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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
Amazonian ethnomedicine, we believe that there is the potential for such a
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
Mechanistic and Clinical Assessment of Zangrado
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-
Miller et al.
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).
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
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.
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).
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).
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,
Miller et al.
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
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.
Mechanistic and Clinical Assessment of Zangrado
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).
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
Miller et al.
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
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
Table 1 Comparison of the Bioactivity Proﬁles of Common Therapeutic Approaches
to Itch and Pain
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
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Reduction in Itch Severity with
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.
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
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
Chemical structure of tacrolimus.