BR102021000600A2 - PHARMACEUTICAL COMPOSITION CONTAINING A PEPTIDE DERIVED FROM PHA1SS TOXIN AND USES - Google Patents

Abstract

PHARMACEUTICAL COMPOSITION CONTAINING A PEPTIDE DERIVED FROM Pha1ß TOXIN AND USES The present technology deals with a pharmaceutical composition containing a 10 amino acid peptide derived from Pha1ß toxin isolated from the venom of the Phoneutria nigriventer spider. The present technology also describes the use of this pharmaceutical composition and the peptide in the production of drugs for the treatment of inflammatory pain.

Description

PHARMACEUTICAL COMPOSITION CONTAINING A PEPTIDE DERIVED FROM PHA1SS TOXIN AND USES

[01] The present technology deals with a pharmaceutical composition containing a peptide of 10 amino acids derived from the Phα1β toxin isolated from the venom of the spider Phoneutria nigriventer. The present technology also describes the use of this pharmaceutical composition and the peptide in the production of drugs for the treatment of inflammatory pain.

[02] Chronic pain is a serious public health problem worldwide, but managing this pain is difficult due to limitations in the efficacy and toxicity of currently available drug options. It is imperative, therefore, to develop new treatment options for chronic pain.

[03] In this context, animal venoms are one of the richest sources of molecules capable of becoming prototypes for the development of these new analgesic drugs. Phα1β toxin (previously called PnTx3-6), for example, is a 55-amino acid peptide that was obtained from the venom of the Phoneutria nigriventer spider, and which has a marked analgesic effect in acute and chronic rodent pain models. Its analgesic action has also been shown to be associated with the ability to block voltage-sensitive calcium channels (with greater potency for subtype N channels) and also to act as an antagonist of TRPA1 channels.

[04] Through comparative studies, the Phα1β toxin was shown to be more potent than morphine in causing analgesia in several models of induced pain in animals. In addition, the toxin has the advantage of not causing dependence, tolerance and intestinal constipation like the opioid molecule.

[05] On the other hand, obtaining the Phα1β toxin, both in its native and recombinant form, is a complex process, which makes its production expensive, slow and low-yield, which prevents adequate control of its quality and characterizes a barrier to the development of a suitable pharmaceutical formulation. An alternative to obtain Phα1β in an economically viable way would be chemical synthesis. However, attempts to obtain Phα1β by chemical synthesis have not been successful so far. Synthetic polypeptides containing the same amino acid sequence as Phα1β did not preserve the biological activity of the native toxin in functional tests, probably due to incorrect structural folding. This is a common problem for chemical synthesis of proteins with a high number of amino acids. Therefore, new strategies to improve productivity and reduce the metabolism of peptides can be applied synthetically, translating a clear advantage over those obtained naturally. In relation to proteins obtained recombinantly, synthetic peptides also prove to have a less immunogenic effect, in addition to lower production cost in their synthesis compared to recombinant technology.

[06] Through bioinformatics, with the development of techniques for the study of drug-receptor interaction, both through the study of the ligand, and also through the study of structural similarities, the therapeutic possibilities for the development of new molecular structures, similar those found in nature, but capable of being synthesized due to their lower complexity and with the potential to be reproduced.

[07] Patent document US8383162B2, whose priority date is 11/21/2006, entitled “PHα1B toxin, CDNA of PHα1B toxin gene, pharmaceutical composition containing PHα1B toxin, process for their production and product”, describes the isolation of the toxin Phα1β, containing 55 amino acids, pharmaceutical compositions containing such a molecule, and its use as inhibitors of ion channel function. Such document claims the use of the toxin in its entirety and not a peptide fragment within the total sequence as claimed in the present invention.

[08] In the state of the art, no document was found that describes a pharmaceutical composition containing the 10 amino acid fragment of the toxin of the Phoneutria nigriventer spider described in the present technology, which preserves its analgesic activity and can be used for the production of drugs for the treatment of inflammatory pain, more efficiently and effectively compared to the native toxin.

[09] The present technology refers to a pharmaceutical composition containing the peptide of 10 amino acids that has a favorable cost/productivity ratio during chemical synthesis compared to obtaining peptides by direct purification of the venom or by recombinant technology, which consequently opens the possibility of large-scale production. In addition, as it is a lower molecular weight peptide, it favors the pharmacokinetics and biodistribution of the molecule between body compartments. Also, as it is a cyclic peptide and amidated in its carboxyterminal region, the peptide of the present technology can resist the action of serum proteases, which favors its bioavailability.

BRIEF DESCRIPTION OF THE FIGURES

[010] Figure 1 shows the regions found by BLASTP that have a degree of similarity to the toxins of the peptidergic pool 3 of Phoneutria nigriventer and are compared with PnTx3-6. Analyzes showing more than one result (PnTx3-4 and PnTx3-5 toxins) are also broken down.

[011] Figure 2 represents the effect of intrathecal administration of Morphine and Phα1β on transient thermal nociception (hot plate). (A) Represents the Morphine time curve (1.6μg/site). (B) Represents the area under the curve of PBS and Morphine. (C) Shows the time curve of the Phα1β toxin (200pmol/site) and (D) Represents the area under the curve of the animals that received Phα1β. Each point represents the mean ± standard error of 5-8 animals. **p<0.05 represents the level of significance when compared to animals treated with PBS (Student's T test).

[012] Figure 3 represents the effects of synthetic peptides on transient thermal nociception (hot plate) in mice. (A) Represents the PEP1 time curve (1000 pmol/site). (B) Represents the area under the curve of PEP1 and PBS. (C) Demonstrates the PEP2 time curve (1000 pmol/site). (D) Represents the area under the curve of PEP2 and PBS. Each point represents the mean ± standard error of 5-8 animals. **p< 0.01 represents the level of significance when compared to animals treated with PBS (Student's T test).

[013] Figure 4 represents the effect of synthetic peptides on transient thermal nociception (hot plate) in mice. (A) Represents the PEP3 time curve (1000 pmol/site). (B) Represents the area under the curve of PEP3 and PBS. (C) Shows PEP4 time curve (1000 pmol/site). (D) Demonstrates the area under the curve of PEP4 and PBS. Each point represents the mean ± standard error of 5-8 animals tested.

[014] Figure 5 represents the effect of intrathecal administration of recombinant Phα1β and PEP2 on capsaicin-induced nociceptive behavior. ***p <0.0001, represents the statistically significant difference of toxin treatment compared to control mice. (one-way ANOVA followed by the Bonferroni test). Results represent the mean SEM of 6 and 8 animals.

[015] Figure 6 represents the effect of intrathecal administration of PEP2 and Phα1β on mechanical allodynia induced by Capsaicin. Animals were pre-treated with drugs or vehicle 15 minutes before capsaicin injection as indicated in the figure. "Basal" represents the mechanical thresholds of the animals prior to capsaicin administration. "1h" and "2h" represent mechanical threshold measurements after capsaicin administration at the indicated times. PEP2 (1000pmol/site) and Phα1β (200pmol/site). Each bar represents the mean ± standard error of 6-8 animals.

[016] Figure 7 demonstrates the effect of intrathecal (i.t.) administration of PEP2 and recombinant Phα1β on the Forced Locomotion Test in Spinning Cylinder (Rotarod). The intrathecal administration of the drugs was not able to change the time of fall of the animals in the RotaRod test. PBS 5μL/i.t.), Phα1β (200pmol/site, i.t.) and PEP2 (1000 - 2 pmol/site, i.t.). Results represent the mean SEM of 6 and 8 animals.

[017] Figure 8 represents the effect of intrathecal (i.t.) administration of PEP2 in the open field test in mice. (A) Represents the horizontal activity of the animals. (B) Represents the total distance traveled. (C) Shows mouse movement time and (D) Represents observed vertical activity. PBS (5μL/i.t.), Phα1β (200pmol/site, i.t.) and PEP2 (1000 - 2 pmol/site, i.t.). Results represent the mean SEM of 6 and 8 animals.

[018] Figure 9 represents the mass spectrum of PEP2 and its mass/charge ratio. PEP2 was subjected to MALDI-TOF/MS mass spectrometry analysis to determine the monoisotopic mass. Spectra were acquired in reflector mode using an external calibrant. m/z: mass over load ratio. AU: arbitrary reading unit.

[019] Figure 10 represents the molecular mass of PEP2 and reduction of the disulfide bonds of PEP2. The molecular mass was determined by MALDI-TOF/MS using the reflected mode (UltraFlex, III, Bruker Daltonics), the observed molecular mass was 1280.68 (in black) and for the peptide reduced with 10mM TCep solution, it was 1282.62 (in red).

[020] Figure 11 shows the MS/MS spectrum assignment for PEP2 fragments. The observed molecular mass was 1282.68 Da. The peptides were fragmented by MALDI-LIFT-MS/MS experiments, demonstrating the same fragmentation profile. The results were manually analyzed using the PepSeq program and Flex Analysis 3.0 (Bruker Daltonics). The loss of 1 Da at the C-terminus indicates the presence of amidation. Precursor ion charge +1. Tolerance: 0.3 Da. The determined amino acid sequence of the peptide appears at the top of the graph in blue.

DETAILED DESCRIPTION OF THE TECHNOLOGY

[021] The present technology deals with a pharmaceutical composition containing a peptide of 10 amino acids derived from the Phα1β toxin isolated from the venom of the spider Phoneutria nigriventer. The present technology also describes the use of this pharmaceutical composition and the peptide in the production of drugs for the treatment of inflammatory pain.

[022] The pharmaceutical composition of the present technology is characterized by comprising the peptide defined by SEQ ID No1 in its cyclic form and amidated in its carboxyterminal region and pharmaceutically and pharmacologically acceptable excipients.

[023] The pharmaceutical composition described above can be used to prepare a medicament for treating inflammatory pain.

[024] The peptide defined by SEQ ID No1 can be used to prepare a medicament for treating inflammatory pain.

[025] The present invention can be better understood through the following non-limiting examples.

EXAMPLE 1 - Obtaining the 10 amino acid peptide defined by SEQ ID No1, herein called PEP2

[026] Of the toxins obtained from the venom of the Phoneutria nigriventer spider, at least five of them have proven antinociceptive action in rodent pain models and are common to the same venom purification pool: the PnTx3 pool. These are the toxins PnTx3-1, PnTx3-3, PnTx3-4, PnTx3-5 and Phα1β (previously known as PnTx3-6). Thus, we performed a sequence similarity analysis, using the BLAST-P tool, comparing the aforementioned toxins, two-by-two, and targeting the Phα1β toxin (Table 1 and Figure 1). The hypothesis considered was that some peptide segment, within the Phα1β sequence, would be responsible for the antinociceptive activity of the toxin and not the entire sequence.

[027] The existence of similarity regions among the PnTx3 pool toxins with analgesic actions would then give indications of where this region would be found. After the similarity analysis performed by the BLAST-P tool, three main criteria were taken into account to carry out the synthesis of the amino acid sequence for further evaluation of activity: sequences with higher similarity scores; E-value less than 0.5 and positivity greater than 50%. With these criteria analyzed, 3 sequences were defined to follow for chemical synthesis and conducting assays of antinociceptive activity in an acute pain model. Chemical synthesis was carried out by an outsourced company, BIOMATIK. As it is an initial screening in the search for peptide fragments with pharmacological activity, we chose to synthesize peptides with an intermediate degree of purity, for a better cost-benefit ratio.

EXAMPLE 2 - The PEP2 peptide has antinociceptive activity in a transient thermal pain model (hot plate).

[028] The hot plate test consists of evaluating the acute nociceptive response of the animals by observing the time taken by the animal to jump or lick its paws after being placed under a plate at 52oC. The cut-off point of our assay was set at 50 seconds to avoid tissue damage in the paw of the animals. Swiss strain mice were used, which were acclimatized for 1 hour in the experimentation room before the tests were performed. Then, these animals were divided into 7 groups to receive the injection of drugs (or controls) intrathecally. The tests were carried out between 30 minutes and 24 hours after the injection of the treatments and at different time intervals. Three groups received control solutions: PBS (5μL/site), negative control, Morphine (5,600 pmol/site) positive control and Phα1β recombinant (200 pmol/site): positive control of toxin activity. The 4 peptides obtained by chemical synthesis (PEP1, PEP2, PEP3 and PEP4) were used at a dose of 1000 pmol/site. The experiments were performed blindly by the evaluator in relation to the drugs that were injected into the animals.

[029] Animals that received intrathecal morphine (5,600 pmol/site) showed increased latency for paw withdrawal, which corresponds to a significant antinociceptive response, at 30 minutes and 1 hour after administration, increases of 37.3 ± 5 .5 and 37.2 ± 4.5, respectively, of latency in relation to PBS (Figure 2A). The area under the curve of the animals that received morphine was also significantly higher (173 ± 48 sec) than the animals that received PBS (65 ± 22 sec; Figure 2B). The same was observed for the recombinant Phα1β toxin. The area under the curve of animals that received Phα1β was significantly greater than that of animals that received PBS (87±15.4 sec) (Figure 2D).

[030] The intrathecal administration of PEP1, PEP3 and PEP4 peptides (1000pmol/site) did not produce antinociceptive action in the hot plate test, that is, there was no significant increase in the latency of response to the thermal stimulus in any of the observed intervals, which may also be observed when quantifying the area under the curve (Figure 3 and Figure 4). Only PEP2 demonstrated a significant effect in increasing paw withdrawal latency more evident between times 3 and 5 h (Figure 3C) as well as when evaluating the area under the curve (Figure 3D) 239.7 ± 21.8 sec for PEP2 versus 87 +15 sec for PBS. In view of these findings, it is concluded that the PEP2 peptide has biological activity indicative of analgesic action. Since these peptide fragments tested had an intermediate degree of purity, a new chemical synthesis was then carried out, from the sequence corresponding to PEP2 with high purity. The new synthesis was carried out by the company GenOne and the degree of purity achieved was 97%.

EXAMPLE 3 - PEP2 attenuates antinociceptive behavior and mechanical allodynia in a model of inflammatory pain induced by intraplantar capsaicin injection

[031] To investigate the effect of PEP2 on nociceptive behavior, the capsaicin test was performed, which consists of intraplantar injection of capsaicin (1nmol/paw). This injection causes an instantaneous nociceptive reaction that can be measured by the time of nociceptive behavior in the animals. It is caused by direct activation of nociceptors, in addition to leading to secondary hyperalgesia, resulting from central sensitization. It is a simple test and well recommended for the screening of drugs with potential analgesic action. Swiss male mice weighing an average of 25 grams were divided into eight groups, each containing ten animals. All animals were acclimatized in the behavior room for thirty minutes before the start of the tests. After this period, the animals received an intrathecal injection of the tested drugs, vehicle (PBS 5μL/site), recombinant Phα1β (200pmol/site), PEP2 (2 to 1000 pmol/site). After 20 minutes of intrathecal injection, the animals received a subcutaneous injection of 20μL of capsaicin (1nMol/paw) in their right hind paw, then the time of their nociceptive behavior was evaluated for 300 seconds (Figure 5). . PEP2 significantly reduced, at all doses tested, the nociceptive response time, thus indicating that PEP2 is pharmacologically active, similarly to recombinant Phα1β.

[032] Mechanical allodynia was assessed using Von Frey filaments. For this, the animals were placed in acrylic compartments on a metal screen and acclimatized for 30 minutes before the test. One hour after the injection of 20μL of Capsaicin (1nMol/paw) in the right paw of the animals, the animals were then evaluated by Von Frey filaments. The mechanical stimulus was directed with the filaments perpendicular to the plantar surface of the animals. Responses were analyzed in relation to tension-response to the various filaments, which in turn produce different degrees of mechanical stimulation (harmless or noxious). The sessions in mice started with the application of the 1.0 gram filament. If the voltage-response was harmful, a filament with a lower gram value from the last response was used. Von Frey filaments were applied for six sessions and paw withdrawal was recorded as a percentage of responses. The formula used to assess the 50% threshold in the mechanical allodynia model (Von Frey's filament) was: 50% threshold = log of the last thread + (mean k.). Where: log of the last yarn = the value of the last filament used in the series of six applications of Von Frey filaments; K = constant based on Dixon's table; mean log filament strength differences for mice = 0.25.

[033] It was observed that, within 1 hour after capsaicin injection (1nMol/paw), pretreatment with PEP2 (1000pmol/site) was able to reverse capsaicin-induced mechanical allodynia (Figure 6). EXAMPLE 4 - PEP2 does not have, at the doses tested, adverse motor effects detectable by the tests of forced movement in a rotating cylinder and spontaneous exploratory activity.

[034] Rotarod method was used to evaluate the effect of PEP2 on the motor coordination of the animals and verify the possibility of a non-specific muscle relaxation or sedative effect. The procedure was performed as described by Scheidt et al. (2002) with some modifications. Mice were trained for three consecutive days before the day of the experiment on Rotarod (Insight Equipment). On the day of the experiment, a baseline was performed before the treatments, then the animals received the injection with the appropriate treatments intrathecally, PBS (5 μL/site), Phα1β (200pmol/site) and PEP2 (2 to 1000 pmol /place). Ten minutes later, the animals were submitted to a new evaluation in Rotatod and their time, until the 1st fall, was recorded. In Figure 7, we can see that none of the tested doses of PEP2, with an antinociceptive effect, was able to cause sedation or reduction in motor activity, which is demonstrated by the lack of change in latency in the 1st fall.

[035] To evaluate the effect of intrathecal administration of PEP2 on spontaneous locomotor activity (horizontal and vertical exploration, distance covered and movement time), the animals were submitted to an open field test (40X12X20cm), divided into four equal areas 20X12X20 cm in dimension (open field for mice). Motor activity was measured using four infrared light detectors, each focused on a photocell. Mice received intrathecal administration of 5 μL of Phα1β (200 pmol/site), PEP2 (2 to 1000 pmol/site) or PBS vehicle. Then they were submitted to the open field and evaluated for 5 minutes. In the open field test, there was no change in the horizontal exploration and in the total distance covered by the animals. (Figure 8A and B, respectively). When evaluated according to movement time, only animals that received the PEP2 dose of 1000pmol/site showed a reduction in time, but not significantly. In the evaluation of vertical activity, both animals that received PBS (5μL/site) and PEP2 2 pmol/site showed a reduction in their activity, but not significantly. However, all other doses tested showed increased activity.

EXAMPLE 5 - The molecular identity of PEP2 reveals its purity, amino acid sequence and the existence of a disulfide bridge.

[036] Given the evidence of antinociceptive activity of PEP2, it was decided to investigate the degree of purity, amino acid sequence and evidence of its secondary structure, given that the proposed preliminary sequence was obtained by in silico analysis. Molecular identity studies were conducted by MALDI-TOF mass spectrometry and de novo sequencing. For this, 1μl of the peptide was mixed with a saturated solution of a-cyano-4-hydroxycinnamic matrix which was applied in duplicate on a Polished Steel MTP384 plate and crystallized at room temperature. MS data were obtained on MALDI-TOF (Matrix Assisted Laser Desorption/Ionization) an AutoFlex III instrument (Bruker Daltonics, Billerica, USA) controlled by FlexControl 3.0 software. Spectra were acquired in reflector mode. The mass/charge ratio of the PEP2 peptide was established at 1280.68 (Figure 9), and corroborates the information sent by the company GenOne, which manufactured the batch of high purity PEP2 used in the antinociceptive activity tests. It can also be observed, by analyzing the same spectrum, that the PEP2 peptide synthesized by GenOne is pure.

[037] As the constructed sequence has two cysteines, we decided to assess whether these would be forming the disulfide bond. Therefore, for this evaluation, PEP2 was previously incubated in a reducing solution containing 10mM [Tris (2-carboxyethyl) phosphine hydrochloride (TCEP) for two hours at room temperature. The oxidized and reduced samples were evaluated by MALDI-TOF (Figure 10). In the presence of TCEP (10mM), the disulfide bonds of the protein of interest were reduced, so that the overall molecular mass of the protein should increase according to the amount of cysteine amino acids present in the protein. In the case of PEP2, which has 2 cysteine residues, the expected mass increase of 2Da was observed in the global mass of the peptide by the presence of TCEP, demonstrating that there is formation of a disulfide bridge in PEP2 (Figure 10).

[038] Subsequently, it was verified if the PEP2 sequence was synthesized correctly. For that, the precursor ions of PEP2 were fragmented in LIFT mode (MS/MS) in a MALDI TOF-TOF/MS and the spectra were manually interpreted. Analysis of the MS/MS spectra allowed the complete recognition of the generated fragments, and of the y and b ion series, confirming a 100% similarity between the PEP2 ions. A sequence of 10 residues was thus observed: FCNRKKEKCK-NH2 (Figure 11). Therefore, the sequence found for PEP2 coincides with the sequence predicted by the in silico similarity assays.

Claims (3)

PHARMACEUTICAL COMPOSITION, characterized by comprising the peptide defined by SEQ ID No1 in its cyclic form and amidated in its carboxyterminal region and pharmaceutically and pharmacologically acceptable excipients. USE OF THE PHARMACEUTICAL COMPOSITION defined in claim 1, characterized in that it is for the production of medicaments for the treatment of inflammatory pain. USE OF THE PEPTIDE DEFINED BY SEQ ID No1, characterized in that it is for the production of medicaments for the treatment of inflammatory pain.

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