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Stimulation-based analgesia techniques – do they have a role to play in post-operative pain management?

STIMULATION-BASED ANALGESIA TECHNIQUES: DO THEY HAVE A ROLE TO PLAY IN POST-OPERATIVE PAIN Abstract
Despite the advances in surgical procedures and analgesia options, post- operative pain (POP) remains a chal enge. Modest efforts have been made to incorporate non-pharmacological, stimulation-based analgesia techniques in POP management with the hope of better control of pain and reduction of analgesic medication and side effects.
Pain is a complex phenomenon, involving both sensory-discriminatory and affective-cognitive processes. Many levels of the peripheral and central nervous system are involved in the transmission, modulation and integration of nociceptive information – the ‘pain matrix’. Recent research has demonstrated that acupuncture and the-like stimulation-based techniques exert their effect via modulation of the ‘pain matrix’ both at the periphery and on spinal cord /brain The majority of RCTs’ and systematic reviews’ results demonstrate acupuncture and TENS to be effective in reducing analgesic intake, side effects and pain intensity as part of standard POP management. The crucial factor for their effectiveness lies in the location, mode, intensity, timing and duration specifics of the stimulation they produce. Future research should concentrate on studying what the most effective and clinical y applicable stimulation characteristics are. Keywords: Post-operative pain, analgesia, acupuncture, TENS, stimulation-
Pain fol owing surgery is an undesired, but extremely common occurrence.
Although surgical techniques and procedures, as wel as analgesia options have improved tremendously in the past few decades, 10-50% of patients are estimated to develop persistent chronic pain, which can be severe in 2-10% of cases (Kehlet et al, 2006). This increases the human (distress and suffering) and economic (higher morbidity and prolonged hospitalisation requirements) costs of surgery both in the short and long run. As the guiding principal of today’s healthcare system is to provide clinical y effective and cost efficient service, there is a strong impetus to optimise and standardise clinical interventions such as post-operative pain (POP) management (Rosenquist & Rosenberg, 2003). Opioid and anti-inflammatory (paracetamol, steroid and NSAID) pharmacological preparations provide the main analgesia in POP management. Although their effectiveness and safety are continuously improving, there are stil serious risks associated with their side effects. Opioids can cause respiratory and GI tract depression, nausea and vomiting, urinary retention and ileus, dizziness, lowered mental awareness and confusion. NSAIDs are associated with GI tract bleeds, renal damage and cardiac problems (Breivik, 1995; Spacek, 2006). These side effects are especial y pronounced in the elderly, which are becoming an ever- larger group of elective surgery patients (Rakel & Herr, 2004; White, 2002). Over the last 20-30 years, with the increase of knowledge of the physiology of pain, temptative efforts have been made to incorporate non-pharmacological analgesia techniques in order to minimise the use of opioids, reduce adverse events and speed-up recovery and hospital discharge (Rowbotham, 2005). The two main adjunct non-pharmacological modalities in POP management are stimulation-based analgesia and psychological intervention. This review focuses only on the most frequently used external (minimal y invasive), peripheral stimulation-based analgesia techniques: acupuncture in its different modes of application - Manual Acupuncture (MAc), Electro-Acupuncture (EAc) and Auricular Acupuncture (AAc), as wel as non-invasive acupuncture and non- acupuncture point stimulations - Transcutaneous Electrical Nerve Stimulation (TENS), Acupuncture-like Transcutaneous Electrical Nerve Stimulation (AlTENS) and acupressure. The criteria for choosing these techniques are based on availability of published articles rather than on assumed higher clinical effectiveness. This is not a comprehensive review – a limited number of recent articles were analysed with the objective to find out if there is sufficient justification for the incorporation of these analgesic techniques in the POP Post-operative pain
The International Association for the Study of Pain (IASP) defines pain as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” (Merskey et al, 1979).
This definition attempts to express the dual nature of pain: sensory-discriminatory and affective-cognitive. In reality the physiological nature of pain (nociception) is very complex, involving multiple interactions of neural networks from the periphery – to spinal cord – to brain and back to spinal cord and periphery – the so-cal ed ‘pain matrix’ (Melzack & Wal , 1965). Pain can be general y classified in few sub-types according to the mechanisms of the underlying pathology and neuro-pathophysiology: inflammatory pain, neuropathic pain, visceral, cancer and chronic non-specific pain (Falquhar-Smith, 2007). Pain generating events can occur at the periphery, involving somatic tissue, viscera or nerve tissue, as wel as in the spinal cord or in higher supra- spinal regions of the CNS along the ‘pain matrix’ neuro-axis. Post-operative pain shares the same general characteristics (Spacek, 2006) with any other somatic pain. It is location and surgical procedure specific and it is different in nature at different stages in the post-operative period (Kahlet et a., 2006). In the immediate stages, the surgical incision and tissue manipulation produces actual tissue damage with ensuing inflammatory responses. Often during surgery nerve fibres get also severed or compressed, so the resulting pain has concomitant characteristics of both inflammatory and neuropathic pain. Taking into account the vulnerable and unstable physical, mental and emotional state of a patient after a surgical procedure (malaise, stress, anxiety and apprehension) and it is easy to see how the acute pain can lead to maladaptive plasticity and development of persistent chronic pain states (Kehlet et al, 2006). In order to avoid that, adequate post-operative analgesia is crucial. The fol owing paragraphs attempt to summarise the main interactions between the different links in the ‘pain matrix’ and identify potential targets for stimulation-based Inflammatory pain is a direct result of activation and excitation of primary
nociceptive afferent (PNA) neurons by tissue damage (mechanical, thermal or chemical) in injury and surgery. A variety of different inflammatory substances get released in the interstitium by damaged cel s, platelets, immune cel s and the nerve endings themselves: prostaglandins, cytokines, histamine, bradykinin, serotonin (5-HT), norepinefrine, substance P (SP), Neurothrophic factors and many more (Farquhar-Smith, 2007). This continuous chemical excitation of the afferent neurons has a two-fold effect on nociception: firstly, it hyper-excites the primary nociceptive afferent (PNA) neurons with corresponding plastic changes (up and down regulation of gene-expression, ion channel and receptor activity and distribution), which lowers their resting potential and causes primary sensitisation and increased firing; secondly, the increased continuous bombardment of the second order neurons in the Dorsal Horn (DH) leads to their sensitisation (neural plasticity changes associated with NMDA, AMPA, GABA and opioid receptors and ion channels pre- and post-synaptical y), which leads to increase of nociceptive signals being conducted to other parts of the Central Nervous System (CNS) – other spinal laminae and segments and higher brain centres. Inflammatory pain is associated with two phenomena – hyperalgesia and al odynia, which result from primary and secondary sensitisation. In the initial stages of tissue damage inflammatory pain directly corresponds in intensity to the underlying pathology, but if the local and descending inhibitory control is ineffective (there could be many reasons for inadequate higher centres pain suppression: psychological and emotional, physiological and metabolic, genetic) the sensation of pain can persists for longer than the actual tissue damage and lead to central sensitisation with plastic changes in the sub-cortical and cortical regions of the CNS (Woolf and Salter, 2000; Kehlet et al, 2006; Dickenson, Pharmacological y, inflammatory pain is treated with paracetamol, NSAIDs and steroids, which form an important part of standard POP management.
Traditional y acupuncture is commonly used to reduce pain and inflammation and there have been few studies confirming its anti-inflammatory action. Electro- acupuncture (EAc) has been shown to increase the plasma concentration of corticosterone in rats (Li et al, 2007), reduce peripheral SP concentration (Cao & Wang, 1989) and enhances the degranulation of mast cel s (Zhang et al, 2007).
Another interesting study (Wang et al, 2006) il ustrated the reduction of hyperalgesia and primary sensitisation by EAc to be connected with down- regulation of expression of NMDA receptors in the central pre-synaptic terminals of smal nociceptive fibres in the Dorsal Root ganglion (DRG). As inflammation is a common consequence of surgery, acupuncture and electro acupuncture demonstrate potential to counteract the overexcitement of the primary nociceptive afferents, reduce the primary sensitisation and contribute to the POP management in the initial stages after surgery. Neuropathic pain is a direct result from damage to nerve tissue – in the
periphery (as is the case of most surgical procedures) efferent and afferent axons get damaged. The ensuing neuroma (the proximal end of the damaged axon) begins to spontaneously fire – ectopic pacemaker-like activity, which leads to bombardment of DH neurons (Kehlet et al, 2006; Dickenson, 2007). The neuroma is also associated with abnormal sprouting and ion channels expressions and migration as wel as recruitment in the Dorsal root ganglion (DRG) of neighbouring unaffected afferent fibres. All this leads to maladaptive plasticity in the DH and secondary sensitisation with symptoms of hyperalgesia, al odynia and concurrent hypoaesthesia (Kehlet et al, 2006).
Chronic (also known as clinical) pain is characterised by nociception in the
absence of clear clinical pathology. Its nature is complex and not ful y understood. It is accepted (Dickens, 2007) that there is maladaptive plasticity involving multiple links in the descending pain control system (both inhibitory and facilitory) from the mid-brain and brainstem as wel as local spinal circuits.
Ultimately, the aim of any analgesic treatment is to prevent the formation of Descending Inhibitory System (DIS), Diffuse Noxious Inhibitory Control
Many nuclei of the medul a, midbrain, thalamus and hypothalamus, limbic system, prefrontal and somato-sensory cortices are involved in the integration and modulation of nociceptive information (Apkarian et al, 2005; Peyron et al, 2000). The different circuits have specific roles to play – discrimination of the spatial, temporal and intensity specifics of the stimulus, affective and emotional response, memory, volition and coordination of motor and autonomic functions and so on. Their main output converges in the midbrain and brainstem – Periaqueductal Grey (PAG), Nucleus Raphae Magnus (NRM), Locus Coeruleus (LC) and Rostro Ventral Medul a (RVM) from where the major serotonergic, opiodergic and noradrenergic inhibitory pathways project directly to DH secondary and primary afferent nociceptive neurons as wel as local spinal inhibitory interneurons (Purves, 2004; Farquhar-Smith, 2007). The DIS is the principal top-down regulation and modulation of pain mechanism in the nervous system. Majority of its inhibitory effect is produced by release of endogenous opioid neurotransmitters in the DH of the spine – β-endorphin, enkephalins and dynorphin, which inhibits both the primary afferents and prevents the firing of the secondary DH neurons. This is the pharmacological rational for the use of opioid analgesics to prevent the sensation of pain. Activation of the DIS and release of endogenous opioids is one of the principal mechanisms of action of stimulation-based analgesia – widely accepted as the mode of action of traditional manual acupuncture (MAc) and electro acupuncture (EAc) (Han et al, 1999; Han, 2003; Han, 2004). There are numerous papers published in the last 10 years of brain imaging (fMRI and PET) studies on brain activation during acupuncture points stimulation (Dhond et al, 2007a; Dhond et al, 2007b; Napadow et al, 2005; Napadow et al, 2007; Dougherty et al, 2008; Hsieh et al, 2001), which confirm that. They share many common findings, which demonstrate areas of activation and deactivation in the ‘pain matrix’, consistent with the understanding of the function of DIS. Another possible mode of activation of the DIS and endogenous opioid release is the Diffuse Noxious Inhibitory Control (DNIC) phenomenon. Le Bars and col eagues (1979) reported suppression of the activity of nociceptive Trigeminal neurons in the medul ary DH after conditioning painful stimulation anywhere on the body, irrespective to the area or tissue type. Bing and col eagues (1991) demonstrated that acupuncture produces similar action to DNIC. Their results show that manual acupuncture stimulation at Zu San Li (S36) acupoint on the lower limb of a rat produced very similar pain conditioning inhibition of the Tigeminal convergent neurons as emerging the hindpaw in hot water. Spinal ‘Gating’ (SG)
In the sixties Ronald Melzack and Patrick David Wal (1965) postulated an exciting new theory of integration and modulation of nociceptive signals in the DH – the spinal ‘gate’. They reasoned that there must be substantial interaction in the DH between PNA (C fibers), non-nociceptive afferents (A β fibers), local inhibitory interneurons and secondary projection neurons. In summary, when thick myelinated fast conducting A β fibers get stimulated, they can over-ride the excitation in the DH produced by signals from the nociceptive slow conducting C fibers and suppress the firing of the secondary projection neurons – close the ‘gate’. Although quite simple, this theory has helped to explain some of the mechanisms of action of stimulation-based techniques such as acupuncture and TENS. Not everybody agrees with the specifics of type of fibers involved.
Kawakita and Okada (2006) point out that acupuncture points’ location is based on traditional theory, which postulates that acupoints can simultaneously be stimulated by needling (mechanical) and burning moxa (thermal). As A β fibers are not associated with transmission of heat signals, they put forward that other A-type fibers (Aδ) connected with polymodal receptors should be considered the main afferents conducting acupuncture and the-like stimulations. As il ustrated in the previous paragraphs, substantial research into the mechanisms of pain as wel as analgesia and the mechanisms of action of acupuncture and similar type of stimulation-based techniques has been done al over the world. Although complete understanding is far from been achieved, there are clear indications of the analgesic potential of stimulation-based techniques. The fol owing paragraphs review the data coming from clinical trials of different types of acupuncture and TENS, used for the treatment of post- Acupuncture and related techniques
Acupuncture is an age-old therapeutic technique, used for mil ennia in East Asia for the treatment of disease and promotion of health. It is based on the traditional anatomo-physiological understanding of the inter-connectedness between the different parts of the human body via the acupuncture meridian system. The principal role of the meridians is to control homeostasis and coordinate the metabolism of al the tissues in the body by distributing Qi (a vital substance closely connected with the blood, which has its origin in the nutrients and water of the food and air, as wel as other regulatory substances produced by the body itself) (Cheng, 1987). Although anatomical y there is no such a structure that resembles the classical acupuncture meridians, recent research (Langevin and Yandow, 2002) has found a strong connection between location of acupuncture points and organisation of the extracel ular connective tissue matrix. From modern anatomo-physiological perspective, acupuncture meridians are better viewed as functional rather than anatomical entities, which closely overlap with the function of the nervous, endocrine and immunological systems - the overarching neuro-endocrine-immune system (Claw and Chrousos, 1997). Nowadays acupuncture is practiced extensively al over the world by classical y trained acupuncturists (Chinese, Japanese, Korean and other traditions), as wel as western-trained doctors, physiotherapist and other health professional. There is considerable variety in both theoretical underpinning and practical application of acupuncture to such a degree, that sometimes the only common ground between different style acupuncturists is the use of solid needles, rather than points and stimulation type specifics. This is wel il ustrated in the latest definition of acupuncture by the Department of Health sponsored Acupuncture Regulatory Working Group in the UK. In their report (2003) they defined acupuncture as ‘’.the insertion of a solid needle into any part of the human body for disease prevention, therapy or maintenance of health. There are various other techniques often used with acupuncture, which may or may not be invasive’ . This lack of unity and consensus on what constitutes a clinical y effective treatment makes acupuncture very difficult to study in an experimental design setting. The blinded, placebo control ed clinical trial, which has become the gold standard, further complicates the situation. No wonder there are many inconsistent and, sometimes, conflicting results in randomised clinical trials (RCT) of acupuncture. This is also reflected in the RCTs of the use of In a recent systematic review of acupuncture and related techniques for POP, Sun and col eagues (2008) identified 126 studies, but due to inadequate quality (not RCT, lack of placebo, no post-operative measurements) only 15 met the inclusion criteria. The included RCTs varied to the type of surgery (abdominal gynaecological, thoracic, knee and hip, and oral/molar), to the mode of acupuncture point stimulation (MAc, EAc, AAc, acupressure and capsicum plaster acupoint application), as wel as to time of initiation and length of treatment (pre-, post- and throughout the whole peri-operative period). Their main outcomes were post-operative opioid consumption and pain intensity for the first 8, 24 and 72 hours, recovery room stay and opioid-related side effects: nausea, sedation, pruritis and urinary retention. The results of their meta-analysis showed that the acupuncture treatment reduced significantly opioid consumption in the first 8, 24 and 72 hours respectively by 21%, 23% and 29%. The pain intensity also was reduced, but reached significance only at 72 hours.
Unsurprisingly the opioid-related side effects were lower in the acupuncture group. Sun and col eagues concluded that acupuncture and related techniques are effective opioid-sparing adjuncts to conventional POP management, as suggested by the evidence. As limitations to their study, they pointed out the variability of acupuncture regiments, stimulation types, placebo/sham controls, timing of intervention, outcome measures and smal size of trials, which in their My search has identified only one recent RCT (Michalek-Sauberer et al, 2007), which has failed to show effectiveness of acupuncture for POP and analgesia consumption in third molar tooth extraction surgery. In this study Michalek- Sauberer and col eagues investigated electrical stimulation and auricular acupuncture peri-operatively from 30 minutes before the operation to 48 hours post-operatively. Their protocol involved three different types of Auricular points stimulation: group one had embedded ear needles plus electric stimulation (EA); group two had only embedded needles plus sham electric stimulation (AA) and group three (the control) had smal metal plates attached with sham electric stimulation (NN). The three different groups varied demographical y only marginal y – the EA had significantly more smokers and female patients. The pain intensity, analgesic consumption (Acetaminophen) and rescue medicine requirements (Mefenamic acid) did not differ significantly between the groups.
Their conclusion was that neither electrical auricular acupuncture, nor auricular acupuncture alone, was effective for reducing pain intensity and analgesic consumption for molar tooth extraction POP.
Two other independent studies (Lao et al, 1995; Tavares et al, 2007) also evaluated the effectiveness of acupuncture on POP in identical type of molar extraction oral surgery. In contrast to Michalek-Sauberer and col eagues’ study, both of them found acupuncture to be effective in reducing pain intensity and analgesic requirements. The acupuncture protocols employed in their studies were, however, quite different from the previous study. Lao and col eagues used MAc on classical acupoints on the legs and face post-operatively against a sham treatment (non-penetrating taped needles) immediately after the operation and again when the pain increased. Tavares and col eagues, on the other hand, combined both 5 classical body acupoints (upper and lower extremities and face) as wel as 2 auricular points, which were stimulated both manual y and electrical y 24 hours prior to the operation and immediately after the operation.
The patients themselves were used as a control in this study – one molar extraction was performed with and the other without acupuncture in a random There are few possible explanations for Michalek-Sauberer and col eagues’ results. As there was no ‘no-treatment’ group in their study it is difficult to confirm that stimulation of the three auricular points was ineffective. As the points on the ear are very superficial (directly under a thin layer of skin against the ear cartilage), auricular acupuncturists often tape seeds to patient’s ears, relying on the direct mechanical stimulation for the therapeutic effect. Michalek-Sauberer and col eagues’ control group had smal metal plates taped to the auricular points, so it is possible that this was not an ‘’inactive’ treatment. The electric stimulation device was worn for over two days by the participants, which almost half of them found uncomfortable and irritating. This could have interfered with the relaxing and analgesic effects of the treatment and distorted the results. It is also possible, that the choice of acupuncture points and/or auricular acupuncture in general are not the most effective method for relieving acute POP oral pain as it is in other acute POP conditions (Usichenko et al, 2005). These are just few examples, which il ustrate the importance of the specifics of the acupuncture and related techniques in respect to point selection, type of manipulation, timing and duration. All these techniques produce their therapeutic effect by feeding information (signal ing) in the system - for example electrical stimulation at different frequencies leads to different opiods release: 2Hz – endorphins and 100 Hz – dynorphin (Han, 2003). It is only logical to assume that the system would respond differently to stimulation with different special, temporal, frequency and intensity characteristics. Transcutaneous Electrical Nerve Stimulation (TENS)
RCTs in the effectiveness of TENSE for POP management, further il ustrate the chal enges of stimulation-based therapeutic techniques research. As with the acupuncture techniques, the crucial factor for the effectiveness of TENS application lies in the specifics of the stimulation it produces. TENS is passing low voltage electrical pulses via surface adhesive electrodes, which has been extensively used in physiotherapy for pain control (Kotze and Simpson, 2007). The electrodes are usual y attached close to the area of pain, on the same segmental dermatome. When the electrodes are placed over an acupoint, TENS is referred to as ALTENS. TENS is thought to exert is analgesic effect via stimulation of predominantly Aβ fibers and control ing the ‘spinal gate’ and release of endogenous opioids (Kotze and Simpson, 2007). In the late nineties, a team at the University of Texas conducted studies (Chen et al, 1998; Hamza et al, 1999; Wang et al, 1997), which investigated how the specifics (location of electrodes placement, frequency and intensity) of the TENS stimulation reflected its effectiveness for POP after abdominal (hysterectomy) surgery. Their results demonstrated TENS to be effective in reducing opioid requirements and related side effects, when used as part of the POP management protocol. They also found that the location of electrodes placement next to the incision is as effective as when placed over Zu San Li (S36) acupoints on the lower legs and significantly more effective (37% and 39% opioid intake reduction) compared to sham TENS and inappropriate location (shoulder) TENS (Chen et al, 1998). Hamza and col eagues’ study (1999) demonstrated that different frequencies (2Hz, 100Hz and mixed 2/100Hz) TENS produce different outcomes. All the TENS stimulations reduced significantly the need for analgesics, but the mixed 2/100Hz stimulation had grater effect (53% versus 32% and 35% in the 2Hz and 100Hz respective reduction compared to sham treatment). Wang and col eagues (1997) examined the effect of the intensity of TENS stimulation. The results of their RCT demonstrated that high (subnoxious) intensity TENS is significantly more effective in reducing opiod requirements (65%) compared to no treatment - low intensity TENS group achieved 34% and In light of the findings of the studies in the previous paragraph, not taking the specifics of the TENS stimulation into account could very easily lead to conflicting results in RCTs. Unsurprisingly, two systematic reviews of TENS in POP management have reached total y opposite conclusions. Carrol and col eagues (1996) in their meta-analysis emphasised predominantly the importance of randomisation in TENS studies of POP, but failed to take into account, that only few of the studies included in their analysis had used stimulation with sufficient intensity. Consequently they concluded that TENS does not provide any benefit for POP in respect to pain intensity and analgesic consumption. Bjordal and col eagues’ systematic review (2002), on the other hand, reached total y opposite conclusions – TENS, when administered with adequate intensity and appropriate frequency could significantly reduce analgesic consumption for POP. These discrepancies further confirm that, when investigating effectiveness of stimulation-based techniques, the specifics of the produced stimulation are of Conclusions
The progress made in the past few decades in the field of neuro-biology and neuro-pharmacology has lead to great improvements in pain management and treatment. The tremendous increase in knowledge of the intimate molecular, cel ular and systemic mechanisms involved in pain processing have also opened the door for the investigation of age-old stimulation-based techniques like acupuncture. Although we are far from understanding al the mechanisms of action of acupuncture (and the-like techniques), research evidence clearly demonstrates the major effect acupuncture stimulation produces on peripheral (PNA), spinal cord (DH) and supra-spinal level of the central nervous system. In the area of POP management, evidence from RCTs and systematic reviews points out that both acupuncture and TENS are useful adjunct to standard POP management, reducing the analgesic requirements, side effects and pain intensity. The crucial factor in their ability to produce significant clinical effect is appropriate and adequate stimulation in respect to location, mode, intensity, The majority of modern research has focused on pharmacological interventions of molecular and cel ular signaling. That has led to the development of new, more effective drugs, but there have always been limitations to the success of transferring new theoretical knowledge into clinical y effective drug treatments.
The main obstacle often originates from the systemic way pharmacological agents are administered in practice (oral, intravenous, intramuscular and subcutaneous injections and so on). Although they target specific cel receptors, they affect many more cel s than intended, producing undesired side effects, Recent research, especial y in acupuncture, has demonstrated that stimulation- based techniques produce their effect also through signaling. The difference is that the signaling produced is direct and specific to the characteristics (location, mode, intensity, timing and duration) of the stimulation, which modulates the many control ing functions of the nervous system. Future research of stimulation- based techniques should concentrate on studying what the most effective and clinical y applicable stimulation characteristics are, both for pain and other clinical References
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SANDRA OUTLAW, PLAINTIFF-APPELLANT vs. SANDRA L. WERNER, M.D., ET AL., DEFENDANTS-APPELLEES No. 92297 COURT OF APPEALS OF OHIO, EIGHTH APPELLATE DISTRICT, CUYAHOGA COUNTY 2009 Ohio 2362 ; 2009 Ohio App. LEXIS 2004 May 21, 2009, Released PRIOR HISTORY: [**1] [*P2] On December 21, 2007, Outlaw originallyCivil Appeal from the Cuyahoga County Court offiled this action

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Prof. Matías Llabrés Facultad de Farmacia Universidad de La Laguna 38200 La Laguna, Tenerife Spain Scientific publications Updated: April, 2007 MANOVA of statistical moments in biopharmaceuticals studies: a numerical example with three equally doses of amoxicillin. Vila, J. L., Matínez-Pacheco, R., Jiménez, J., Llabrés, M. Journal of Pharmacokinetics and Biopharmaceutics 8

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