Mechanistic and Clinical Assessment of ZangradoR, an Extract of the Amazonian Ethnomedicine Sangre de Grado, for the Treatment of Itch

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Miller et al.



may evoke an itch response. Hence, there is substantial need for new

therapeutic approaches that have a broad therapeutic potential. Given that

the activation of sensory afferent nerves is the final pathway in eliciting an

itch, any agent that suppresses sensory afferent nerve activation would fulfill

the criteria of this search. In zangrado , a botanical extract derived from an

k

Amazonian ethnomedicine, we believe that there is the potential for such a

therapeutic innovation.

Zangrado is an extract of sangre de grado or sangre de drago, a latex

obtained from various Croton species from the jungles of the upper

Amazon, primarily Peru and Ecuador. Zangrado has reduced proanthocyandin content, which allows it to be topically useful because the inherent

ethnomedicine has a rich burgundy color that accounts for its reference to

blood in its ethnic name. The ethnomedical uses for sangre de grado are

diverse but include wound healing, anti-itch, and analgesia. In addition,

taken orally, it alleviates nausea and diarrhea and promotes the healing of

gastrointestinal ulcers (1). We have been researching the scientific basis for

these ethnomedical reports, and have noted that a suppression of sensory

afferent nerves appears to underlie all of these actions (2,3).

Sangre de grado is collected ethnomedically by slashing the tree and

applying the sap or resin (Fig. 1), as required, to the afflicted area. Relief is

very rapid, usually within seconds. Thus, historically, this would not impair

the native hunters from pursuing their activities. Commercially, the tree is not

suitable for tapping (as maple or rubber is) because repeated slashing of the

tree results in opportunistic fungal infections and compromised productivity.

Additionally, the sap is quite viscous and flows slowly. Hence, harvesting

usually involves cutting down the tree and making incisions every 9 in.—from

these cuts the sap is collected.

One of the key applications of sangre de grado in the Amazon is

topical administration to relieve the symptoms of insect bites, stings, and

plant reactions. These are numerous and extensive problems. In order to

assess the efficacy of sangre de grado for these disorders under more controlled conditions, we set about to compare a balm with sangre de grado vs.

a placebo in pest management workers. These individuals are prone to these

types of complications as a work hazard. Specifically, the study was conducted in New Orleans, LA, in the spring when fire ants are a major pest.

Fire ants (Solenopsis invicta) are derived from South America and inflict a

painful but intensely pruritic response. The itch persists for a week and

scratching can lead to secondary complications of infection.

Current therapies for fire ant stings are noneffective and largely

confined to nonprescription pharmaceuticals. Interestingly, heat exacerbates

the itch further, highlighting the interactive components of thermal hyperalgesia and itch for this condition. Fire ants represent a major problem of



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307



Figure 1 The resin of Amazonian Croton lechleri is collected by cutting the bark

and the red bloodlike sap (sangre de grado or sangre de drago) can then be applied

topically, or collected for oral administration.



epidemic proportions in the southern United States. One third of all

residents and 50% of all children in the southern United States are stung

each year and an estimated 27 million people are stung annually.

Fire ants (Solenopsis spp.) are exceptional among the arthropods in

producing venoms that are rich in alkaloids and unusually low in proteins

(4,5). The venoms are stored in the poison sac and delivered through the

stinger in microgram quantities. In humans, injection of venom leads to

pronounced necrosis of the epidermis with the formation of pruritic

pustules. Coma and death have even been reported in a few cases involving

multiple stings coupled with anaphylactic shock (6). The incidence of allergic/anaphylactic reactions is approximately 0.5% for those suffering repeat bites. As the pustules develop, the first fluid is clear, and then necrotic

polymorphonuclear cells and lymphocytes abound, and by 72 hr, the most

common cells are necrotic plasma cells. The pustule floor disintegrates as the

lesion begins to heal, and the fluid spreads into connective tissues (7). Non-



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allergic victims have suffered thousands of stings with no lasting adverse

consequences other than the pustules and associated annoying symptoms,

although repeated scratching can establish infected, slow-to-heal wound

sites (8). There are virtually no studies that have addressed the types and

amounts of inflammatory mediators released in response to fire ant stings,

let alone therapies that negate these events.

The venoms are composed mainly of 2-alkyl or alkenyl-6-methyl piperidines, alkaloids also known as the solenopsins (9). As only the alkyl or

alkenyl group differs among the various piperidines, it is used to identify

the alkaloids readily (10). For example, C11 : 0 denotes an alkaloid with an

11-carbon chain and no double bond, and C13 : 1 denotes an alkaloid with a

13-carbon chain and one double bond. Both cis and trans isomers of the

solenopsins are usually present, but their relative proportions are speciesspecific. For instance, cis forms predominate in S. xyloni and S. geminata,

whereas trans forms predominate in S. richteri and S. invicta (11). Considering the extent of the problem that fire ants pose and the need for new

therapeutic approaches, the pest management worker study was designed to

address this potential application among pest management workers.

Another difficult-to-manage pruritic condition is itch associated with

opioid analgesia. Opioid narcotics are commonly associated with the induction of nausea and vomiting, as well as itch. The current understanding

suggests that this complication results from a spinal disinhibition. Specifically, the sensory afferent nerves responsible for mediating itch are activated

subsequent to removal of the inhibitory influence of pain pathways. In contrast, the same scenario is thought to be responsible for the ability of capsaicin creams to block itch, thereby promoting a pain-induced inhibition of

itch pathways at the spinal level.

Currently, therapy is focused on reversing the opioid effects with receptor antagonism, but there is little research directed at the potential benefits

of agents that directly suppress the traffic of itch signals, largely because of the

lack of available pharmacological tools. Zangrado was tested as a potential

therapy in an appropriate animal model, morphine-induced itch, and emesis

in ferrets. Ferrets are particularly responsive to opioids in this regard (12).



II.



METHODS



A.



Clinical Trial—Insect Bites and Stings



We designed a double-blind placebo-controlled trial with Zangrado in pest

management workers. These individuals routinely suffer from insect bites and

stings as part of their occupation and were asked to apply either placebo or

zangrado balms (coded) to the skin following ‘‘events’’ and to document the



Mechanistic and Clinical Assessment of Zangrado



309



symptoms and time taken to achieve relief. Ten participants were also asked

to describe their preference using the two test balms. The trial was performed

in New Orleans, LA, in the spring when the primary hazard is fire ants.



B.



Inhibition of Sensory Afferent Nerve Pathways



To assess directly the effects of zangrado on sensory afferent nerves and their

activation, we evaluated the capsaicin-induced increases in gastric blood flow

in anesthesized rats using a laser Doppler flow probe. Previously, we have

reported that zangrado negates the capsaicin-induced secretory responses in

intestinal epithelia in a manner that was independent of neurokinin receptors,

as well hyperalgesia responses to protease-activated 2 receptors and prostaglandin E2 (3).



C.



Inhibition of Opioid-Induced Itch



We evaluated whether zangrado could alleviate opioid-induced licking in ferrets. Morphine-6-glucuronide (15 mg/kg, i.p.) was administered to ferrets,

with either zangrado (3 mg/kg, i.p.) or vehicle administered 15 min before the

opioid. Ferrets were videotaped for behavioral analysis and the number of

grooming or licking episodes was counted over the course of 1 hr as an index

of itch response. With the knowledge that opioid-induced emetic responses

are attenuated by cannabinoids, we also examined whether this opioidinduced licking/itch response could be reversed by the cannabinoid receptor

1 antagonist (AM 251).



III.



RESULTS



In the pest management workers evaluated over the course of 3 months, fire

ants were the most common event, with all 10 participants suffering from

at least one episode. The remaining events were in order of incidence: wasps =

other ants (six), cuts, bee = mosquito = plant reactions = abrasions (all

one). For all applications, the participants preferred the zangrado balm to the

placebo. Relief was reported for all symptoms, including itch and pain, on

average, in less than 2 min. Given the current difficulties in treating the

itching response to fire ants, this result was regarded as being remarkable,

consistent with the ethnomedical experience from the Amazon. Of interest to

note is that the active balm only needed to be reapplied once or twice,

suggesting that, in addition to rapidly ameliorating the pruritic condition,



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zangrado curtailed the duration of this response to fire ant stings. One individual reported an itch response to a plant reaction, and this was also

effectively blocked by zangrado.

Opioid administration to ferrets resulted in a significant licking/grooming response, which was virtually abolished by zangrado (Fig. 2). Coadministration of the cannabinoid receptor 1 antagonist, AM 251, failed to reverse the

actions of zangrado. Additionally, zangrado did not produce any sedation or

hypothermia characteristic of cannabinoids. Collectively, these results suggest that the antipruritic action of zangrado is pharmacologically distinct

from cannabinoids.

In anesthetized rats, topical capsaicin evoked a dramatical increase in

gastric blood flow (Fig. 3). This response was abolished by topical pretreat-



Figure 2 Induction of itch in ferrets is prevented by zangrado but not reversed by

cannabinoid antagonists. Morphine-6-glucuronide (15 mg/kg, i.p.) resulted in a

substantial induction in licking and grooming in ferrets, indicative of itch. Zangrado

given as a 15-min pretreatment (3 mg/kg, i.p.) prevented this response ( p < 0.01).

This action of zangrado was not mediated by cannabinoid receptors because the

cannabinoid antagonist, AM 251, was without effect.



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311



Figure 3 Zangrado blocks the hyperemia response to topical capsaicin. Blood flow,

measured by a laser Doppler flow meter, was increased by topical capsaicin as a means

of activating sensory afferent nerves. Zangrado completely prevented this response

( p < 0.01), indicating its ability to suppress sensory afferent nerve activation.



ment with zangrado, consistent with its analgesic actions and its actions on

capsaicin-induced intestinal epithelial secretion (2,3).



IV.



DISCUSSION



From these results and our previous studies (2,3), we have determined that

zangrado has a profound ability to suppress the activation of sensory afferent nerves. As a result, zangrado provides relief for itch, pain, and neurogenic inflammation. The topical application of a zangrado balm for fire

ant bites highlights the significant anti-itch actions. Indeed, participants

reported that only one to three applications in total were required to almost

immediately relieve itching, which otherwise would persist for a week. It

appears that zangrado promoted rapid healing of fire ant stings, and

negated an otherwise persistent response. This suggests that the actions of



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zangrado may extend beyond immediate relief but to a termination of a

positive feedback cycle that may maintain these conditions.

Given this therapeutic efficacy, it is critical to determine how these

actions are achieved as it will provide important information as to the basic

mechanisms of itch and pain. Currently, zangrado defies definition. Therapeutic approaches such as antihistamines and 5-HT3 receptor active agents

cannot explain zangrado’s analgesic actions or its reported effects on protease-mediated neurogenic inflammation. We have previously documented

that sangre de grado is a very effective inhibitor of the thermal hyperalgesia

and edema associated with intradermal application of peptide agonists for

proteinase-activated 2 receptors (PAR-2). PAR-2 receptors are located

directly on sensory afferent nerves (13) and mediate the itch and pain response following mast cell release of tryptase. Apart from peptide ligands

that function as receptor antagonists, there is no other therapy known to

block the actions of PAR-2 activation. This highlights the unique functional

profile of zangrado.

The prevention of morphine-induced itch could indicate that zangrado

is an opioid antagonist, but that is not compatible with zangrado’s analgesic

actions. With inhibition of capsaicin-induced hyperemia responses, zangrado

could be acting as a vanilloid receptor 1 (VR1) antagonist. However, that is

not consistent with the ability of zangrado to prevent protease-activated 2

receptor responses on sensory afferent nerves; furthermore, capsaicin, a VR1

agonist, is often used to block itch largely by evoking concurrent pain

responses and short-circuiting the itch sensation. Thus, this pathway is also

unlikely.

Cannabinoids have been found to negate the skin responses of capsaicin

(14,15), as well as attenuate the emetic responses of opioids (8,16). The itch

response induced by morphine, which was blocked by zangrado, was not

reversed by cannabinoid receptor antagonists, indicating that zangrado is not

working through cannabinoid-dependent mechanisms. These comparisons

are detailed in Table 1.

Collectively, although it is clear that zangrado is effective in blocking

itch and pain in response to a variety of stimuli, these effects are not consistent with known pharmacological approaches. Indeed, the best way of

describing these actions is the activation of a yet-to-be-identified receptor

that evokes a generalized inhibitory action on sensory afferent nerves.

Possible candidates for action are sodium channels or tetrodotoxin-resistant

voltage channels, but this remains to be explored. There is a possibility that

because it is a natural product with multiple chemical constituents, the

bioactivity reflects diverse mechanisms mediating by distinct components.

For example, zangrado has some similarities to vanilloid receptor antagonists in terms of signal transduction mechanisms, and because zangrado



Mechanistic and Clinical Assessment of Zangrado



313



Table 1 Comparison of the Bioactivity Profiles of Common Therapeutic Approaches

to Itch and Pain

Itch



Agent

Antihistamine

5-HT3

compounds

Capsaicin

Opioids

Cannabinoids

Zangrado



Pain



Capsaicin

responses



PAR2

responses



Central

complications



Opioid

responses



#

Mild #



p!

p!



p!

p!



p!

p!



z

z



Mild #

p!



#

z

#

#



#

z

#

#

#



p!

#

#



?

p!

?

#



p!

z

z

p!



?

#

#



This panel highlights the unique profile of bioactivity of zangrado among known agents that either

relieve or induce itch and pain.



attenuates capsaicin responses, it may block vanilloid (capsaicin) receptors.

There are a substantial number of papers that suggest that numerous vanilloid receptors exist, or at least that the current receptor classification does

not explain the plethora of observations (17,18). Given that endogenous

chemicals that interact with cannabinoid and vanilloid receptors have been

found to exist (19,20), zangrado may also provide insight into potential

endogenous anti-itch chemicals. However, until that time, zangrado deserves further exploration as a therapeutically useful tool for a variety of

pruritic conditions.



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1.

2.



3.



4.

5.



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Miller MJS, MacNaughton WK, Zhang X-J, Thompson JH, Charbonnet RM,

Bobrowski P, Lao J, Trentacosti AM, Sandoval M. Treatment of gastric ulcers

and diarrhea with the Amazonian medicinal, sangre de grado. Am J Physiol

2000; 279:G192–G200.

Miller MJS, Vergnolle N, McKnight W, Musah RA, Davison CA, Trentacosti

AM, Thompson JH, Sandoval M, Wallace JL. Inhibition of neurogenic inflammation by the amazonian herbal medicine, sangre de grado. J Invest Dermatol 2001; 117:725–730.

Baer H, Liu TY, Anderson MC, Blum M, Schmid WH, James FJ. Protein

components of fire ant venom (Solenopsis invicta). Toxicon 1979; 17:397–405.

Attygalle AB, Morgan ED. Chemicals from the glands of ants. Chem Soc Rev

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Rhoades RB. Medical Aspects of the Imported Fire Ant. Florida: The University Presses of Florida, 1977.

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Diaz JD, Lockey RF, Stablein JJ, Mines HK. Multiple stings by imported fire

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venoms: chemotaxonomic correlations with alkaloidal compositions. Toxicon

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Van Sickle MD, Oland LD, Ho W, Hillard CJ, Mackie K, Davison JS, Sharkey

KA. Cannabinoids inhibit emesis through CB1 receptors in the brainstem of

the ferret. Gastroenterology 2001; 121:767–774.

Steinhoff M, Vergnolle N, Young SH, Tognetto M, Ennes HS, Trevisani M,

Hollenberg MD, Wallace JL, Caughey GH, Mitchell SE, Williams LM, Geppetti

P, Mayer EA, Bunnett NW. Agonists of proteinase-activated receptor-2 induce

inflammation by a neurogenic mechanism. Nat Med 2000; 6:151–158.

Ko M-C, Woods JH. Local administration of D9-tetracannabinol attenuates

capsaicin-induced thermal nociception in rhesus monkeys: a peripheral cannabinoid action. Psychopharmacology 1999; 143:322–326.

Li J, Daughters RS, Bullis C, Benjamin R, Stucky MW, Brennan J, Simone

DA. The cannabinoid receptor agonist WIN 55,212-2 mesylate blocks the

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Simoneau II, Hamza MS, Mata HP, Siegel EM, Vanderah TW, Porreca F,

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Di Marzo V, Bisogno T, Melck D, Ross R, Brockie H, Stevenson L, Pertwee R,

De Petrocellis L. Interactions between synthetic vanilloids and the endogenous

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30

Reduction in Itch Severity with

Topical Immunomodulators:

A New Approach for Patients

with Inflammatory Disease

Alan B. Fleischer, Jr.

Wake Forest University School of Medicine, Winston-Salem,

North Carolina, U.S.A.



I.



INTRODUCTION



The most common pruritic disorders are caused by inflammatory skin diseases. Atopic dermatitis is a chronic, relapsing form of eczema characterized

by scaling, itchy, inflamed skin that can be triggered by an interplay of genetic, immunological, and environmental factors. Along with asthma and allergic rhinitis, atopic dermatitis is part of a larger family of allergic diseases

(1). Immune dysregulation appears to play an important role in the etiology

of atopic dermatitis (2,3). Bone marrow-derived cells may play a primary

role because sensitivity to antigens has been transferred to recipients of bone

marrow cells from patients with atopic dermatitis (4). Altered T-cell function appears to be the primary immunological abnormality present in atopic

dermatitis and patients have elevated levels of IgE. Agents that decrease the

inflammation may indirectly improve the sensation of itch.

Topical corticosteroid agents have been the mainstay of therapy for

atopic dermatitis due to their broad immunomodulatory effects. Topical

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corticosteroid agents are not ideal agents because when used over the long

term, they may cause cutaneous atrophy (5–7). Not infrequently, patients

display disease refractory to short-term topical corticosteroid agents. Indeed, the majority of patients with atopic dermatitis fail to clear their skin

by the end of the approved durations of treatment. Clinicians and patients

must then decide whether the benefits of ongoing topical therapy outweigh

its associated ever-increasing risks. Clinicians have been searching for corticosteroid-sparing agents that can be used when long-term topical therapies

are required, yet lack the expense, inconvenience, and monitoring required

of phototherapy and systemic immunosuppressive therapy. The recent development of topical tacrolimus may fill this role.

Tacrolimus is a 23-member macrolide of molecular mass 822 Dalton

(Fig. 1) produced by Streptomyces tsukabaensis, a fungus found in the soil of

Mount Tsukuba, Japan (8). The drug’s name is derived as follows: t—Mount

Tsukuba, acrol—macrolide, imus—immunosuppressant. It is frequently also



Figure 1



Chemical structure of tacrolimus.