mg/kg/day, 250 mg/kg/day, and 300 mg/kg/day) were administered daily (i.p.). However, on the day of PTZ administration, animals were treated with the Z-acid 30 minutes before administering PTZ. After PTZ was injected, each animal was placed separately in a clear plexi-glass cage for close observations for 1 hour. The drug control groups received daily valproic acid. The Racine scoring categorization of epileptic seizures pattern was used to monitor the animals (Table 1). The animals showing score 4-5 were considered to be fully kindled. The cumulative kindling score was then calculated. Experiments were terminated once the animals were fully kindled. The treatment regimen is shown in table 2. At the end of each experiment, animals were humanely sacrificed.
The manifestation of neurotoxicity of Z-acid was determined by inverted screen acute neurotoxicity test. A platform of a rectangular metal screen was inverted through an arc of 180° was employed in our study. Mice were pre-tested on the apparatus the day preceding the experiment, and those failing the task were not used for the subsequent drug test. Testing was carried out at 5-, 30-, 60- and 120-minutes following i. p. administration of Z-acid. Mice unable to climb to an upright position for 1 min duration were rated as failures.
Animals were transferred into individual cages the day before the experiments to allow them to acclimatize to the new environment. The toxicity profiles were established by slightly modifying the procedure of Irwin as described by Turner, 1972. The behavior (locomotion, head weaving, biting, licking or grooming, hyper excitability, ataxia and sedation, writhing, jumping etc of the animals were observed for 1-2 hr after they were injected with vehicle, standard drug and test samples. These effects on behavior were recorded using a scoring system (scores were allocated according to the intensity of the symptoms from (0-4) as described by Turner, 1972.
This was examined by Traction test. The forepaws of a mouse were placed on a small twisted wire rigidly supported above a bench top. Normal mice grasped the wire with forepaws and when allowed to hang free, placed at least one hind foot on the wire within 5 seconds. Inability to put up at least one hind foot constituted failure to the traction. The test was conducted at 30 min and 1 h after the injection of saline, diazepam, valproic acid or Z-acid.
Once the animals were observed for behavioral analysis, they were anesthetized, dissected and the gross anatomy of internal organs such as kidneys, liver, spleen, pancreas and heart were closely observed in order to see if there were any possible changes in the gross appearance of these organs.
To evaluate the sub-chronic toxicity profile of Z-acid the compound was administered daily into mice at the dose of 300 mg/kg (i. p.) for a period of 3 months. The animals were divided into 5 groups (8/group) as outlined below:
At the end of the study, animals were anaesthetized, dissected and gross examination of vital organs were performed, along with serum and whole brain samples collection. Serum and brain sample were stored in −20° C. for further examination.
The pharmacokinetics of Z-acid in both plasma and brain after a single i.p. dose of Z-acid (300 mg/kg) was studied in mice. Animals were sacrificed at 5 min, 15 min, 30 min, 60 min, 120 min, 180 min, and 240 min after the dose administration (n=4 for each time point). Blood samples were obtained in heparin containing vacutainers. The control group consists of the animals receiving no other treatment and this serve as blank plasma and aided in optimization and validation of the GC/MS method. The blood samples were later centrifuged to obtain plasma which was stored at −20° C. for further processing. Likewise, the brain samples were also collected from the sacrificed animals and stored at −20° C. for further processing on GC-MS.
In this set of experiment, effect of Z-acid was investigated on PTZ-induced epileptiform activity. Rats (210±10 g) were divided into six groups as control, diazepam, Z-acid, PTZ, PTZ+diazepam, PTZ+Z-acid with 6 sample size in each group. PTZ was injected at the dose of 100 mg/kg. Z-acid was intraperitoneally injected in anesthetized rats at the dose of 150 mg/kg whereas drug control group was treated with 5 mg/kg dose of diazepam. Saline was used as placebo. PTZ was prepared in saline whereas Z-acid was dissolved in 0.1 N NaOH. EEG was recorded from cortical region of the rat brain.
The protocol was started by anesthetizing rat followed by fixing the animal in stereotaxic apparatus for surgery. The skin was removed to expose the bregma and lambda region of the skull. Three holes of 0.6 mm diameter were drilled with the help of a manual drill. One hole was made on the parietal lobe for the placement of active electrode whereas two were made on the occipital lobes for reference and ground electrodes. A screw with the length of 5 mm and diameter of 0.5 mm was inserted on each hole and electrodes were then connected with the EEG recording system. The recording was started by monitoring the baseline EEG recording for 5 min. After 5 min of noise-free recording, PTZ was injected to induce acute seizures. The EEG recording after the injection of PTZ was continued for 10 min to observe the induction of epileptiform activity. During this duration, spikes which are known as epileptiform were observed with high amplitude which was synchronized with seizures observed phenotypically in rat. After 10 min of recording, Z-acid was injected intraperitoneally to observe its effects on PTZ-induced epileptiform activity.
In this set of experiment effects of Z-acid was investigated on PTZ-induced epileptiform activity through nasal route of administration. Rats (210±10 g) were divided into three groups as control, PTZ and PTZ+Z-acid with 6 sample size in each group. PTZ was injected at the dose of 100 mg/kg. Z-acid formulation was administered through intranasal route in anesthetized rats at the dose of 50 mg/kg. Pharmaceutical composition of Z-acid for one rat (˜200 g) contain 10 mg Z-acid in 30 μl vegetable oil. Pharmaceutical composition of 10 mg Z-acid in 25 μl vegetable oil/5 μl Tween 80 was also used and we observed same results for both formulations. Z-acid-Lys-Lys-OH dissolved in water was tested through nasal route. EEG was recorded from cortical region of the rat brain.
The protocol for the preparation of the animals for treatment was same as described above. The nasal application of Z-acid was done 30 min before PTZ injection. The recording was started by monitoring the baseline EEG recording for 5 min. After 5 min of noise-free recording, PTZ was injected to induce acute seizures in rat. The EEG recording after the injection of PTZ was continued to observe the induction of epileptiform. During this duration, spikes which are known as epileptiform were observed with high amplitude which was synchronized with seizures observed phenotypically in rat.
Data was analyzed by one-way ANOVA with followed by Tukey's post-hoc analysis. Values P<0.05 were considered as significant.
NMRI male mice weighing between 18-22 g were selected for the study. PTZ 110 mg/Kg was administered to the PTZ group and observed the first myoclonic jerk and HLTE. Z-acid was administered at a dose of 300 mg/Kg, 400 mg/Kg and 500 mg/Kg i.p. to 3 different groups (n=6) and after 30 minutes, PTZ 110 mg/Kg was administered to these animals to evaluate the anti-seizure potential of the test compound at these doses. It was noted that Z-acid at all the three doses significantly delayed the onset of myoclonic seizure and prevented HLTE in all the animals compared to the PTZ group thus preventing the seizures in PTZ-induced seizure animal model (
2.2.2. scPTZ-Induced Chemical Kindling Model of Epileptogenesis
A gradual increase in the seizure score was displayed reaching a score of 5 after 18 treatments by the untreated scPTZ control group animals with an average seizure score of 4.9. The valproic acid treated group compared to the PTZ-kindled control group did not exhibit any seizure pattern till the end of the kindling protocol. We observed that the active dose which was able to inhibit the process of epileptogenesis was 300 mg/kg body weight. At this test dose, Z-acid exhibited a complete inhibition in the development of kindling induced by scPTZ administration.
Sub-chronic toxicity studies were performed for 3 months, daily dosing of 300 mg/kg. All vital organs were intact, and no abnormal marks and spots were observed in gross examination. All animals survived until the end of experiments, Gross anatomy of the organs after treatment revealed no signs of toxicity. Blood samples were processed for LFT profile, CBC and LDH, creatinine, and urea levels which found in normal range after 3 months, daily dosing at the dose of 300 mg/kg
Plasma drug concentration was estimated through area of peak of the Z-acid obtained through GC/MS. The graph between plasma concentrations versus time was plotted. Similarly, brain Z-acid concentration was estimated by analyzing brain samples. It was observed that Z-acid rapidly appeared in both plasma and brain and peak concentration was achieved within 5 min and 15 min of intraperitoneal administration in plasma and brain, respectively. Thereafter, it rapidly disappeared from plasma (Figure-2) and brain (Figure-3) in parallel within 2-2.5 h and 1 h, respectively. Apparent volume of distribution was 10 L in plasma. The half-life was found to be 60 min in plasma. Thereafter, Z-acid was likewise eliminated in parallel from both compartments. No evidence was found for persistence or sequestration of Z-acid in brain.
Effects of Z-Acid on PTZ-Induced Epileptiform Activity in Rats Followed by Intraperitoneal Administration of Z-Acid (EEG Protocol)
Effects of intraperitoneal administration of Z-acid in animal model of PTZ-induced epileptiform. Seizures were monitored in term of spike discharge/min during three sessions of EEG recording including baseline, after PTZ injection and after drug injection. The drug was administered after 10 min of PTZ injection. It was observed that the spikes induced by PTZ was significantly reduced after 5 min of administering Z-acid (i.p.) and then completely diminished after 7-10 min of Z-acid treatment. The recording was continued for 55 min from the start point. At the end of the experiment, number of spikes was counted from the oscilloscope with the help of e-probe software manufactured by Science beam Institute, Iran. The data was collected from three time points of experiments including baseline, PTZ injection and compound injection (
In
Effect of intranasal administration of Z-acid formulation in animal model of PTZ-induced epileptiform was studied. Seizures were monitored in term of spike discharge/min. The drug was administered before 30 min of PTZ injection. The recording continued for 55 min from the start of experiment (
2.2.6. In
Male Wistar rats weighing between 200-220 g were selected for the study. Sub-anesthetic dose of sodium thiopental was given to animals. After 20 minutes, PTZ 120 mg/kg was administered to the PTZ group and seizure scores were observed. It was seen that in PTZ group score 4 seizure appeared within 2 minutes of PTZ administration and thereafter frequent score 3 jerks occurred. In test animals, after sub-anesthesia, 10 mg dose of Z-acid dissolved in olive oil was given to the animal through intranasal route. After 5 minutes of the administration of the compound, pentylenetetrazole (PTZ) at a dose of 120 mg/kg was administered intraperitoneally and epileptic seizures were observed. It was observed that Z-acid formulation intranasally prevented and delayed seizures and only scores 2 appeared in the animals.
