The Development and Application of Methodology of Reverse Pharmacology Illustrated with the Research on Analgesic Effect of Resina Draconis

Generally speaking, new situation was opened up for drug discovery when modern science has got a lot of progress in biotechnology, such as the high throughput screening, combinatorial synthesis, asymmetric synthesis, et al. But the pharmaceutical industry is still facing serious challenges because the drug discovery has become more expensive, more risk and lower efficiency. Therefore, a significant change that focus shift from single-target drugs to multi-target drugs have been occurring based on the knowledge of traditional medicine. Strategic choice which based on the drug discovery from natural products, ethnic pharmacology and traditional medicine is expected to overcome the main obstacle that caused by three factors of time, cost, and toxicity during the previous drug discovery. It has been coming to the fore as another drug discovery engine. New strategic choice spawned an interdisciplinary research field called reverse pharmacology [3,9] which provides a paradigm shift from random tracking accidental discovery to organizing a route for seeking lead compound in drug development.

In the study of reverse pharmacology, traditional drugs which have been proved curative in the long-term medical practice were selected for exploratory research aimed at standardization and safety. The confirmation of traditional drugs in the different biological levels, the clarification of its pharmacological mechanism and the identification of its corresponding active components or effective substance will provide scientific basis for the clinical application of traditional drugs, lay the foundation for safety assessment and quality control of traditional drugs, find new leads for creating new chemical drugs and turn the leads to drug candidate through preclinical and clinical research. In this process, safety is the most essential starting point and the effect becomes a thing to be confirmed. Previous "laboratory - clinical" mode in the drug discovery process was reversed to "clinical - laboratory" by reverse pharmacology inspired by the traditional knowledge. The innovativeness of this research mode is that combines the traditional knowledge and modern science and technology, to provide better and safer lead compounds more efficiently. Thus, the traditional herbal medicines which have appeared high efficacy and low side effects in long-term clinic practice got more attention.

The traditional medicine, a rich resource to obtain new lead compounds, generally has complex and diverse chemical composition. In case of analgesic drugs, based on past clinical experience and experimental observation, single herb analgesic are rhizoma ligustici wallichii [10], corydalis [11], panax notoginseng [12], radix angelicae pubesentis [13], clematis [14], etc. However, the analgesic activity component of these herbs has few developments so far. One reason for this situation is although these drugs and their ingredients have analgesic effect, the mechanism and targets have been not elucidated in the modern technological level. Another reason is which ingredients produce the analgesic effect of original drug has not been confirmed as the original drug is a mixture of a variety of chemical constituents and it usually has a variety of pharmacological effects.

Different from single chemical entity drugs, the pharmacological study of traditional medicine need not only clarify the pharmacological mechanism of the traditional drug but also identify the effective substances which produce specific pharmacological effects. The mechanism of the pharmacological effects was revealed by identifying the effective substances, while identifying the effective substances depend on clarifying the pharmacological mechanism of the traditional drug. This is a key issue that clarify scientific basis of clinical curative effect of traditional medicine through the correlation of effective substances and pharmacological mechanism. This is also a methodology question about how to get lead compounds which can reflect original drug’s good performance.

Usual research method of traditional drug is the effects of the chemical composition of the original drug and those of the original drug itself were studied separately. This method do not link the effects of the original drug and its composition, and do ignore a problem that the effect of the original drug and its composition may not be completely consistent, which should give full attention in the research of traditional drug. Therefore, the past method does little help to clarify the pharmacological mechanism of the original drug and to identify the compositions which have the specific pharmacological effect of the original drug and its degree. It is more difficult to determine whether the isolated compositions still retain the relationship between the ingredients in original drug when they produce specific pharmacological effect (Few studies on the combined effects of ingredients in original drug). Aiming at these unsolved questions in research and development of traditional medicine, based on the biological background of traditional medicine Resina Draconis, the basic principle of reverse pharmacology methodology that seeking the component/combinations as primer of new drug should compare the effects of the component/combination with those of the traditional medicine itself with the reference of the pharmacological effects of the traditional medicine were put forward.

In order to solve the problem, the operational definition of the material basis for the efficacy of traditional drug was established as follows [15]. If some component/combination extracted from the traditional drug could produce some pharmacological effect which could also be produced by the traditional drug, the component/combination can be regarded as the effective component/combination of the traditional drug corresponding to the pharmacological effect (We’d like to point out that the concept of active component is used particularly to refer to the component extracted from nature medicine with some pharmacological effects). When the quantity of the effective component/combination is correlative with the quantity of the component/combination contained in the traditional drug and when the pharmacological effect of the effective component/combination could replace that of the traditional drug, the effective component/combination can be named as the material basis for the efficacy of the traditional drug. Whether the traditional drug and its component/combination have pharmacological effects is determined by the values of their pharmacodynamic parameters. The effective degree of the component/combination is determined by the degree of agreement between the values of pharmacodynamic parameters of the component/combination and those of the traditional drug. According to the operational definition, a corresponding research strategy was designed: when the material basis for the efficacy of the traditional drug corresponding to some pharmacological effect is identified, the pharmacological research on the traditional drug should be combined with that on its component/combination. Besides, the study of material basis and the research on pharmacological mechanism of the traditional drug should also be combined. The pharmacological effect of the traditional drug as a whole should be identified first, and by using it as the reference, whether the examined component/combination of the traditional drug has similar effect should be subsequently identified. And then the values of pharmacodynamic parameters of the component/combination should be compared with those of the traditional drug. The comparison determines whether the component/combination is the effective component/combination or the material basis for the efficacy of the traditional drug. Conforming to the operational definition, the component/combination whose pharmacological effect could embody the pharmacological characteristics of the traditional drug and replace the pharmacological effect of the original traditional drug should be found out from various components of the traditional drug to act as the material basis for its efficacy.

The operational definition of the material basis for the efficacy of the traditional drug reflects the relationship between the pharmacological effect of original drug and that of the component/combination, and it helps to quantitatively characterize effectiveness of the component/combination. By the establishment of operational definition of effective substance, searching for the effective substance of traditional drug was converted to a problem that is how to detect, express and analyze the relationships between the pharmacological effects of the component/combination and the traditional drug itself, which could be solved by modern technology. This strategy makes the drug discovery based on traditional medicine resource can be really implemented. After the introduction of the operational definition of the material basis for the efficacy of the traditional medicine, the research model of reverse pharmacology is as follows. At first, based on the reliable data and clinical observations of the therapeutical effect of the traditional drug, the hypothesis of pharmacological mechanism of the traditional drug should be raised. Aiming at the target, exploratory study was carried out to confirm the effect of traditional drug at the molecular and cellular levels. According to the above operational definition and referring to the effects of the original drug, the component/combination having pharmacological activity were screened out. Through the qualitative and quantitative analysis of the pharmacological effects between the original drug and its component/combination, the material basis for the efficacy of the traditional drug which could replace (or approximation to replace) the original traditional drug can be identified. If the material basis for the efficacy consists of several components, the interaction between the components should also be analyzed. Afterwards, more extensive pharmacological and pharmacodynamic studies on the material basis for the efficacy should be carried out at the tissue, organ and individual levels.

Follow the methodology of reverse pharmacology above, a well-known traditional drug Resina Draconis which has been proved has good analgesic effect and no significant side effect in the long-term clinical practice was carried out a series of studies.
 

Pain is a kind of subjective feeling which is from unpleasant to unbearable. This kind of feeling is due to some physical and chemical stimulation on peripheral nerve endings, such as high threshold of mechanical stimulation, too high or too low temperature stimulation, and variety of harmful chemical stimulation, etc. These kinds of stimulation are called nociceptive/noxious stimuli because they can lead to tissue damage. In the process of biological evolution some cells differentiation into sensory neurons. A part of neurons were classified as nociceptor which specifically receive nociceptive stimulation [16]. DRG neurons transmit nociceptive information from the peripheral region to central nervous system. The sensory nerve fibers derived from the abdominal region and the visceral nerve fibers transmitting pain gather together in DRG neurons. There are a lot of membrane protein (membrane receptor /channel protein) molecules in the membrane of the DRG neurons [17], such as Sodium channels, potassium channels, calcium channels, TRPV1, ASICs, nAChRs, etc.

Voltage-gated sodium channel, a kind of membrane glycoprotein, is widely distributed in excitable cells. Each channel comprises one a-subunit and two ß-subunits. All sodium channel a-subunits consist of four homologous domains that form a single, voltage-gated aqueous pore [18]. Based on different sensitivity to tetrodotoxin (TTX), sodium channels in DRG neurons are classified into two types, one is TTX-S sodium channel and the other is TTX-R sodium channel.

TTX-S sodium channels are responsible for action potential initiation and propagation in excitable cells [19], including nerve, muscle, and neuroendocrine cell. Because the stimulus intensity inducing tactile allodynia could activate afferent Aß fiber, there is a great possibility that TTX-S sodium channels are also involved in the transmission of nociceptive information [20]. It has been reported that the application of TTX at low doses (12.5-50 nM, far less than those needed for blocking action potential conduction) produced a significant elevation of mechanical threshold in the paw for foot withdrawals [21]. This change was a sign of reduced allodynic behaviors and suggested that TTX-S subtypes of sodium channels played an important role in maintaining allodynic behaviors in an animal model of neuropathic pain.

The involvement of TTX-R sodium channel has long been known in pain transmission, especially in chronic inflammatory pain and neuropathic pain. Hyperalgesic agents directly increased the magnitude of TTX-R sodium current, decreased its threshold for activation and increased its rate of activation and inactivation, which may underlie nociceptor sensitization [22]. In a model of neuropathic pain, drugs selectively decreased the expressions and the current amplitudes of TTX-R sodium channels, which could reverse the neuropathic pain induced by spinal nerve injury [23]. TTX-R sodium channel has been an attractive therapeutic drug target for inflammatory and neuropathic pain on the basis of its specific distribution in primary sensory neurons and its modulation induced by inflammatory mediators.

TRPV1 receptor is widely distributed in the central and peripheral nervous systems. It is activated not only by chemical factors such as capsaicin and acids, but also by a physical factor such as hot temperature (= 42^°C) [24,25]. TRPV1 receptor is considered an integrator of noxious stimuli in peripheral nociceptor terminals and therefore may be at a crossroads for pain transmission pathways. The logical strategy for development of novel analgesics is to target the beginning of pain transmission pathway and aim potential treatments directly at nociceptors. The TRPV1 receptor antagonists may deliver broad spectrum efficacy in nociceptive pain via silencing pain signaling pathways [26]. Therefore, the discovery of new TRPV1 receptor antagonists becomes an attractive destination in the treatment of pain.
 

Based on the understanding of physiology and pathological mechanisms of pain in modern medicine, TTX-S and TTX-R voltage-gated sodium channels and TRPV1 in rat dorsal root ganglion (DRG) neurons were selected as drug targets. By using whole-cell patch-clamp technique, the effects of Resina Draconis and its flavonoids on these targets were observed. Resina Draconis’s effects on TTX-S/TTX-R sodium currents and capsaicin-induced TRPV1 currents on rat DRG neurons were used as the reference for the evaluation of the pharmacological effects. The extracted components/combinations which have pharmacological activities were screened out, then the pharmacological parameters of the components/combinations and Resina Draconis were compared. According to the operational definition of the material basis for the efficacy of the traditional drug, it is to be determined that the material basis for the effects of Resina Draconis on TTX-S/TTX-R sodium currents and capsaicin-induced TRPV1 currents. The concentrations of various components in the combinations were determined by the mass ratio of these components in final products of Resina Draconis, which guaranteed that the quantity of each component existent in the combination was in agreement with that in Resina Draconis.

Cochinchinenin A, cochinchinenin B and loureirin B extracted from Resina Draconis which have significant pharmacological activity were screened out. Experimental results suggest that only loureirin B could produce approximate inhibition on the TTX-S sodium currents as Resina Draconis. Loureirin B could be regarded as the material basis of Resina Draconis for the inhibition on the TTX-S sodium currents [27]. Although cochinchinenin A, cochinchinenin B and loureirin B can inhibit the TTX-R sodium currents when they was used alone, none of them had approximate effect of Resina Draconis. The combination of cochinchinenin A, cochinchinenin B and loureirin B (molar ratio is 38:19:8) could produce approximate inhibition on the TTX-R sodium currents as Resina Draconis. So it is considered that the combination of cochinchinenin A, cochinchinenin B and loureirin B (38:19:8) was the material basis of Resina Draconis for the inhibition on the TTX-R sodium currents (Figure 1) [15].

None of the three components had approximate inhibition of Resina Draconis on the capsaicin-induced TRPV1 receptor currents on rat DRG neurons. But the combination of cochinchinenin A, cochinchinenin B and loureirin B (molar ratio is 38:19:8) could produce approximate inhibition on the capsaicin-induced TRPV1 receptor currents as Resina Draconis (Figure 1). Therefore it was considered that the combination containing cochinchinenin A, cochinchinenin B, and loureirin B (38:19:8) was also the material basis of Resina Draconis for inhibiting capsaicin-induced TRPV1 receptor current [28]. This inhibition could relief pain by intervening in the transmission of pain messages.
 

The above results that the material basis for inhibition on the TTX-R sodium currents in DRG neuron of Resina Draconis was the effective combination of the three components showed that sometimes the pharmacological effect of the traditional drug was produced not only by one component but by the effective combination of several components. It is likely that the pharmacological effect of the single component in the combination was influenced by other components in the combination, which required that the interaction of the components of the material basis for the efficacy of the traditional drug should be assessed if the material basis for the efficacy of the traditional drug was not a single component. This is also an indispensable step in making clear the pharmacological mechanism of the material basis for the efficacy of the traditional drug.

Drug interactions are usually divided into three types: antagonism, synergism and additivity. One mathematical model used to assess drug-drug interactions was developed by Greco et al [29,30] and could be used to characterize the interactions of two or three drugs when the dose-response curve of each drug follows Hill equation. All the dose-response curves of cochinchinenin A, cochinchinenin B and loureirin B could be fitted well with Hill equation. The interactions of cochinchinenin A, cochinchinenin B and loureirin B in modulating the TTX-R sodium currents in DRG neurons were analyzed by using the Greco model. The calculation suggested that the interaction of cochinchinenin A, cochinchinenin B and loureirin B was synergistic when the combination inhibited the TTX-R sodium currents on DRG neurons [15]. This marked synergistic interaction of the three components may explain the experimental phenomenon that the three components used in combination at lower dose could produce the same effects on the TTX-R sodium currents as the three components used alone at higher doses.

A visual model of the interaction of three components was established according to the equivalent surface formula derived by Guo et al. [31]. As shown in Figure 2, point Q1 was a mixed concentration point of combination (38 µmol/l of cochinchinenin A, 19 µmol/l of cochinchinenin B, and 8 µmol/l of loureirin B). The coordinate values of Q1 were statistically smaller than those of their corresponding theoretical additive points. Therefore, the point Q1 was below the respective corresponding three additive surfaces, which indicated that interactional effect of the three components in combination on capsaicin-induced TRPV1 currents were synergistic.
 

The drug effectual procedure in vivo is very complex so that it is necessary to design the in vivo experiment to confirm the material basis for the analgesic effect of Resina Draconis at systemic level. In pain transmission pathways, spinal dorsal horn (SDH) is a pivotal center for integrating and relaying pain-sensing signals (nociceptive) from periphery to brain. In SDH neurons, it was wide dynamic range (WDR) neurons that play a fundamental role in the transmission and processing of pain signals and in the coding for the stimulus intensity [32].

Extracellular microelectrode recordings were used to observe the effects of Resina Draconis and its component/combination on the discharge activities of WDR neurous in SDH of intact male Wistar rats evoked by electric stimulation at sciatic nerve. The effect of 0.5 mg/mL Resina Draconis on the discharge activities of WDR neurons were taken as a reference for the comparison between the pharmacological effect of Resina Draconis and the effects of the component/combination. The concentration of each component in various component combinations was determined according to the percentage content of each component in final products of Resina Draconis, which ensured that the quantity of each component existent as a single or in combination was in agreement with that in Resina Draconis. The comparative results showed that although the evoked discharge of WDR neurons could be inhibited to different extent by the three components (cochinchinenin A, cochinchinenin B and loureirin B), none of them could produce the same inhibition as Resina Draconis when their quantities were correlative with their quantities contained in Resina Draconis. In the various combinations of the three components, only the combination of cochinchinenin A, cochinchinenin B and loureirin B (molar ratio is 38:19:8) could produce the approximate inhibition on the evoked discharge of WDR neurons as Resina Draconis (Figure 3).