WO2011086422A1

WO2011086422A1 - Pharmaceutical oral product obtained from parts of heliotropium plants

Info

Publication number: WO2011086422A1
Application number: PCT/IB2010/050197
Authority: WO
Inventors: Luiz Francisco Pianowski; João Batista CALIXTO; Claudio Paulino Chaves
Original assignee: Amazonia Fitomedicamentos Ltda
Priority date: 2010-01-15
Filing date: 2010-01-15
Publication date: 2011-07-21

Abstract

The present invention concerns a pharmaceutical product for oral administration, standardized, safe and efficacious in the treatment of inflammation, both acute and chronic, particularly inflammatory diseases of the intestines, as well as a process to obtain it from parts, particularly leaves, of plants of the genus Heliotropium, particularly Heliotropium indicum.

Description

PHARMACEUTICAL ORAL PRODUCT OBTAINED FROM

PARTS OF HELIOTROPIUM PLANTS

Field of the invention

The present invention concerns a pharmaceutical product for oral administration, standardized, safe and efficacious in the treatment of inflammation, both acute and chronic, particularly inflammatory diseases of the intestines, as well as a process to obtain it from parts, particularly leaves, of plants of the genus Heliotropium, particularly Heliotropium indicum.

Background of the invention

Inflammation can be defined as a complex biological response of the vascular tissue to harmful stimulus, characterized by symptoms of pain, swelling, redness, etc. It may be classified in two different types, according to the onset speed: acute and chronic.

It is known that inflammatory processes play a role in a large number of diseases, for many of which until recently the causing mechanisms were unknown. In others, previously considered as degenerative or associated with aging, inflammation has became the most important explanation.

Inflammatory diseases of the intestine comprise a specific group of inflammatory conditions of the colon and small intestine. The main ones are Crohn's disease, ulcerative colitis and irritable bowel syndrome (also known as irritable colon syndrome or functional colonic disease). In general, such diseases are characterized by recurrent abdominal discomfort, and abnormal intestineal function. It has been suggested that part of the population presents some of those diseases in determined moments of life, predominantly women, mainly young women.

The majority of available treatment up to now is merely palliative, that is, they only bring relief to the classic symptoms, such as diarrhea, rectal bleeding and abdominal pain. Furthermore, patients with ulcerative colitis, for instance, may have remission periods between flare-ups. Treatment of ulcerative colitis involves medication that induces remission, that is, by reducing abnormal inflammation on the colon coating, they control symptoms. Among such medication there are mainly the amino-salicilates, the corticosteroids and the immunomodulators, that may present undesirable immunosuppressing effects.

And yet there is a need for a pharmaceutical product that is safer and efficient for the treatment of inflammation, both acute and chronic, particularly concerning inflammatory diseases of the intestine.

Several species of plants have been used along time by popular medicine for the treatment of all kinds of diseases. In that sense, the study of plants with immunomodulating potential has become an interesting research field with a view to treating several diseases, specially those with chronic character.

Heliotropium is a genus of flowering plants in the borage family, Boraginaceae. There are 250 to 300 species in this genus, which are commonly known as heliotropes. Particularly, the Heliotropium indicum (family Boraginaceae, genus Heliotropium), also known as Indian Heliotrope (or "crista de galo", in Portuguese), is a plant found in several temperate and tropical regions around the world, particularly in India. Studies about it were never extensive, and topic antimicrobial and/or antitumor activity of plant extract have been briefly discussed in the academic ambit.

The Japanese patent document JP2000095663 teaches its topic use for whitening of the skin, and patent document EP1915997 teaches its use as capillary products that may comprise extracts of several other plants. Patent application US20050266105 teaches that the plant had already been studied for the treatment of AIDS and its effect on inflammation (Indian Journal of Pharmacology 2000; 32: 37-38).

Little is known about the chemical content and the plant extract. There are reports of it containing an isoflavone (Pandey et. AL: "A new isoflavone glycoside from Heliotropium indicum", Journal of the Indian Chemical Society, 2007) and an alkaloid (patent documents US20060236421 , US3717710 and US4458082). Those reports, though, were never related to an anti-inflammatory action. Additionally, there are reports of possible side effects (Paulo Nascimento: "Plantas que fazem mal", Jornal da Unicamp, Brazil, number 163 June 2001 ).

Though only scarce studies have been performed with Heliotropium indicum, little is known about the extract contents and effects, what has so far kept it from becoming a commercial standard pharmaceutical product, safe and efficient in the treatment of inflammation. The specific mention to Heliotropium indicum is also a non-limitative indication of a preferred alternative of the invention.

Description of figures

The present invention is additionally illustrated by the following non-limitative examples. In the attached figures, the product identified as AM100 is the product according to the invention.

Figure 1 presents the chromatogram of the CG-MS analysis of the standardized pharmaceutical product of the invention.

Figure 2 shows the effect of treatment with the product according to the invention (200 - 400 mg/kg, oral administration, 1 h) or with dexamethasone (0,5 mg/kg, subcutaneous administration, 4 h), upon the paw edema induced by carrageenan in mice. Each point represents an average of 4- 5 animals and the vertical bars the error standard deviation. Significant differences from the control ^*P < 0,05 and ^**P < 0,01.

Figure 3 shows the effect of long term treatment with the product according to the invention (200 mg/kg, twice a day, for 8 days) or with dexamethasone (1 mg/kg, subcutaneous administration, twice a day, for 8 days) about the microscopic score of the colon of mice with colitis induced by TNBS (2,4,6-trinitrobenzene sulfonic acid). Each point represents the average of 5 animals and the vertical bars the error standard deviation. Significant differences from from the control (saline) **P < 0,01. Significant differences from from the TNBS group *P < 0,05. Figure 4 presents the effect of long term treatment with the product according to the invention (400 mg/kg, oral administration, twice a day, for 8 days), or with dexamethasone (1 mg/kg, subcutaneous administration, twice a day, for 8 days) about the microscopic score of the colon of mice with colitis induced by TNBS. Each point represents the average of 5 animals and the vertical bars the error standard deviation. It significantly differs from the control (saline) **P < 0,01. Significant differences from the TNBS group *P < 0,01.

Figure 5 presents the effect of long term treatment with the product according to the invention (400 mg/kg, oral administration, twice a day, for 8 days), or with dexamethasone (1 mg/kg, subcutaneous administration, twice a day, for 8 days) about the microscopic score of the colon of mice with colitis induced by TNBS. Each point represents the average of 5 animals and the vertical bars the error standard deviation. It significantly differs from the control (saline) **P < 0,01. Significant differences from the TNBS group ^mP < 0,01.

Figure 6 presents the effect of the treatment with the product of the invention (400 mg/kg, oral administration, 1 hour) or HOE-140 (icatibant, 50 nmol/paw, subcutaneous) about the nociception induced by the intraplantar injection of BK (bradykinin) in mice. Each group represents an average of 6-8 animals, and the vertical lines the error standard deviation. Significant differences from the control group, **P < 0,01. Figure 7 presents the response frequency of right hindpaw withdrawal for animals treated with the product according to the invention (400 mg/kg, oral administration, 1 h) according to different time intervals after the 4th day of the SNPC (sciatic nerve partial constriction) in mice. Each group represents an average of 5-7 animals, and the vertical lines the error standard deviation. B (basal threshold before surgery). Significant differences from the control group, ***p < 0,001 and **p < 0,01. B (basal threshold before surgery).

Description of the Invention

The present invention concerns a standardized pharmaceutical product, for oral administration, safe and efficacious in the treatment of inflammation, both acute and chronic, particularly inflammatory diseases of the intestine.

Said pharmaceutical product is obtained from parts of the plant Heliotropium, preferably Heliotropium indicum, particularly from the leaves of the plant, and contains non-reducing carbohydrates, namely saccharose and β- galactose linked to the glycerol of the monogalactosyl-diacyl-glycerol (MGDG) molecule. Additionally, the product comprises reducing and non-reducing sugars, such as arabinose, xylose, mannose, galactose and glucose. In a particular embodiment, such sugars represent the following concentrations: 4- 5%, 3,5-4,5%, 2,5-3,5%, 40-45% e 45-55%, respectively. Other Heliotropium plants are obviously encompassed by the invention, provided the results obtained with them are similar to the ones obtained with Heliotropium indicum. A non-limitative list is given below:

Heliotropium amplexicaule, Heliotropium arborescens, Heliotropium curassavicum, Heliotropium europaeum, Heliotropium pannifolium, Heliotropium atlanta, Heliotropium corymbosum, Heliotropium peruvianum, Heliotropium suaveolens, Heliotropium hybriden, etc.

Another object of the present invention is the process to obtain a standardized pharmaceutical product, comprising the steps of:

a) Macerating 1 -1 .5kg of parts of Heliotropium, preferably Heliotropium indicum, particularly dry leaves, in 6-7 liters of ethanol and 500-1000 ml of water (10: 1 ), for 90-100 hours.

b) Filtrating and evaporating the macerate in an evaporator, particularly a rotoevaporator.

c) Filtrating to obtain a final product.

The standardized pharmaceutical product according to the invention shows efficacy in different inflammation models, both acute and chronic. Additionally, the anti-inflammatory and anti-nociceptive actions of the standardized pharmaceutical product according to the invention are comparable to the ones observed for a steroidal anti-inflammatory known as a market reference (dexamethasone), but without the undesirable immunosuppressant effects related to the use of said corticoid or side effects provenient from the plant. Moreover, the standardized pharmaceutical product according to the invention presents surprising anti-inflammatory effect in the specific treatment of inflammatory diseases of the intestines, particularly Crohn's disease, ulcerative colitis and irritable bowel syndrome.

The pharmaceutical product obtained according to the invention is useful to be administered to a patient or utilized in the preparation of pharmaceutical compositions, in doses varying from about 0,01 to about 1 ,000 mg/kg of patient weight, in one or more doses per day. Said compositions may additionally comprise pharmaceutically acceptable excipients, with no particular limitation, such as those mentioned in the following publications: Remington's Pharmaceutical Sciences (Mack Publishing), European Pharmacopeia or Brazilian Pharmacopeia, or new excipients to be developed.

The pharmaceutical product according to the invention, or pharmaceutical composition containing same, is adequate for oral administration in the form of solids, liquids or semi-solids, for example tablets, capsules, pill, powder, granules, suspensions, emulsions, dispersions and other forms known in the art.

The examples that follow aim to illustrate aspects of the present invention without possessing any limitative character.

Examples

Example 1 - Preparation process

1 ,270 g of fresh leaves of Heliotropium indicum were finely cut and subject to maceration in 7 liters ethanol and 700 ml water, for 4 days. After this period, the macerate was subject to filtration in a Buchner funnel. The hydro- alcoholic product was subject to a rotoevaporator to withdraw some of the alcohol until lumps were obtained. The precipitate (lumps) was then filtrated and the resulting final product subject to the tests that are discussed further on.

Example 2 - Analysis for the identification and quantification of components present in the dry extract of Heliotropium indicum

According to the NMR (nuclear magnetic resonance) analysis in fig. 1 , there are non reducing carbohydrates in the dry extract of the plant Heliotropium indicum (identified as AM100), namely saccharose and β- galactose linked to the glycerol of the monogalactosyl-diacyl-glycerol (MGDG) molecule. Such components, saccharose and MGDG were visualized by CCD (charge-couple device) camera when compared to their respective Rfs (Retention Faction) and their color development with orcinol/H₂S0₄. The NMR analysis also showed the presence of reducing sugars mannose and glucose, and the presence of fatty acid esters. The confirmation of such sugars in the NMR experiment was achieved with GC-MS (gas chromatography-mass spectrometry) in the form of alditol-acetate after total hydrolysis, where one observed the corresponding peaks and fragments of EI-MS (electron impact- mass spectrometry) in fig. 1 with the proper concentrations.

Example 3 - efficacy tests

Animals

25 - 35 g Swiss mice or 150-250g Wistar rats were employed in the experiments. The animals were kept in 22 ± 1 °C constant temperature chambers and 60-7-% controlled humidity, 1 h dark-light cycles with free access to water and food. Before the experiments, the animals were acclimatized in the laboratory for a minimum of 1 hour. Each animal was used only once in each test. All experiments were performed between 8 and 18 h.

Carrageenan-induced paw edema in mice

The paw edema was performed according to the methodology described by Cunha et al. (Additional evidence for the anti-inflammatory and anti-allergic properties of the sesquiterpene polygodial. Life Sci., 70: 159-169). The animals were lightly sedated with 2,2,2-tribromoethanol (0.125 g/kg, intraplantar), and received in the right paw 50 μΙ of 0.9% saline comprising carrageenan (300 μg/paw). The left paw receive the same volume of saline and was used as control. The increase in paw volume was measured with a pletismometer (Ugo Basile Co. - Italy) along various time intervals after the intraplantar injection of carrageenan (30 min - 72h). The difference between left and right paw volumes was quantified (in μΙ) and taken as edema index. The animals were treated systemically with the product of example 1 (200-400 mg/kg, oral administration, 1 h before the intraplantar carrageenan shot) or with dexamethasone (0,5 mg/kg, subcutaneous administration, 4 hours before). The responses obtained with animals treated with the product according to the invention or with dexamethasone were compared to those observed with the control animals (treated only with carrageenan).

TNBS-induced colitis in mice Mice were subject to 18-24 h solid fasting, with free access to a 5% glucose solution, and then randomly divided in groups. After anesthesia with 2,2,2-tribromoethanol (0,125 g/kg, intraplantar), a catheter was carefully inserted in the colon of the animals up to 4 cm (taking all colon). The control animals received a 0.9% saline solution that was slowly administered to the colon cavity. For the induction of colitis 0.1 ml TNBS (2.5 mg diluted in 50% ethanol), aiming to break the epithelial intestine barrier, was slowly administered. The saline or the TNBS solution reached all colon. The animals were kept in a 45° semi-vertical position for two minutes and after induction returned to their boxes. After 4 hours the animals received food and filtered water. 72h after induction the animals were sacrificed and the distal colon was removed.

Macroscopic evaluation of TBNS-induced colitis

After the induction of colitis the animals were sacrificed, the colons removed (horizontally), opened (along all extension) and carefully cleaned with 0.9% saline solution, preheated at 37°C. The severity of damage of the colon was evaluated using a scale of macroscopic damage (table 1 ). This damage evaluation system includes characteristics of inflammation presence such as: hyperemia, visible damage (ulcers), diarrhea, thickening and thinning of the wall. Table 1 : Gr|d used for

in TNBS-induced colitis

From the observation of each colon, the colitis was evaluated and quantified.

Microscopic Evaluation of TBNS- induced colitis

The colons were collected as previously described (but not opened for the establishment of the macroscopic score), the fecal bolus removed, and then immediately put in a 10% formol solution for later blocking with paraffin and mounting of histologic sections. The sections were colored with hematoxylin and eosin and the microscopic score was established, with values from 0 to 4 (table 2)

Table 2: Grid used for the evaluation of the microscopic damages in

TNBS- induced colitis

Colitis induction for curative treatment

In the experiments for the evaluation of curative effect of the product according to the invention, the treatment of the animals started before the induction of colitis, and lasted up to the collection day. The animals received the product according to the invention (200-400 mg/kg, oral administration, twice a day, for 8 days) or dexamethasone (1 mg/kg, twice a day, for 8 days). The colitis was induced as previously described, and the colons were collected 72h after induction. For the experiments run to evaluate only the curative effect of the product according to the invention, the colitis was induced and the treatment with the product according to the invention or with dexamethasone was initiated 24 h after the induction of colitis, and lasted 2 days. The collection of colons was performed 72 h after the induction of colitis.

Spontaneous Nociception induced by bradykinin (BK)

To evaluate the participation of the kinin system in the nociceptive activity of the product according to the invention, the bradykinin-induced spontaneous nociception test was run as described by Bland-Ward and Humphrey (Bland-Ward, P.A.; Humphrey, P.P. (1997). Acute nociception mediated by hindpaw P2X receptor activation in the rat. Br. J. Pharmacol., 122: 365-371 ). The mice were treated with the product according to the invention (400 mg/kg, oral administration), HOE-14- (50 nmol/kg, subcutaneous) or vehicle (10 ml/kg, oral administration). After 1 h, the animals received a 20 μΙ/paw intraplantar injection of bradykinin (100 nmol/paw) in the right hindpaw, and then were put in individual glass funnels and observed for 15 minutes. The time the animals spent licking or biting the BK-injected paw was cumulatively quantified, and considered as indicative of nociception.

Analysis of the mechanical threshold with the

von Frey Hair (VFH) filament

To assess the mechanical hypersensitivity, the mice were individually put in 9 X 7 X 1 1 cm transparent acrylic compartments on an elevated wire platform to allow access to the hind paws plantar surface. The animals were acclimatized for 30 min before the behavioral evaluation. The frequency of paw withdrawal was evaluated after 10 stimulations (1 second duration each) with the Von Frey Hair filament (VFH, Stoelting, Chicago, USA). The stimuli were done on the surface of the right hindpaw plantar surface. The 0.6g VFH produced withdrawal frequency basal average of about 15%, what is considered an adequate value for the evaluation of mechanical hypersensitivity. Thus, the 0.6g filament was employed during this study. Aiming to determine the basal mechanical threshold of paw withdrawal, all groups were subject to a pre-evaluation with the filament before any experimental procedure.

Lesion by sciatic nerve partial constriction iSNPC) The animals were anesthetized with 7% chloral hydrate (0.6g/kg, intraplantar). Then an incision of aprox. 3mm was made in the posterior gluteous region. After its exposition, the sciatic nerve was ligated at 1/3 to 1/2 of its dorsal portion with n° 8 silk thread. In the false-operated group, the sciatic nerve was exposed without the ligature. On the 4th day after surgery, the animals were treated with the product according to the invention (400 mg/kg, oral administration), and the control group as well as the false-operated group received vehicle (10 ml/kg, oral administration). After 1 h treatment the mechanical hypersensitivity was evaluated at different time intervals, with the use of Von Frey Hair filaments. Statistical Analysis

The results are expressed as average ± mean standard deviation of at least for experiments per group. The statistic differences among the groups were evaluated with the T test for pared or non-pared samples, or still with analysis of variance (ANOVA) followed by the tests Dunnet, Newmann- Keuls or T test modified by Bonferroni when appropriate. Values of P below 0.05 (P<0,05) were considered as indicative of statistical significance.

Effects of the product according to the invention upon the carraqeenan-induced paw edema

The results presented in fig. 2 show that the systemic treatment with the product according to the invention (200-400 mg/kg, oral administration, 1 h) produced significant inhibition of paw edema induced by carrageenan (300 μg/paw) in mice, in time intervals of 30, 120, 240 min and 24h. The percentages of inhibition were: 31 .4 ± 9.7, 38.2 ± 13.3 e 22.5 ± 3.1 % for a dose of 200 mg/kg (120 min, 240 min e 24 h after the carrageenan injection, respectively) and 57.9 ± 6.7, 50.5 ± 14.3, 70.9 ± 5.3 e 42.9 ± 4.0 %, for the dose of 400 mg/kg (30, 120, 240 min e 24 h after the injection of carrageenan, respectively). In the same way, the paw edema induced by carrageenan in mice was significantly reduced with the treatment with dexamethasone (o.5mg/kg, subcutaneous, 4h) with percentages of inhibition 62 ± 1 1 , 61 ± 7, 65 ± 8, 75 ± 5 e 61 ± 4 %, in 30, 60, 120, 240 min e 24 h after the injection of carrageenan, respectively.

Curative effect of the product according to the invention upon colitis As demonstrated in figures 3 and 4, the product according to the present invention (200-400 mg/kg, oral administration, twice a day, for 8 days) was able to reduce the macroscopic score of TBNS-induced colitis in 85.8 ± 5.1 % and 94.5 ± 2.1 % (for doses of 200 and 400 mg/kg, respectively), visually showing an improvement on the colitis when compared to the group treated only with TNBS (2,5 mg). In the same way, dexamethasone (1 mg/kg twice a day, for 8 days) also conferred protection, reducing the macroscopic score in 66.8 ± 1 1.2% and 81.3 ± 3.6. The microscopic evaluation of the colon (fig. 5) demonstrated that the product according to the invention was able to reduce (97± 0.4%) the tissue damage provoked by the treatment with TNBS (animals with colitis).

Effect of the product according to the invention upon BK-induced spontaneous nociception

As observed in fig. 6, the product according to the invention (400 mg/kg, oral administration, 1 h) was able to cause significant inhibition of the nociception induced by intraplantar injection of BK (100 nmol/paw), with percentage of inhibition of 70 ± 5 %. Similar result was observed in the animals treated with HOE-140 (50 nmol/paw, subcutaneous administration), a drug used as positive control, with percentage of inhibition of 47 ± 12 %.

Effect of the product according to the invention upon SNPC

As can be observed in figure 7, a SNPC, a neuropathic pain animal model, caused significant reduction in the basal threshold in response to stimulation with the Von Frey filament (0.6g; p < 0.001 ) when compared to the false-operated group. The product according to the present invention (400 mg/kg, oral administration, 1 h) significantly decreased the SNPC-induced mechanical hypersensitivity, with a percentage of inhibition of 54 ± 5 % (figure 7). This effect remained for up to 4 hours after treatment.

Results

According to the result in figure 1 , related to the GC-MS chromatogram of the evaluated sample, it can be seen that in the product according to the invention there are different reducing and non-reducing sugars, namely arabinose, xylose, mannose, galactose and glucose. Those sugars are present in the dry extract in the following concentrations 4.27%, 4.14%, 3.04%, 42.05%, 49.50%, respectively. In this way, it can be concluded that galactose and glucose are the major components in the sample in question, that is, they represent the largest fraction in the chemical composition of the product according to the present invention.

The product according to the present invention shows important anti-inflammatory action and anti-nociceptive (analgesic effect) activity.

It must be understood that the embodiments described hereinbefore are merely illustrative, and that any modification to them may occur to a person skilled in the art. Consequently, the present invention is not limited to the specific embodiments described herein, equivalents being encompassed by the claims attached.

Claims

1 . Standardized pharmaceutical oral product obtained from parts of the Heliotropium plant characterized in that it comprises non-reducing carbohydrates selected from saccharose and β-galactose linked to the glycerol on the monogalactosyl/diacyl/glycerol (MGDG) molecule.

2. Product, according to claim 1 , characterized in that the Heliotropium plant is Heliotropium indicum.

3. Product according to claims 1 or 2 characterized in that it comprises arabinose, xylose, mannose, galactose and glucose, with the concentrations of 4-5%, 3.5-4.5%, 2.5-3.5%, 40-45% and 45-55%, respectively.

4. Process to obtain the product according to one of claims 1 to 3, characterized by the fact that it comprises the steps of:

a. Macerating parts of Heliotropium indicum in a mixture of aprox. 10:1 of ethanol and water, for 90-100 hours.

b. Filtrating and evaporating the macerate in an evaporator, particularly a rotoevaporator.

c. Filtrating to obtain a final product.

5. Process according to claim 4 characterized in that the ratio among the weight in kg of plant parts, volume in liters of ethanol and volume in liters of water is 1-1 .5:6-7:0.5-1 .

6. Process according to claim 4 characterized in that said plant parts are leaves.

7. Oral pharmaceutical composition characterized in that it comprises from 0.001 to 99.9% of the standardized pharmaceutical oral product according to one of claims 1 to 3.

8. Use of a standardized pharmaceutical oral product according to one of claims 1 to 3 characterized by being in the manufacture of an oral pharmaceutical composition useful in the treatment of inflammation.

9. Use, according to claim 8, characterized in that said inflammation concerns inflammatory diseases of the intestines.

10. Use, according to one of claims 8 or 9, characterized in that said inflammation include the Crohn's disease, ulcerative colitis and irritable bowel syndrome.

1 1 . Method of treating inflammatory processes characterized in that it comprises administering to a patient in need of such treatment from 0.01 to 1 ,000 mg/kg of patient weight, one or more times a day, a pharmaceutical product according to one of claims 1 to 3.