Use of selected single cobrotoxin molecule as an analgesic

Abstract

A composition of matter for an analgesia and its method of use is disclosed. The composition comprises selected single cobrotoxin molecule which is characterized by its high affinity binding to nicotinic acetylcholine receptors, rapid onset, and better safety profile comparing to a cobrotoxins complexes. The method of use is for the treatment of pain, especially for the treatment of refractory pain as associated with advanced cancer, rheumatoid arthritis, chronic neuropathic, migraine, and viral infections.

Claims

1. A method for treating pain in a mammal. Said method comprising administering to a mammal in need thereof a pharmaceutical composition of a therapeutically effective amount of Chain A, Cobrotoxin, or Chain A, Cobrotoxin B; and a pharmaceutically acceptable carrier base for use in alleviating or controlling pain.

2. The method of claim 1 where Chain A, Cobrotoxin and Chain A, Cobrotoxin B can be from elapid, sea snake, recombination resource, or from chemical synthesis.

3. The method of claim 1 for parenteral (intravenous, intramuscular, Intraarticular, intrathecal or subcutaneous), nasal, oral, sublingual or rectal administration ranging from 1 g/Kg to 350 g/Kg.

4. The method of claim 1 for topical administration comprising between 50 g and 500 g per gram of base.

5. The method of claim 1 comprising administering the composition ranging from once per year to several times a day.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 is a photograph of the separation of proteins from Naja Atra by electrophoresis using a discontinuous polyacrylamide gel as a support medium and sodium dodecyl sulfate (SDS) to denature the proteins.

[0031] FIG. 2 is the result of the isolation of Naja Atra venom by cation-exchange chromatography on an open column and reverse-phase HPLC; among a total of 12 fractions, fractions A and B were identified as high-affinity cobrotoxins molecules binding to nAChR in 125I labeled-Btx-nAChR binding inhibition test.

[0032] FIG. 3 is an illustration of the quantitative micro-dialysis.

[0033] FIG. 4 the brain time-concentration curves of the selected single cobrotoxin molecules and a cobrotoxins complexes.

DETAILED DESCRIPTION OF THE INVENTION

[0034] 1) Separation and Purification of cobra venom toxins Based on lyophilized venom powder from Naja atra, a total of 12 fractions were isolated by cation-exchange chromatography on an open column (502.5 cm I.D.) packed with TSK CM-650(M). The process was performed and described in the following sequence: [0035] i. Venom powder was dissolved in 10 ml of 0.025 M ammonium acetate (pH6.0). [0036] ii. Starting buffer (20-50 mg/ml) was applied to TSK CM-650 column equilibrated with the same buffer. [0037] iii. After the column had been washed with 300 ml of the initial buffer, the proteins adsorbed were eluted with a two-stage linear gradient (0.1-0.5 M and 0.7-1.0 M ammonium acetate buffer) as indicated in the FIG. 2. [0038] iv. A reverse-phase HPLC (RP-HPLC) was performed on a Hitachi' liquid chromatograph with a model L-6200 pump. The column eluates (6 ml/tube/7.5 min) were monitored for absorbance at 280 nm.

[0039] As shown in FIG.-2, a total of 12 fractions from the aforementioned ion-exchange chromatography were further desalted and purified by a reverse-phase HPLC (RP-HPLC) with Vydac RP-C18 (4.6250 mm, 5.0 um). Fractions A and B were identified as high-affinity cobrotoxins with enhanced binding ability to nAChR in a later 125I labeled-Btx-nAChR binding inhibition test. [0040] 2) Screening and selection of single cobrotoxin molecule with high affinity binding to nAChR for better drug target engagement The 125I labeled--bungarotoxin(Btx)-nAChR binding inhibition test was performed to evaluate the affinities of different cobrotoxins molecules. Both -bungarotoxin and cobrotoxin bind to nAChR, and competitively inhibits counter part's binding ability. -bungarotoxin is considered to have very high affinity with nAChR, it can competitively block nAChR at the acetylcholine binding sites in a relatively irreversible manner, so cobrotoxin's ability to inhibit -bungarotoxin's binding to nAChR can represent cobrotoxin's affinity with nAChR. Higher the affinity of cobrotoxin with nAChR, greater the inhibitory rate toward -bungarotoxin's binding to nAChR. In our radio-immunoprecipitation test, only nAChR bonded -bungarotoxin will be precipitated with nicotinic acetylcholine antibody (mAb 35) and Rabbit Anti-Rat IgG, whilst unbounded -bungarotoxin will be washed out. By testing the concentration of bonded -bungarotoxin, we can determine the inhibitory rates of different cobrotoxins molecules isolated in step 1).

[0041] Calculation of 125I-Btx-nAChR binding inhibitory rate (%)

=100(C.sub.BSAC.sub.cbx)(C.sub.BSAC.sub.Btx),

[0042] Where

[0043] C.sub.BSA means concentration using beef serum albumin to inhibit 125I-Btx-nAChR binding, 0% inhibited.

[0044] C.sub.Btx means concentration using -bungarotoxin to inhibit 125I-Btx-nAChR binding, 100% inhibited.

[0045] C.sub.cbx means concentration using isolated cobrotoxin in step 1) to inhibit 125I-Btx-nAChR binding.

[0046] The 125I-Btx-nAChR binding inhibition experiment process is performed and described in following sequence: [0047] i. A total of 0.5 ml nAChR crude extract from rat skeletal muscle was added to different purified cobrotoxin, then mixed with 1 ul mAb35 (5.9 mg/ml) and 1 ul 125I-Btx (0.18 g/ml), then blended at 4 C., and stored overnight for at least 10 hours. [0048] ii. About 10 l rabbit anti-rat IgG (4.5 mg/ml) was added to the aforementioned mixture on the next day and kept at 4 C. for two hours. [0049] iii. The resultant from the previous steps was then centrifuged for five minutes at 13,000 rpm and the sediment was washed thrice with Triton X-100 lotion. The (Bq) value per second was measured using the gamma radioimmunoassay counter. [0050] iv. Result:

[0051] Fractions A and B were in the range of approximately 40%-50% binding inhibition rates whilst other fractions were approximately between 0%-20% binding inhibition rates. Fractions A and B were purified and desalinated by RP-HPLC column (4.6250 mm, VYDAC RP-C18) and were selected for further sequencing process.

[0052] 3) Sequencing the primary structures of the selected cobrotoxin molecules (fractions A and B) to establish rigorous quality control standards to eliminate synergism and to improve consistency of target engagement Edman degradation method has been used to determine the amino acid sequence of fractions A and B, the process is performed and described in the following sequence: [0053] i. A total of 5 nmol of proteins were dissolved in 100 l of 0.1% trifluoroacetic acid (TFA). [0054] ii. These samples were reduced with -mercaptoethanol then reacted with iodoacetic acid. [0055] iii. These reduced and carboxylmethylated (RCM)-proteins were treated with Glu-C and Arg-C endoproteinases then separated on RP-C18 HPLC. [0056] iv. The resulting RCM-proteins were then subjected to automated Edman degradation to obtain the N-terminal sequences using a protein sequencer (Model 491A-ABI). [0057] v. By comparing the amino acid compositions and primary sequences determined by the sequencer, the sequence assignment can be determined.

[0058] In our test, the amino acid sequence of fraction A protein was: lechnqqssq tptttgcsgg etncykkrwr dhrgyrterg cgcpsvkngi einccttdrc nn The name according to NCBI Data Bank is Chain A, Cobrotoxin.

[0059] The amino acid sequence of fraction B protein was: lechnqqssq tpttktcsge tncykkwwsd hrgtiiergc gcpkvkpgvn Inccttdrcn n The name according to NCBI Data Bank is Chain A, Cobrotoxin B.

[0060] 4) Respiratory inhibition test Chain A, Cobrotoxin and Chain A, Cobrotoxin B were separately administered to 10 Kunming mice of 202 g. No respiratory inhibition was observed at effective dose of 60 g/kg.

[0061] 5) Comparison of the ability to cross the BBB and the time to reach effective brain concentration of Chain A, Cobrotoxin, Chain A, Cobrotoxin B with a cobrotoxins complexes

[0062] The estimated molecular weights of Chain A, Cobrotoxin, Chain A, Cobrotoxin B are 6.90.6 KDa; the estimated molecular weights of a cobrotoxins complexes extracted from crude venom ranged from approximately 6.5 KDa to 15 KDa. Quantitative microdialysis (QMD) is used to determine the absolute unbound brain extracellular fluid (bECF) concentration of Chain A, Cobrotoxin, Chain A, Cobrotoxin B, and the cobrotoxins complexes. QMD is the only technique that enables sampling of bECF in conscious animals and provides direct evidence of exposure at extracellular target sites (Kielbasa and Stratford, Jr. 2015)

[0063] QMD consists of implanting a probe in rat (FIG.-3). The membrane of the probe will allow the exchange of perfusate with bECF, so the concentration of Chain A, Cobrotoxin, Chain A, Cobrotoxin B, and a cobrotoxins complexes in bECF could be measured through dialysate. However the absolute drug concentration needs to be adjusted by taking into account the recovery rate of the probe as the recovery rate is less than 100% due to the fact that flow rate of perfusate through the probe does not allow sufficient time for the solute to equilibrate between the perfusate and bECF across the probe membrane (Li Di and Edward H. Kerns. 2015). In response, an in vivo recovery rate test of the probe (MD-2200) was performed on 125I-labeled Chain A, Cobrotoxin, Chain A, Cobrotoxin B, and a cobrotoxins complexes, each with three different concentrations of 5, 25, and 50 ng/ml as perfusate, with a flow rate of 2 L/min was perfused through the probe. During microdialysis, the dialysate was collected every 10 minutes for an hour, then tested using SN629B-radioimmunoassay counter to calculate cpm. Finally, cpm was converted to cobrotoxin concentration. The recovery rate (by loss) % was calculated as:

=100=(C.sub.perfusateC.sub.dialysate)C.sub.perfusate,

[0064] where C.sub.perfusate is the drug concentration in the perfusate, and .sub.Cdialysate is the drug concentration in the dialysate. In our test, the average recovery rate (by loss) was 15%.

[0065] Microdialysis was performed to assess the free drug concentration of Chain A, Cobrotoxin, Chain A, Cobrotoxin B, and the cobrotoxins complexes in rats brain. A total of 15 rats of 32020 g were categorized into three groups. After implantation of probes(MD-2200), they were perfused with artificial cerebrospinal fluid with a flow rate of 2 L/min. Same dosage of 120 g; 1.61107 Bq/kg 125I-labeled Chain A, Cobrotoxin, Chain A, Cobrotoxin B, and the cobrotoxins complexes were administered respectively into the three groups by IV injection. The dialysate was collected on an interval of 10 minutes for 360 minutes, followed by the testing of the dialysate with SN629B-radioimmunoassay counter. Converting cpm to cobrotoxin concentration, adjusted by the recovery rate (by loss), the brain concentrations curves of Chain A, Cobrotoxin, Chain A, Cobrotoxin B, and the cobrotoxins complexes at different time points in bECF were obtained.

[0066] As shown in FIG.-4, during any time course, Chain A, Cobrotoxin and Chain A, Cobrotoxin B were always ahead of the cobrotoxins complexes in reaching a specific concentration. A composition of molecular size of 6.90.6 KDa had a faster ability to cross the BBB than the complexes of molecular weights ranging from 6.5 KDa to 15 KDa, and reached higher brain concentrations sooner. Another reason that might impact larger molecule cobrotoxins in reaching higher brain concentration is rapid renal clearance, because it takes the cobrotoxins 10 times longer to cross the BBB compared to the renal clearance speed. (Lin and Lu, 2009). Cobrotoxins of larger molecular weight has a higher chance to be excreted from circulating blood due to lower speed crossing the BBB.

[0067] Sufficient concentration is the first pillar for a drug to show its expected pharmacology activity (Morgan P et al., 2012). Insufficient brain exposure leaves many central nervous system (CNS) diseases untreated or without optimum drugs. In past years, a high percentage of promising CNS drug candidates have failed. A major cause of this failure is the restricted access of many drug candidates circulating in the blood to penetrate into the brain owing to the BBB (Li Di and Edward H. Kerns, 2015).

[0068] Cobrotoxins complexes, due to the difference in molecular size and in affinity binding to nAChR, cause not only delays in onset but also inconsistency in clinical effect. However, by identifying, isolating, and using the selected single cobrotoxin molecule with high-affinity binding to a target receptor and better ability to penetrate the blood-brain barrier, an new analgesic with a high safety profile, rapid onset, and constant clinical effect was formulated that could greatly improve the pharmacology activities.

EXAMPLES

[0069] Effects of Chain A, Cobrotoxin and Chain A, Cobrotoxin B on Pain Responses in Mice/Rats

Example 1

[0070] Tail flick test for evaluation of the analgesic effect of selected cobrotoxin molecule at CNS level

[0071] Most commonly, an intense light beam is focused on the animal's tail and a timer starts. When the animal flicks its tail, the timer stops and the recorded time (latency) is a measure of the pain threshold.

[0072] METHODS: Periaqueductal Gray (PAG) was injected with selected cobrotoxin molecule in rats, and the central analgesic effect was evaluated by the tail flick due to thermal radiation. 20 Wistar rats with a weight of 20020 g were divided into 2 groups, 10 per group. Both Chain A, Cobrotoxin and Chain A, Cobrotoxin B, at 1.5, 3.0, and 6.0 g/kg exhibited a dose-dependent increase in the latency induced by thermal radiation. The analgesic effect of Chain A, Cobrotoxin and Chain A, Cobrotoxin B appeared in 1015 minutes and peaked at 2530 minutes after drug administration. The maximum increase of pain threshold was over 200%.

[0073] The ED50 of the antinociceptive effect of the thermal radiation-induced tail flick for Chain A, Cobrotoxin and Chain A, Cobrotoxin B was 3.34.0 g/kg in the tail flick test.

Example 2

[0074] Writhing test for the evaluation of analgesic effect by parenteral injection The writhing test is a chemical method used to induce pain of peripheral origin by injection of irritant principles like acetic acid in mice. Analgesic effect of the test compound is inferred from the decrease in the frequency of writhing.

[0075] METHOD: A total number of 60 Kunming mice were used, 10 per group, with a weight of 202 g. Chain A, Cobrotoxin and Chain A, Cobrotoxin B at 30, 45 or 60 g/kg (iv) exhibited a dose-dependent decrease in the frequency of writhing induced by acetic acid. At dose of 60 g/kg (iv), The analgesic effect started at 30 minutes, and could reach 6070% inhibitory rate of mice writhing at 90 minutes after administration. The ED50 of the anti-nociceptive effect of acetic acid induced writhing was 4050 g/kg in the writhing test for Chain A, Cobrotoxin and Chain A, Cobrotoxin B.

Example 3

[0076] Writhing test for the evaluation of analgesic effect by nasal administration METHOD: A total number of 60 Kunming mice were used, 10 per group, with a weight of 202 g. Nasal administration of Chain A, Cobrotoxin and Chain A, Cobrotoxin B at 30, 45 or 60 g/kg exerted a dose-dependent decrease in the frequency of writhing induced by acetic acid. The analgesic effect started at 30 minutes and lasted at least for 6 hours after drug administration. ED50 of the anti-nociceptive effect of acetic acid induced writhing was 3846 g/kg in the writhing test for Chain A, Cobrotoxin and Chain A, Cobrotoxin B.

Example 4

[0077] Writhing test for the evaluation of analgesic effect by oral administration METHOD: A total number of 60 Kunming mice were used, 10 per group, with a weight of 202 g. Oral administration of Chain A, Cobrotoxin and Chain A, Cobrotoxin B at 160, 320 or 480 g/kg exerted a dose-dependent decrease in the frequency of writhing induced by acetic acid. ED50 of the anti-nociceptive effect of acetic acid induced writhing was 300350 g/kg in the writhing test for Chain A, Cobrotoxin and Chain A, Cobrotoxin B.

[0078] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is, therefore, to be understood that numerous modifications may be made to the illustrative embodiments and that other arrangements may be devised without departing from the spirit and scope of the present invention as defined by the appended claims.

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