Development of Pharmaceutical Products from Medicinal Plants (TWNSO)

1. Section One: General Information:a. Title of Innovative Experience: “Molecular- and Clinical-Based

Support for the Use Of Peruvian Natural Product Extract as an anti-inflammatory agent”

b. Keywords: Anti-inflammatory agents; Phyto-extracts; Cat’s claw; Uncitolina; NF-kB; cytokines; Phyto-pharmaceuticals.

c. Implementing Institution: Section of Immunology, Department of Microbiology, Faculty of Sciences. Universidad Peruana Cayetano Heredia. Address: Sectioin of Immunology, Department of Microbiology, Faculty of Sciences. Universidad Peruana Cayetano Heredia, Box 4314. Lima 100. Perù. Phone: (511) 319-0014; Fax: (511) 319-0003, E-mail: jaguilar@upch.edu.pe, Website: www.upch.edu.pe/facien/microweb/inmuno/principal.htm

d. Head of Institution : Oswaldo Zegarra, M.D., Rector Universidad Peruana Cayetano Heredia. Phone : (511) 319-0000

e. Person responsible : Jose L. Aguilar, M.D., Head of Section of Immunology, Faculty of Sciences. Universidad Peruana Cayetano Heredia.

f. Other individuals involved: Biologist Percy Rojas, Associate Investigator at the Section of Immunology. He has ended his Master Degree studies on Immunology. He is responsible of animal experiments with Cat´s claw; Biologist Adolfo Marcelo, Associate Investigator at the Section of Immunology. He has ended his Master Degree studies on Immunology. He is responsible of cellular and molecular studies with Cat´s claw; Patricia Hurtado, M.D. Associate Investigator at the Section of Immunology. She has ended his Master Degree studies on Epidemiology. She participate in clinical experiences with Cat´s claw; Fidel Barrantes, medical student. He participated in clinical experiences with Cat´s claw.

g. Implementation Period and Total Costs: It has been several different studies developed during around 3 years. Total cost will be very difficult to establish because it involved some participation of pre-graduate and post-graduate students which spend time and costs for the experiments. However, it may be projected to around US $25,000. It has been contributions from governmental funds around US $2,000; from private funds around US $15,000 and foreign funds US $3,000; and own funds around US $5,000.

2. Section two: Summary of the Innovative Experience:

Chronic inflammatory diseases are a big public health problem all around the world, worsened because there is an increasing percentage of older people in developed and developing countries. Traditionally exists several natural products with attributed anti-inflammatory properties, like Cat´s claw in Peru. We pay attention to an alcoholic extract of Cat´s claw and developed studies in the cellular and molecular bases to explain its properties and to develop clinical studies to demonstrated the scientifically probed useful of this extract.

3. Section three: Background and Justification:

The augmenting amount of people with chronic inflammatory diseases need to be pointed. In developed countries this is a currently programmed situation. However, in developing countries this will turn on a critical issue because of the economical crisis that reserve all resources for “productive issues”, but there is not resources for older people. This situation will maintain a big part of the population outside of pharmaceutical products. Also the develop of side effects on pharmaceutical products chemically synthesized is a consideration. Then is critical to develop therapeutic alternatives. Used the regional resources also will reduce the spend costs to imported substrates. Traditional information is useful, however, in a global word, scientifically proved data is mandatory to support wide use of a natural product. This data must be hard and provide molecular, cellular and clinical solid information to be accord with the current concepts of evidence-based medicine.

4. Section four: Description:

We start this project with the wide investigation of the properties of Peruvian natural products. We select Uncaria tomentosa (Cat´s claw) and decided to evaluate a standardized hidro-alcoholic extract (Uncitolina®). First we evaluate the possible toxicity of this extract in two models: animal lethal doses 50 (LD50), and in cell culture. In both systems the extract demonstrated no toxicity. (Capcha R., Rojas PA, Aguilar JL. Acute toxicity (LD50) for two standardized extracts of Uncaria tomentosa Willd DC and an extract of Lepidium meyenii (Maca) rich in glucosinolatos. Published on Proceedings of the First International Congress FITO 2000, First Peruvian Congress of Medicinal Plants and Phytotherapy. American Phytotherapy Institute. Lima, September 2000). Then we evaluate the capability of the extract to induce anti-inflammatory effect on an animal model (Carragenin induced paw edema), demonstrating good effect. Then we developed molecular and cellular scientific experiments: inhibition of the production of a pro-inflammatory cytokine: tumour necrosis factor alpha (TNF) in the air pouch model in mice, inhibition of the activation of the transcription factor NF-kB in animal Jurkat T cell culture, inhibition of the production of the inflammatory enzyme cyclo-oxigenase in cell culture (Aguilar JA, Rojas PA, Marcelo A, Plaza A, Bauer R, Reininger E, Klaas CA, Merfort I. Anti-inflammatory activity of two different extracts of Uncaria tomentosa (Rubiaceae). Journal of Ethnopharmacology 2002;81(2):271-6), inhibition of the adhesion molecular ICAM-1 expression on human macrophages, experiments which corroborate the anti-inflammatory effect and provide hard data of the molecular steps where this extract act. Also clinically we demonstrated security of the extract on humans on a phase I clinical study (Rojas PA, Aguilar JL, Timoteo OS, Capcha R, Marcelo A, Plaza A, Merfort I, Bauer R. Effect of an extract of Uncaria tomentosa (Uña de gato) upon the production of the Tumoral Necrosis Factor - in a model in vivo and its action as an anti-inflammatory agent. Published at Proceedings of the VIth International Congress of Immunology. La Hababa, Cuba 2002. It received one of the awards of OPS-Wellcome Trust 2002), and demonstrated a clinical response of patients with acute inflammatory process when use pharmacological doses do the extract, with no side effects (Aguilar JL, Barrantes F. Open Clinical Study of the Anti-inflammatory Effect of Uncitolina in articular diseases. Published at the Proceedings of the Ist International Congress of Immunology. Lima, August 2001.

5. Section five: Partnerships:

The community has been involved in clinical studies with a previous informed consentient. Also community will be improved and stimulate to produce a product with scientific support. Academic part has been involved by the UPCH. Private has supported in part this investigations.

6. Section Six: Replicability:

This methodology may easily be reproduced and applied to other natural products from different areas. The idea is maintain local resources to avoid spend on imported products. Also an interchange between developing countries may be planned.

7. Section Seven: Impact:

Right now there is more scientific hard data to support the incorporation of this product in the pharmaceutical strategies provided to medical doctors and general population. It need to be quantified, to offer this alternative to local pharmaceutical corporations, to introduce natural product extracts on its plans. The sustainability will depend on the introduction (marketing) in the medical programs not only in my country but all around the region.

8. Section Eight: Lessons Learnt:

The main obstacle to the develop of this project was the absence of economical supports to develop the studies. Finally this was obtained slowly, with great enforce. Another obstacle was the difficulty to introduce a natural product extract in health professionals, which prefer synthetic marketed products.

9. Section Nine: Future Plans:

Now, a great enforce is been developed to obtain economical support to develop a clinical study phase III, with a big number of patients to demonstrate again clinically the benefits of the product.

Also, it need to continue basic investigation to show the mechanisms by which work this plant, not only as an anti-inflammatory agent, but as a therapy for cancerous cells.

World wide institutions or corporations are welcome to participate in the final developing of this product.

10. Section ten: Tables and graphics:

Table 1. Anti-inflammatory effect of an hydro-alcoholic and an aqueous cat’s claw extract in mice’s paw oedema after 4 hours.

Dose (mg/kg) n

Anti-Inflammatory Activity (%)

Hydro-alcoholic extract Aqueous extract Indomethacin

7 8     37,8

50 8 35,3 21,9  

100 8 37,3 23,8  

200 8 43,0 33,0  

500 8 51,7 26,7  

Table 2. Inhibition of cyclooxygenase-1 and -2 by an hydro-alcoholic and an aqueous cat’s claw extract at a concentration of 50 g/mL (standard deviation in brackets)

Extract Cyclooxygenase-1 Cyclooxygenase-2

Hydro-alcoholic 7,8 ( 10,4) 21,7 ( 3,1)

Aqueous 32,7 ( 6,6) 12,2 ( 6,5)

Table 3. Variation on functional class on patients treated with Uncitolina.

Functional Class

Time of evaluation

Begining 3 weeks 6 weeks

III 70,5 % 40,0 % 16,6 %

II 29,5 % 35,0 % 33,3 %

I 0 25,0 % 50,1 %

Figure 1

HPLC analysis of oxindole alkaloids in hydro-alcoholic extract

_ + + + + +++ + +TNF

1 2 3 4 5 6 7 8 9 10

µg/mL Extract: 50 100 200 500 50 100 200

500Extract A Extract B

Figure 2

The effect of the hydroalcoholic (A) and the aqueous (B) Cat's claw bark extracts on NF-kB DNA binding.

Lane 1 shows unstimulated Jurkat cells, in lane 2 cells were treated with 200 U/mL TNF- alone. In the other lanes (3-10) cells were pretreated for 1 h with various concentrations of the respective bark extract and subsequently stimulated with TNF- for 1 h. Equal amounts of protein from cell extracts were analysed for NF-B activity by EMSA. The filled arrowhead () indicates the position of NF-B DNA complexes. The open circle () denotes to non-specific activity binding to the probe and the open arrowhead () shows unbound oligonucleotide.

Figure 3. Inhibition of the Amount of TNFalpha in the air pouch model using Cat

´s claw extract.

Figure 4

Evolution of the visual scale analogue in patients dealt with Uncitolina

Object 2