Pharmacokinetics of multiple doses of co-crystal of tramadol-celecoxib: findings from a four-way randomized open-label phase I clinical trial.

AIM: We compared the pharmacokinetic (PK) profiles of co-crystal of tramadol-celecoxib (CTC) vs. each reference product (alone and in open combination) after single (first dose) and multiple dosing.
METHODS: Healthy adults aged 18-50 years received, under fasted conditions, 15 twice-daily doses of the following treatments (separated by ≥14-day washout): 200 mg immediate-release (IR) CTC (equivalent to 88 mg tramadol and 112 mg celecoxib; treatment 1); 100 mg IR tramadol (treatment 2), 100 mg celecoxib (treatment 3); and 100 mg IR tramadol and 100 mg celecoxib (treatment 4). The treatment sequence was assigned by computer-generated randomization. PK parameters were calculated using non-compartmental analysis. Parameters for CTC were adjusted according to reference product dose.
RESULTS: A total of 30 subjects (20 males, mean age 35 years) were included. Multiple-dose tramadol PK parameters for treatments 1, 2 and 4, respectively, were 551, 632 and 661 ng ml-1 [mean maximum plasma concentration (Cmax )]; 4796, 4990 and 5284 ng h ml-1 (area under the plasma concentration-time curve over the dosing interval at steady state); and 3.0, 2.0 and 2.0 h (median time to Cmax at steady state). For treatments 1, 3 and 4, multiple-dose celecoxib PK parameters were 445, 536 and 396 ng ml-1 ; 2803, 3366 and 2897 ng h ml-1 ; and 2.0, 2.0 and 3.0 h. Single-dose findings were consistent with multiple-dose data. Types of adverse events were consistent with known reference product safety profiles.
CONCLUSION: After single (first dose) and multiple dosing, PK parameters for each active pharmaceutical ingredient in CTC were modified by co-crystallization compared with reference products alone or in open combination.

RCT Entities:   Population Interventions Outcomes

What is Already Known about this Subject

Development of pharmaceutical co‐crystals containing two active pharmaceutical ingredients (APIs) may confer each API with distinctive physicochemical, pharmacokinetic (PK) and clinical profiles compared with the reference products.

Co‐crystal of tramadol–celecoxib (CTC) is a novel API‐API co‐crystal under development for the treatment of pain.

In a previous single‐dose phase I study of CTC, the PK parameters of each API were modified by co‐crystallization compared with the reference products (immediate‐release tramadol or celecoxib) alone and in open combination.

What this Study Adds

After multiple dosing, the PK profiles of tramadol and celecoxib from CTC are modified by co‐crystallization compared with reference products alone or in open combination.

The types of adverse event observed during multiple‐dose treatment were as expected, based on the reference product labels.

These observations are consistent with the CTC mechanistic effect that can translate into favourable PK changes.

Tables of Links

Introduction

Pain is a complex, multifaceted phenomenon, originating from various sources and involving multiple physiological pathways 4. A multimodal approach to analgesia is often considered necessary 5, 6. Combining drugs with complementary mechanisms of action can amplify pain relief when additive, supra‐additive or ‘synergistic’ interactions occur 7, 8. Multimodal therapy may also permit use of lower drug doses and thereby improve the safety and tolerability of treatment 9, 10, 11. For example, combining non‐steroidal anti‐inflammatory drugs (NSAIDs) with opioids to treat acute pain can improve efficacy compared with opioids alone and also reduce opioid consumption 12, 13 and opioid‐associated adverse events (AEs) 14, 15. Traditionally, multimodal analgesia has involved the administration of multiple separate drug formulations in ‘open’ combination, or use of fixed‐dose combinations (FDCs) in which component active pharmaceutical ingredients (APIs) are contained in a single formulation at a fixed ratio often representing the doses of the individual approved drugs 16.

A new approach to multimodal therapy is the development of ‘co‐crystal’ drugs 17. Within a co‐crystal, the physicochemical properties of an API may be modified compared with other solid‐state forms, although the API's molecular structure is unchanged. This may result in enhanced bioavailability and changes in other pharmacokinetic (PK) properties. For example, co‐crystallization of carbamazepine with the co‐former saccharin significantly improves its physical stability and dissolution. This translated to a higher mean maximum plasma concentration (C max) and similar median time to C max (T max) compared with the marketed form of carbamazepine in preclinical models 18. Similarly, co‐crystallization of ibuprofen and nicotinamide enhances the solubility of ibuprofen 19, 20. Although their physicochemical properties may be altered, APIs retain their biological activity within the co‐crystal structure as they are not modified covalently 21.

Co‐crystals containing two or more APIs represent the next generation in co‐crystal technology and offer a novel approach to multimodal therapy 22. A number of such co‐crystals have been identified, although only one (a complex comprised of anionic forms of sacubitril and valsartan, sodium cations and water molecules) licensed for use in chronic heart failure (Entresto®; Novartis, Basel, Switzerland) 23 has so far been approved. As described in the label, the valsartan in Entresto is more bioavailable than the valsartan in other marketed tablet formulations – i.e. ‘26 mg, 51 mg and 103 mg of valsartan in Entresto is equivalent to 40 mg, 80 mg and 160 mg of valsartan in other marketed tablet formulations, respectively’ 23.

Co‐crystal‐associated changes in physicochemical properties that may modify PK parameters do not necessarily lead to an improved clinical benefit over the single API. In the examples above, increases in exposure are reflected in dose adjustments to maintain efficacy and safety. The first‐in‐class co‐crystal of tramadol–celecoxib (CTC) presents a different concept supported by in vitro and phase I data after a single dose 24 ; neither tramadol nor celecoxib from CTC show increased exposure levels compared with the individual authorized tramadol or celecoxib, but rather show a change in profile that may translate into clinical benefits per se. One aspect of CTC based on its individual API PK and pharmacodynamic profiles is the hypothesis of CTC‐associated improved efficacy with reduced doses of each API. In this vein, to understand the safety of CTC, we will have to wait for the results of efficacy clinical trials. We will then know the real benefit–risk relationship for CTC and therefore its real therapeutic benefit.

CTC is in development by Esteve Pharmaceuticals (as E‐58425) and Mundipharma Research (as MR308) for the treatment of acute pain. The final immediate‐release (IR) tablet formulation of CTC combines racemic tramadol hydrochloride (rac‐tramadol.HCl) and celecoxib at an intrinsic 1:1 molecular ratio (1:1.27 weight ratio) conferred by the co‐crystal structure. CTC represents a rational approach to multimodal analgesia, combining four mechanisms of action. Tramadol is a weak mu‐opioid receptor agonist and inhibits the reuptake of serotonin and noradrenaline, and its main active metabolite, (+)‐O‐desmethyl‐tramadol (M1), has a much greater affinity for the mu‐opioid receptor than tramadol itself 25, while celecoxib selectively inhibits cyclooxygenase‐2 26. In a rat model of postoperative pain, a form of CTC co‐crystal without additives in suspension (‘CTCsusp’) demonstrated synergistic analgesia (i.e. efficacy greater than that predicted by the addition of the individual analgesic effects of rac‐tramadol.HCl and celecoxib alone). In addition, CTCsusp displayed comparable efficacy to the morphine and oxycodone in this model but with an improved safety profile 27. Intrinsic dissolution studies have shown that the release profiles of tramadol and celecoxib from the co‐crystal are modified compared with those from each reference drug 28. Such effects have the potential to optimize the PK profiles, and thus efficacy and safety, of each API in CTC. In a single‐dose phase I study of CTC, the PK characteristics of tramadol and celecoxib from CTC were modified by co‐crystallization relative to IR tramadol or celecoxib alone and the open combination of these reference products 24. These changes in PK parameters could translate into a real therapeutic benefit, to be determined in phase II and III clinical trials.

The main objective of the present phase I study was to compare the PK profile of CTC with that of each authorized reference product alone and in open combination after single (first dose) and multiple dosing. The safety and tolerability of CTC following single (first dose) and multiple dosing were also evaluated.

Methods

Study subjects

Males and nonpregnant, nonlactating females aged 18–50 years with a body mass index of ≥18.5 and <29.0 kg m−2 were eligible for inclusion in the study if they were non‐ or ex‐smokers and in good general health, as determined by medical history, physical examination, electrocardiogram (ECG) and standard clinical laboratory tests. Individuals were excluded if they had a history of significant hypersensitivity to tramadol, celecoxib, opioids, sulphonamides or any related products; a history of severe hypersensitivity reaction to any drug; a significant history of drug dependency or alcohol abuse; a condition that may have affected the PK profile of the study drugs; or used systemic contraception, hormone replacement therapy, monoamine oxidase inhibitors or enzyme‐modifying drugs within 4 weeks of the start of the study (see Appendix S1 for full inclusion and exclusion criteria).

Study design and treatments

This was a randomized, open‐label, four‐period, four‐sequence, crossover, single‐ and multiple‐dose study performed in a single centre in Canada. Four treatments were administered under fasting conditions (Figure 1). The order in which treatments were received by each subject was assigned from a computer‐generated randomization list. The four treatments were: treatment 1: 2 × 100 mg IR CTC tablets (200 mg; equivalent to 88 mg rac‐tramadol.HCl and 112 mg celecoxib; proposed marketed formulation); treatment 2: 2 × 50 mg IR tramadol capsules (rac‐tramadol.HCl; 100 mg; Adolonta®, Grünenthal GmbH, Germany); treatment 3: 1 × 100 mg celecoxib capsule (100 mg; Celebrex®, Pfizer Manufacturing Deutschland GmbH, Karlsruhe, Germany); and treatment 4: open combination of 100 mg IR tramadol (rac‐tramadol.HCl; 2 × 50 mg capsules) and 100 mg celecoxib (1 × 100 mg capsule).

Figure 1

Study design. Treatment 1, 2 × 100 mg CTC tablets; treatment 2, 2 × 50 mg IR tramadol capsules; treatment 3, 1 × 100 mg celecoxib capsule; treatment 4, 100 mg IR tramadol (2 × 50 mg capsules) plus 100 mg celecoxib (1 × 100 mg capsule). CTC, co‐crystal of Ttramadol–celecoxib; IR, immediate‐release

Each treatment period was separated by a washout period of ≥14 days. Treatments were administered orally twice daily with 240 ml water (12 h apart, in the morning and evening) for 7 days, with a final dose in the morning of day 8 (15 doses in total). Morning doses were administered following a fast of at least 10 h and evening doses following a fast of at least 2 h. On days 1 and 8, fasting continued for at least 4 h following the first and 15th treatments. Standardized meals and snacks were provided postdose at approximately the same time throughout the study. Water was provided ad libitum until 1 h predose and allowed approximately 1 h after each dose. Volunteers were instructed not to take any non‐investigator‐approved prescription medications or over‐the‐counter products during the study and to avoid alcohol, and grapefruit‐, pomelo‐ or xanthine‐containing food or drink. Strenuous activity was restricted.

The study protocol was approved by an institutional review board (project number 2167, approved on 24 November 2011 by ETHIPRO; Montreal, QC, Canada) and the study was performed in compliance with Good Clinical Practice, the Declaration of Helsinki and relevant US, European and Canadian standards. Written informed consent was provided by all subjects.

PK sampling and analytical methods

For the single‐dose part of the study, blood samples for PK measurements were collected prior to drug administration and at the following times postdose: 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10 and 12 h. For the multiple‐dose part, samples were also collected: (i) within 5 min before the third (day 2), fifth (day 3), seventh (day 4), ninth (day 5), 11th (day 6), 13th (day 7) and 14th (day 7) drug administrations; and (ii) within 5 min before and at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 12, 24, 48 and 72 h following the 15th (day 8) administration.

Blood samples were collected and centrifuged (1500 g for 10 min at 4°C) to obtain plasma, which was separated into duplicate tubes and frozen until assayed. Samples from all subjects who received at least one study treatment were assayed. Plasma concentrations of tramadol, M1 and celecoxib were measured using validated high‐performance liquid chromatography with tandem mass spectrometry methods. Sample pretreatment involved the solid phase extraction of tramadol, M1 and celecoxib and their internal standards (propranolol and E‐6087, respectively) from 0.050 ml of human plasma. These compounds were identified and quantified over a theoretical concentration range of 4.00–640.00 ng ml−1 for tramadol, 1.00–160.000 ng ml−1 for M1 and 2.50–1000.00 ng ml−1 for celecoxib. Assay inter‐run precision (coefficient of variation) and accuracy (nominal values) were 8.3% and 102.5%, respectively, for tramadol; 10.1% and 105.2% for M1; and 10.5% and 107.8% for celecoxib. Assay specificity was assessed by employing six independent sources of matrix and verifying for the absence of interference, compared with the respective limit of quantifications at the retention times and mass transitions of analytes and internal standards. Quantitation was carried out using peak area ratios, and back‐calculated concentrations were determined using least squares regression analysis employing a weighted (1/x2) linear regression.

Safety assessments

Safety was assessed by monitoring AEs and by evaluation of standard clinical laboratory parameters, physical and neurological examinations, and 12‐lead ECG. AEs were classified by system organ class and preferred term using the Medical Dictionary for Regulatory Activities version 15.1.

Data and statistical analyses

Sample size determination

As this was a descriptive study to characterize the PK of CTC following multiple doses, no hypothesis testing was planned. A sample size of 32 was estimated to be adequate based on judgements regarding PK properties and accounting for potential dropouts.

PK parameters

For the single‐dose part of the study (first dose), the main PK parameters, calculated using noncompartmental analysis, were C max, cumulative area under the plasma concentration–time curve (AUCτ), measured concentration at the end of the dosing interval (C t) and T max. For the multiple‐dose part of the study (last dose), the main PK parameters, calculated using noncompartmental analysis, were C max at steady state (C max,ss), C t at steady state (C τ,ss), average plasma concentration during dosing interval (C avg), AUCτ at steady state (AUCτ,ss), trough concentration or predose concentration measured at a specific time (C pd) and T max at steady state (T max,ss.). Additional PK parameters were calculated following the last drug administration in each study period and provided for information purposes only.

Statistical analyses of PK data were performed in SAS® version 9.0 (SAS Institute, Cary, NC, USA). Parameters were calculated, as above‐mentioned, using noncompartmental analysis (non‐adjusted and adjusted to the 100 mg doses of the reference products) with a log‐linear terminal phase assumption. An analysis of variance (ANOVA) model was used to analyse all PK parameters with subject effect (nested within sequence), treatment, period and sequence as fixed factors. The natural logarithmic transformation of C max, AUCτ and C t adjusted to dose for the single‐dose part of the study, and of C max,ss, C τ,ss, C avg and AUCτ,ss adjusted to dose for the multiple‐dose part of the study were used for all statistical inference. Additional PK parameters were calculated following the last drug administration in each study period and provided for information purposes only. The 90% confidence interval (CI) for the exponential difference in least squares (LS) means between each comparison was calculated for the ln‐transformed parameters. Subjects who provided evaluable PK data for a particular treatment were included in the descriptive analysis of that treatment; those who provided evaluable PK data for both treatments under comparison were included in the PK and statistical analysis. An additional ANOVA model was used to determine if each analyte had reached steady state after multiple doses of CTC.

Safety

Safety data were analysed using descriptive statistics. Safety was assessed in subjects who received ≥1 dose of study treatment.

Results

Subjects

Thirty‐two subjects were enrolled between 10 October and 23 December 2013. The majority were male (62.5%) and white (71.9%); mean age was 35 years (standard deviation: 9) (Table 1). Eight subjects withdrew/were withdrawn before the end of the study for personal reasons unrelated to clinical events (n = 4), personal reasons related to clinical events (n = 2) or a positive amphetamine test (n = 2). Twenty‐nine, 30, 28 and 28 subjects received CTC, tramadol alone, celecoxib alone and the open combination of tramadol and celecoxib, respectively.

Table 1

Subject demographics (n = 32)

Characteristic
Age, years	35 (9)
Gender, n (%)
Male	20 (62.5)
Female	12 (37.5)
Race, n (%)
White	23 (71.9)
Black	6 (18.8)
Asian	2 (6.3)
Other	1 (3.1)
Weight, kg	73.9 (10.8)
Height, cm	169.7 (9.0)
Body mass index, kg m −2	25.57 (2.24)

Data are mean (standard deviation) unless otherwise stated

PK of tramadol, M1 and celecoxib after administration of CTC

PK parameters for tramadol, its M1 metabolite and celecoxib after single and multiple doses of CTC are summarized in Table 2. Plasma concentration–time profiles for these analytes during multiple dosing are shown in Figure 2. For tramadol, it was not proven statistically that steady state had been reached after the final dose of CTC. However, this finding was not considered clinically relevant. It was proven that steady state had been reached for M1 and celecoxib.

Table 2

Summary of pharmacokinetic parameters for tramadol, M1 and celecoxib following single and multiple doses of 200 mg CTCa (n = 29)

Parameter		Tramadol		M1		Celecoxib
		Mean	CV (%)	Mean	CV (%)	Mean	CV (%)
Single dose	C max (ng ml−1)	219.99	25.2	41.37	44.5	275.86	37.4
	T max (h)b	3.00	41.3	4.00	48.2	2.00	53.6
	AUCτ (ng h ml−1)	1767.33	26.8	360.13	42.1	1436.43	32.1
	C τ (ng ml−1)	98.38	44.1	23.67	37.4	47.59	38.7
Multiple dose	C max,ss (ng ml−1)	484.02	22.4	67.31	37.3	487.08	28.6
	T max,ss (h)d	3.00	32.4	3.00	37.1	2.00	55.9
	AUCτ,ss (ng h ml−1)	4200.73	31.6	653.70	36.4	3088.81	28.6
	C τ,ss (ng ml−1)	243.13	45.1	43.03	37.3	124.10	40.7
	C avg (ng ml−1)	350.06	31.6	54.47	36.4	257.40	28.6
	Fluctuation (%)c	77.42	56.8	44.48	48.5	144.10	26.1
	AUC0–last (ng h ml−1)	7427.52	49.9	1204.01	36.8	5431.19	34.8
	T ½el (h)	8.93	24.8	9.76	20.1	12.32	30.8
	AUC∞ (ng h ml−1)	7667.96	49.6	1271.11	35.3	5686.07	32.9
	AUC0–last/∞ (%)	96.30	3.8	94.45	5.6	95.10	5.9
	K el (h‐1)	0.08	22.9	0.07	20.1	0.06	29.5
	C pd − 24 (ng ml−1)	225.95	41.4	43.05	37.6	170.44	33.2
	C pd − 12 (ng ml−1)	226.40	45.4	42.27	35.5	138.18	39.2
	C pd 0 (ng ml−1)	237.65	42.8	43.58	37.6	176.18	40.1
	C pd 12 (ng ml−1)	243.13	45.1	43.03	37.3	124.10	40.7
	RA(Cτ)	2.55	22.7	1.90	23.4	2.68	27.3
	RA(AUC)	2.39	17.4	2.02	35.0	2.21	22.7

AUC∞, area under the plasma concentration–time curve extrapolated to infinity; AUC0–last, area under the plasma concentration–time curve calculated from 0 to last observed quantifiable plasma concentration; AUC0–last/∞, relative percentage of AUC0–last with respect to AUC∞; AUCτ, area under the plasma concentration–time curve over the dosing interval after single dosing; AUCτ,ss, area under the plasma concentration–time curve over the dosing interval at steady state; C avg, average plasma concentration during dosing interval; C max, maximum observed plasma concentration; C max,ss, maximum observed plasma concentration at steady state; C pd, trough concentration or predose concentration measured at a specified time following a repeated dose regimen; CV, coefficient of variation; C τ, measured concentration at the end of the dosing interval; CTC, co‐crystal of tramadol–celecoxib; C τ,ss, measured concentration at the end of the dosing interval at steady state; K el, apparent elimination rate constant; M1, (+)‐O‐desmethyl‐tramadol; RA(Cτ), accumulation ratio C τ,ss/Cτ; RA(AUC), accumulation ratio AUCτ,ss/AUCτ; T ½el, terminal elimination half‐life; T max, time to reach maximum observed plasma concentration; T max,ss, time to reach maximum observed plasma concentration at steady state

Equivalent to 88 mg tramadol and 112 mg celecoxib

Median values shown

Calculated from ([C max,ss–Cτ,ss]/C avg)*100

Figure 2

Mean plasma concentration–time curves for tramadol, M1 and celecoxib following multiple doses of 200 mg CTC (equivalent to 88 mg tramadol and 112 mg celecoxib). CTC, co‐crystal of tramadol–celecoxib; M1, (+)‐O‐desmethyl‐tramadol

Comparison of tramadol PK after different treatments

Single dose (first dose)

Figure 3A shows mean plasma concentration–time curves for tramadol after a single dose of each tramadol‐containing treatment. Key single‐dose PK parameters for tramadol are summarized and statistically compared in Table 3. After dose adjustment, for C max, 90% CIs for the ratio of geometric LS means (CTC vs. tramadol alone or vs. tramadol plus celecoxib) were outside the equivalence range of 80–125%. For AUCτ and C t, 90% CIs were within this range for both treatment comparisons (Table 3). Median T max for tramadol from CTC was delayed (3.5 h) relative to tramadol alone and the open combination of tramadol and celecoxib (1.75 and 2.00 h, respectively).

Figure 3

Mean plasma concentration–time curves for tramadol after single (A) and multiple (B) doses of CTC, tramadol alone and the open combination of tramadol and celecoxib. CTC, co‐crystal of tramadol–celecoxib

Table 3

Summary and statistical comparison of tramadol pharmacokinetic parameters following single doses of CTC, tramadol alone or the open combination of tramadol and celecoxib

Parameter	Treatment 1: 200 mg CTCa (n = 29)		Treatment 2: 100 mg tramadol (n = 30)		Treatment: 4: 100 mg tramadol + 100 mg celecoxib (n = 28)		Ratio of geometric LS means (90% CI)
	Mean	CV (%)	Mean	CV (%)	Mean	CV (%)	Treatment 1 vs. treatment 2	Treatment 1 vs. treatment 4
C max (ng ml −1 )	249.72b	25.7b	330.27	15.7	331.41	20.8	74.39 (69.95–79.11)	75.58 (71.16–80.29)
AUC τ (ng h ml −1 )	2011.47b	27.3b	2220.16	24.9	2299.81	23.8	90.35 (86.26–94.64)	88.20 (84.09–92.50)
C t (ng ml −1 )	113.04b	44.0b	102.38	45.7	113.73	38.7	112.43 (105.52–119.79)	100.08 (93.37–107.28)
T max (h) c	3.50	40.8	1.75	39.7	2.00	29.3

AUCτ, area under the plasma concentration–time curve over the dosing interval; CI, confidence interval; C max, maximum observed plasma concentration; C t, measured concentration at the end of the dosing interval; CTC, co‐crystal of tramadol–celecoxib; CV, coefficient of variation; LS, least squares; T max, time to reach maximum observed plasma concentration

Equivalent to 88 mg tramadol and 112 mg celecoxib

Parameters for treatment 1 were adjusted according to reference dose

Median values shown

Multiple dose (last dose)

Mean tramadol plasma concentration–time profiles after multiple dosing are shown in Figure 3B. Key multiple‐dose PK parameters for tramadol are summarized and statistically compared in Table 4. After dose adjustment, for the comparison of C max,ss between CTC and the open combination, the 90% CI of the LS means ratio fell outside the 80–125% range. For all other statistical comparisons (including those of C τ,ss, C avg and AUCτ,ss), 90% CIs were within this range. Median T max,ss for tramadol was delayed by 1 h with CTC compared with both of the other tramadol‐containing treatments (3.0 vs. 2.0 h). Tramadol accumulation ratios were similar across treatments (Table 4).

Table 4

Summary and statistical comparison of tramadol pharmacokinetic parameters following multiple doses of CTC, tramadol alone or the open combination of tramadol and celecoxib

Parameter	Treatment 1: 200 mg CTCa (n = 29)		Treatment 2: 100 mg tramadol (n = 30)		Treatment 4: 100 mg tramadol + 100 mg celecoxib (n = 28)		Ratio of geometricLS means (90% CI)
	Mean	CV (%)	Mean	CV (%)	Mean	CV (%)	Treatment 1 vs. treatment 2	Treatment 1 vs. treatment 4
C max,ss (ng ml −1 )	551.19b	22.7b	631.97	23.8	661.24	25.2	87.55 (80.41–95.32)	84.42 (77.59–91.84)
C τ,ss (ng ml −1 )	279.89b	44.8b	257.13	44.1	274.51	43.3	108.38 (103.42–113.57)	103.62 (99.39–108.04)
C avg (ng ml −1 )	399.62b	31.9b	415.85	29.9	440.35	32.1	95.71 (92.47–99.06)	91.91 (88.50–95.45)
AUC τ,ss (ng h ml −1 )	4795.47b	31.9b	4990.17	29.9	5284.17	32.1	95.71 (92.47–99.06)	91.91 (88.50–95.45)
T max,ss (h) c	3.0	33.0	2.0	34.7	2.0	35.9
AUC 0–last (ng h ml −1 )	7502.19	50.1	8579.33	48.3	9003.85	46.5
AUC ∞ (ng h ml −1 )	7749.40	49.6	8749.72	48.1	9350.52	49.3
AUC 0–last/∞ (%)	96.20	3.8	97.70	2.5	96.54	4.3
Fluctuation (%) d	76.65	58.2	96.25	34.5	93.83	31.0
K el (h −1 )	0.08	23.2	0.08	24.8	0.08	25.8
T ½el (h)	9.00	24.7	8.82	22.5	8.94	27.9
C pd − 24 (ng ml −1 )	227.35	41.8	272.22	43.4	286.89	41.8
C pd − 12 (ng ml −1 )	229.26	45.2	254.10	42.2	267.08	45.4
C pd 0 (ng ml −1 )	239.42	43.1	267.08	40.6	277.22	43.3
C pd 12 (ng ml −1 )	246.31	44.8	257.13	44.1	274.51	43.3
RA (Cτ)	2.56	22.9	2.65	30.0	2.48	26.1
RA (AUC)	2.40	17.6	2.25	18.4	2.28	17.4

AUC∞, area under the plasma concentration–time curve extrapolated to infinity; AUC0–last, area under the plasma concentration–time curve calculated from 0 to last observed quantifiable plasma concentration; AUC0–last/∞, relative percentage of AUC0–last with respect to AUC∞; AUCτ,ss, area under the plasma concentration–time curve over the dosing interval at steady state; C avg, average plasma concentration during dosing interval; CI, confidence interval; C max,ss, maximum observed plasma concentration at steady state; C pd, trough concentration or predose concentration measured at a specified time following a repeated dose regimen; CTC, co‐crystal of tramadol–celecoxib; C τ,ss, measured concentration at the end of the dosing interval at steady state; CV, coefficient of variation; K el, apparent elimination rate constant; LS, least squares; RA(Cτ), accumulation ratio C τ,ss/Cτ; RA(AUC), accumulation ratio AUCτ,ss/AUCτ; T ½el, terminal elimination half‐life; T max,ss, time to reach maximum observed plasma concentration at steady state

Equivalent to 88 mg tramadol and 112 mg celecoxib

Parameters for treatment 1 were adjusted according to reference dose

Median values shown

Calculated from ([C max,ss–Cτ,ss]/C avg)*100

Comparison of M1 PK after different treatments

Dose‐adjusted M1 C max was lower after a single dose of CTC compared with tramadol alone or in open combination with celecoxib (Figure 4A; Table 5). Similar values for C t and AUCτ were observed across treatments. The 90% CIs of the LS means C max ratio for M1 were outside the equivalence range for both treatment comparisons. All other statistical comparisons were within this range (Table 5). Median T max for M1 from CTC was delayed at 4.00 h (vs. 2.03 h for tramadol alone and 3.00 h for tramadol plus celecoxib).

Figure 4

Mean plasma concentration–time profiles for M1 following single (A) and multiple (B) doses of CTC, tramadol alone and the open combination tramadol and celecoxib. CTC, co‐crystal of tramadol–celecoxib; M1, (+)‐O‐desmethyl‐tramadol

Table 5

Summary and statistical comparison of M1 pharmacokinetic parameters following single doses of CTC, tramadol alone or the open combination of tramadol and celecoxib

Parameter	Treatment 1: 200 mg CTCa (n = 29)		Treatment 2: 100 mg tramadol (n = 30)		Treatment 4: 100 mg tramadol + 100 mg celecoxib (n = 28)		Ratio of geometric LS means (90% CI)
	Mean	CV (%)	Mean	CV (%)	Mean	CV (%)	Treatment 1 vs. treatment‐2	Treatment 1 vs. treatment 4
C max (ng ml −1 )	45.97b	44.6b	55.66	48.1	52.46	46.1	83.46 (76.81–90.67)	86.94 (79.97–94.52)
AUC τ (ng h ml −1 )	400.93b	42.3b	439.70	40.4	428.25	41.0	90.25 (85.86–94.86)	91.86 (86.14–97.97)
C t (ng ml −1 )	26.53b	37.9b	23.58	36.8	24.87	40.4	112.66 (107.27–118.33)	107.12 (100.14–114.59)
T max (h) c	4.00	46.7	2.03	56.4	3.00	53.4

AUCτ, area under the plasma concentration–time curve over the dosing interval; CI, confidence interval; C max, maximum observed plasma concentration; C t, measured concentration at the end of the dosing interval; CTC, co‐crystal of tramadol–celecoxib; CV, coefficient of variation; LS, least squares; M1, (+)‐O‐desmethyl‐tramadol; T max, time to reach maximum observed plasma concentration

Equivalent to 88 mg tramadol and 112 mg celecoxib

Parameters for treatment 1 were adjusted according to reference dose

Median values shown

Findings for M1 after multiple dosing (Figure 4B; Table 6) were similar to those observed with single‐dose treatment. Similar M1 accumulation ratios were obtained with each treatment.

Table 6

Summary and statistical comparison of M1 pharmacokinetic parameters following multiple doses of CTC, tramadol alone or the open combination of tramadol and celecoxib

Parameter	Treatment 1: 200 mg CTCa (n = 29)		Treatment 2: 100 mg tramadol (n = 30)		Treatment 4: 100 mg tramadol + 100 mg celecoxib (n = 28)		Ratio of geometric LS means (90% confidence limits)
	Mean	CV (%)	Mean	CV (%)	Mean	CV (%)	Treatment 1 vs. treatment 2	Treatment 1 vs. treatment 4
C max,ss (ng ml −1 )	74.71b	36.6b	87.23	34.9	82.32	31.7	84.66 (78.18–91.67)	88.75 (82.34–95.66)
C τ,ss (ng ml −1 )	47.79b	36.7b	47.45	38.2	47.74	35.0	100.82 (95.01–106.99)	99.26 (93.97–104.85)
C avg (ng ml −1 )	60.33b	35.3b	65.97	34.6	63.48	31.3	91.20 (86.32–96.35)	93.53 (89.38–97.87)
AUC τ,ss (ng h ml −1 )	723.91b	35.3b	791.68	34.6	761.81	31.3	91.20 (86.32–96.35)	93.53 (89.38–97.87)
T max,ss (h) c	3.00	37.6	2.00	42.2	2.00	28.9
AUC 0–last (ng h ml −1 )	1178.39	36.4	1428.34	38.2	1402.01	35.3
AUC ∞ (ng h ml −1 )	1246.81	35.0	1467.77	37.0	1447.35	33.7
AUC 0–last/∞ (%)	94.31	5.6	96.73	2.8	96.18	4.1
Fluctuation (%) d	44.51	49.4	60.96	34.5	54.20	37.2
K el (h −1 )	0.07	20.2	0.08	23.3	0.08	22.2
T ½el (h)	9.84	19.9	9.47	23.5	9.49	22.0
C pd − 24 (ng ml −1 )	42.15	37.3	51.67	36.3	48.96	36.2
C pd − 12 (ng ml −1 )	41.44	35.2	49.35	37.1	46.94	37.6
C pd 0 (ng ml −1 )	42.43	36.4	50.81	38.1	47.99	34.8
C pd 12 (ng ml −1 )	42.05	36.7	47.45	38.2	47.74	35.0
RA (Cτ)	1.89	23.8	2.10	25.9	2.07	31.8
RA (AUC)	2.02	35.6	1.98	30.0	2.00	36.9

AUC∞, area under the plasma concentration–time curve extrapolated to infinity; AUC0–last, area under the plasma concentration–time curve calculated from 0 to last observed quantifiable plasma concentration; AUC0–last/∞, relative percentage of AUC0–last with respect to AUC∞; AUCτ,ss, area under the plasma concentration–time curve over the dosing interval at steady state; C avg, average plasma concentration during dosing interval; CI, confidence interval; C max,ss, maximum observed plasma concentration at steady state; C pd, trough concentration or predose concentration measured at a specified time following a repeated dose regimen; CTC, co‐crystal of tramadol–celecoxib; C τ,ss, measured concentration at the end of the dosing interval at steady state; CV, coefficient of variation; K el, apparent elimination rate constant; LS, least squares; M1, (+)‐O‐desmethyl‐tramadol; RA(Cτ), accumulation ratio C τ,ss/Cτ; RA(AUC), accumulation ratio AUCτ,ss/AUCτ; T ½el, terminal elimination half‐life; T max,ss, time to reach maximum observed plasma concentration at steady state

Equivalent to 88 mg tramadol and 112 mg celecoxib

Parameters for treatment 1 were adjusted according to reference dose

Median values shown

Calculated from ([C max,ss–Cτ,ss]/C avg)*100

Comparison of celecoxib PK after different treatments

Mean celecoxib plasma concentration–time curves after single doses of each celecoxib‐containing treatment are shown in Figure 5A. Single‐dose PK parameters for celecoxib are summarized and compared in Table 7. After dose adjustment, the 90% CI of the LS means ratio for celecoxib AUCτ were within the 80–125% range for CTC vs. the open combination of tramadol and celecoxib (Table 7). The 90% CIs for all other comparisons were outside this range. Median celecoxib T max after single‐dose CTC was 2.00 h, compared with 3.00 h for celecoxib alone and 4.00 h for tramadol plus celecoxib.

Figure 5

Mean plasma concentration–time profiles for celecoxib following single (A) and multiple (B) doses of CTC, celecoxib alone and the open combination tramadol and celecoxib. CTC, co‐crystal of tramadol–celecoxib

Table 7

Summary and statistical comparison of celecoxib pharmacokinetic parameters following single doses of CTC, celecoxib alone or the open combination of tramadol and celecoxib

Parameter	Treatment 1: 200 mg CTCa (n = 29)		Treatment‐3: 100 mg celecoxib (n = 28)		Treatment 4: 100 mg tramadol + 100 mg celecoxib (n = 28)		Ratio of geometric LS means (90% CI)
	Mean	CV (%)	Mean	CV (%)	Mean	CV (%)	Treatment 1 vs. treatment 3	Treatment 1 vs. treatment 4
C max (ng ml −1 )	246.52b	38.8b	358.23	36.6	202.26	36.9	69.45 (59.52–81.03)	123.20 (101.83–149.06)
AUC τ (ng h ml −1 )	1287.36b	32.9b	1928.95	34.8	1255.78	30.0	67.26 (61.88–73.11)	102.68 (90.99–115.87)
C t (ng ml −1 )	42.79b	39.7b	71.97	37.5	83.03	60.7	58.58 (53.63–63.98)	54.17 (46.49–63.11)
T max (h) c	2.00	51.9	3.00	45.3	4.00	72.5

AUCτ, area under the plasma concentration–time curve over the dosing interval after single dosing; CI, confidence interval; C max, maximum observed plasma concentration; C t, measured concentration at the end of the dosing interval; CTC, co‐crystal of tramadol–celecoxib; CV, coefficient of variation; LS, least squares; T max, time to reach maximum observed plasma concentration

Equivalent to 88 mg tramadol and 112 mg celecoxib

Parameters for Treatment‐1 were adjusted according to reference dose

Median values shown

PK findings for celecoxib after multiple dosing are shown in Figure 5B and Table 8. After dose adjustment, the 90% CIs of the LS means ratio for C max,ss and C τ,ss were outside the equivalence range for CTC compared with tramadol plus celecoxib, as were those of C max,ss, C avg and AUCτ,ss for CTC vs. celecoxib alone. Accumulation ratios for celecoxib were similar across treatments (Table 8).

Table 8

Summary and statistical comparison of celecoxib pharmacokinetic parameters following multiple doses of CTC, celecoxib alone or the open combination of tramadol and celecoxib

Parameter	Treatment 1: 200 mg CTCa (n = 29)		Treatment 3: 100 mg celecoxib (n = 28)		Treatment 4: 100 mg tramadol + 100 mg celecoxib (n = 28)		Ratio of geometric LS means (90% CI)
	Mean	CV (%)	Mean	CV (%)	Mean	CV (%)	Treatment 1 vs. treatment 3	Treatment 1 vs. treatment 4
C max,ss (ng ml −1 )	444.76b	27.4b	536.21	32.6	396.28	34.4	85.06 (78.89–91.72)	115.66 (105.43–126.88)
C τ,ss (ng ml −1 )	112.65b	41.0b	123.45	40.9	144.52	32.1	91.37 (83.68–99.78)	76.26 (71.65–81.17)
C avg (ng ml −1 )	233.59b	28.4b	280.48	26.5	241.44	31.0	83.22 (79.11–87.54)	97.83 (93.07–102.83)
AUC τ,ss (ng h ml −1 )	2803.12b	28.4b	3365.80	26.5	2897.29	31.0	83.22 (79.11–87.54)	97.83 (93.07–102.83)
T max,ss (h) c	2.00	57.4	2.00	35.4	3.00	45.1
AUC 0–last (ng h ml −1 )	5544.34	34.5	5196.90	34.6	5564.37	34.3
AUC ∞ (ng h ml −1 )	5810.35	32.4	5342.87	33.5	5823.14	34.1
AUC 0–last/∞ (%)	94.93	6.1	96.96	2.6	95.57	5.2
Fluctuation (%) d	146.07	25.6	148.07	28.0	103.28	22.7
K el (h −1 )	0.06	28.5	0.07	21.4	0.06	23.2
T ½el (h)	12.55	30.0	9.99	29.9	11.93	30.4
C pd − 24 (ng ml −1 )	173.35	33.3	165.95	41.6	197.57	40.6
C pd − 12 (ng ml −1 )	139.99	39.9	133.8	35.6	172.99	45.9
C pd 0 (ng ml −1 )	180.81	39.3	181.68	39.6	216.32	39.1
C pd 12 (ng ml −1 )	126.17	41.0	123.45	40.9	144.52	32.1
RA (Cτ)	2.71	27.4	1.74	24.8	1.99	32.4
RA (AUC)	2.24	22.6	1.82	23.1	2.45	35.3

AUC∞, area under the plasma concentration–time curve extrapolated to infinity; AUCo–last, area under the plasma concentration–time curve calculated from 0 to last observed quantifiable plasma concentration; AUC0–last/∞, relative percentage of AUC0–last with respect to AUC∞; AUCτ,ss, area under the plasma concentration–time curve over the dosing interval at steady state; C avg, average plasma concentration during dosing interval; CI, confidence interval; C max,ss, maximum observed plasma concentration at steady state; C pd, trough concentration or predose concentration measured at a specified time following a repeated dose regimen; CTC, co‐crystal of tramadol–celecoxib; C τ,ss, measured concentration at the end of the dosing interval at steady state; CV, coefficient of variation; K el, apparent elimination rate constant; LS, least squares; RA(Cτ), accumulation ratio C τ,ss/Cτ; RA(AUC), accumulation ratio AUCτ,ss/AUCτ; T ½el, terminal elimination half‐life; T max,ss, time to reach maximum observed plasma concentration at steady state

Equivalent to 88 mg tramadol and 112 mg celecoxib

Parameters for treatment 1 were adjusted according to reference dose

Median values shown

Calculated from ([C max,ss–Cτ,ss]/C avg)*100

Safety

The number of subjects who reported ≥1 AE after administration of CTC, tramadol alone, celecoxib alone and the open combination of tramadol and celecoxib was 29 (45%), 26 (87%), 11 (39%) and 20 (71%), respectively. At least one treatment‐related AE was reported by 12 (41%), 25 (83%), 10 (36%) and 19 (68%) subjects. Most AEs (78%) were mild in severity. AEs experienced by two or more subjects are shown in Table 9. By system organ class, gastrointestinal and nervous system disorder events were the most frequently reported AEs. Constipation was the most common individual AE, occurring in seven subjects with CTC and 10, two and eight subjects with tramadol alone, celecoxib alone, and tramadol plus celecoxib, respectively. Other AEs included somnolence, headache, dizziness and, less commonly, nausea and vomiting, hiccups and insomnia. No serious AEs or deaths occurred during the study.

Table 9

Adverse events reported in at least two subjects

System organ class	Adverse event	Treatment 1: 200 mg CTCa (n = 29)	Treatment 2: 100 mg tramadol (n = 30)	Treatment 3: 100 mg celecoxib (n = 28)	Treatment 4: 100 mg tramadol + 100 mg celecoxib (n = 28)
Gastrointestinal disorders	Constipation	7 / 8	10 / 10	2 / 2	8 / 8
	Severe constipation	0 / 0	2 / 2	0 / 0	4 / 4
	Dry lips	1 / 1	1 / 1	0 / 0	0 / 0
	Nausea	1 / 1	5 / 5	1 / 1	5 / 6
	Vomiting	0 / 0	2 / 2	0 / 0	2 / 2
General disorders and administration site conditions	Fatigue	0 / 0	1 / 1	1 / 1	1 / 1
	Feeling hot	0 / 0	1 / 1	0 / 0	1 / 1
Injury, poisoning and procedural complications	Vessel puncture site pain	1 / 1	1 / 1	0 / 0	1 / 1
Nervous system disorders	Dizziness	0 / 0	6 / 7	0 / 0	4 / 5
	Headache	0 / 0	7 / 7	0 / 0	3 / 3
	Somnolence	5 / 7	7 / 12	6 / 6	8 / 11
Psychiatric disorders	Insomnia	1 / 1	1 / 1	0 / 0	2 / 2
Respiratory thoracic and mediastinal disorders	Hiccups	0 / 0	0 / 0	0 / 0	3 / 3

Data shown are number of subjects/number of events. CTC, co‐crystal of tramadol–celecoxib

Equivalent to 88 mg tramadol and 112 mg celecoxib

There were no notable findings from other safety assessments. All abnormal laboratory values, with one exception, were not considered to be clinically significant. One subject showed an abnormal alkaline phosphatase result at a poststudy visit that was considered clinically significant and reported as a mild AE. The last treatment administered to this subject was the open combination of celecoxib and tramadol.

Discussion

As pain is multifactorial, it is generally accepted that multimodal analgesia is optimal, targeting different physiological mechanisms 29. This has led to an interest in the development of co‐crystals as an innovative approach for multidrug delivery 22. The present phase I study aimed to compare single‐ (after first dose) and multiple‐dose (after last dose) PK profiles of CTC, a co‐crystal of tramadol and celecoxib, with those of each reference product (IR tramadol and celecoxib) alone and in open combination. The study demonstrated that tramadol, its M1 metabolite and celecoxib accumulate within plasma after multiple doses of CTC to an extent similar to that observed after multiple doses of IR tramadol and celecoxib alone or in an open combination. Although it was not proven statistically that steady state was reached for tramadol after multiple doses of CTC, it can be assumed that this finding was not clinically relevant due to the high percentage of steady state reached for tramadol (based on C pd values), the known elimination half‐life of tramadol, the duration of treatment and the fact that the M1 metabolite of tramadol achieved steady state (as did celecoxib).

Following multiple doses, and after dose adjustment, a co‐crystal effect on both tramadol and celecoxib was observed. Tramadol C max,ss for CTC was lower compared with tramadol alone or in open combination with celecoxib, yet remained above levels required for efficacy 30. Analysis of C τ,ss, C avg and AUCτ,ss for tramadol from CTC compared with the other tramadol‐containing treatments suggested that similar levels of exposure between treatments were achieved. The reduction in tramadol C max,ss observed with CTC is consistent with a lower intrinsic dissolution rate (as observed in vitro) and, consequently, a slower absorption of tramadol. Indeed, T max,ss, was slightly prolonged for tramadol from CTC compared with other treatments. Similar observations were made for the M1 metabolite. Tramadol PK parameters following multiple doses of 100 mg of tramadol alone were comparable with those observed when given in open combination with celecoxib. Tramadol C max and T max values were also similar to those reported in the literature 25, 31.

After multiple dosing, celecoxib from CTC showed a reduced AUC. Lower C max,ss and similar T max,ss compared with celecoxib alone was obtained, indicating a lower rate and extent of exposure of celecoxib after co‐crystal administration. However, when celecoxib from CTC was compared with the co‐administration of celecoxib and tramadol, absorption was faster and C max,ss was greater, albeit without a concomitant increase in exposure. This observed reduction in the C max of celecoxib when it was administered as a free combination with tramadol may be due to the effects of tramadol on gastrointestinal motility (slowing down), and this is minimized with CTC. Therefore, this suggests that co‐crystallization of the two APIs improves the PK profile of celecoxib and avoids the apparent effects on dissolution and absorption that occur when the two drugs are co‐administered. This is also consistent with changes in the intrinsic dissolution rate of celecoxib from CTC. As expected, intrasubject variabilities in measured PK parameters (quantified as the coefficient of variation) were greater for celecoxib than for tramadol. It is unlikely, however, that this greater variability reduces the certainty of conclusions drawn around the celecoxib data in the present study. Of note, all multiple‐dose PK findings, including those for celecoxib, were consistent with the single‐dose data collected in the present study and in a separate phase I study of CTC 24.

The potential clinical implications of the PK profile for CTC observed in the present study remain to be determined. However, in theory, a reduced tramadol C max may translate into improved safety and tolerability. CTC was well tolerated in the present study, and the AEs observed were consistent with the safety profile of tramadol 30. In aggregate, based on a descriptive analysis, a reduction in opioid‐related AEs (e.g. dizziness, nausea, vomiting and severe constipation) was observed with CTC compared with other tramadol‐containing treatments. This effect could potentially be attributed to the lower tramadol C max, as there is a dose–response effect on the incidence of AEs for tramadol 32. Also, the faster celecoxib T max with CTC could translate into an earlier onset of analgesia and warrants further investigation. Our results demonstrate that simple co‐administration of authorized tramadol and celecoxib does not replicate the PK profile of the co‐crystal, and therefore its clinical effects are also likely to be different.

There were some limitations to the study. To have used noncompartmental analysis could limit the ability to determine C max and T max accurately. Another limitation was the requirement to perform dose adjustments prior to statistical comparison of treatments. This was due to the fact that the doses of APIs in CTC (88 mg tramadol and 112 mg celecoxib) differed from those in the commercially available formulations of tramadol and celecoxib. In addition, as treatments were given under fasting conditions, the effects of food on PK parameters were not evaluated. Another limitation arises from the fact that we do not know the role that the formulation of CTC played in the results obtained. In fact, we have no data based on comparative PK clinical trials in healthy volunteers between formulated CTC vs. unformulated CTC. However, based on our dissolution profile studies (internal data), unformulated CTC provides a unique dissolution profile, clearly different from the open combination, where CTC dissolves twice as fast as the open combination. This dissolution profile suggests that the outcomes obtained in the present study were due to CTC per se.

In conclusion, the study demonstrated that after single (first dose) and multiple dosing, the PK parameters of each API in CTC were modified by co‐crystallization compared with reference products alone or in open combination. The potential implications of this unique profile should become clearer as clinical development progresses. A phase II trial comparing CTC with tramadol in patients with moderate to severe acute pain after oral surgery has been completed 33, and several phase III trials are ongoing.

Competing Interests

S.V., A.V., M.S., M.E., A.S., N.G., G.E. and C.P. are employees of Laboratorios del Dr Esteve, S.A.U. L.S. was an employee of Laboratorios del Dr Esteve, S.A.U. when the study was performed. M.L. and E.S. are employees of the clinical research organization Algorithme Pharma.

The authors acknowledge the following employees from Laboratorios del Dr. Esteve for their contribution to this study: Carmen Santano, Noemí Álvarez, Imma Cervera (Pharmaceutical Innovation); Esther Ortiz, Alberto Puyada (Clinical Investigation); Carmen Garcia (R&D Portfolio Management); Mercedes Encabo, Santiago Puig, Josep Maria Cendrós (ADME and Bioanalysis); and Fina Camacho (Quality Assurance). The study was funded by Laboratorios del Dr. Esteve, S.A.U. (Barcelona, Spain) and carried out by Algorithme Pharma, a clinical research organization (Laval, QC, Canada). Medical writing support was provided by Alice Wareham PhD (Aspire Scientific Ltd, Bollington, UK) and was funded by Mundipharma Research GmbH & Co. KG (Limburg, Germany) . Monitoring support was provided by Vahe Bartekian and Vatché Bartekian (Vantage Biotrials, Montreal, QC, Canada).

Contributors

S.V., M.L., A.V., M.S., L.S., N.G., G.E. and C.P. were involved in the conception and design of the study and the analysis and interpretation of data. A.S., M.E. and E.S. were involved in the acquisition of data. All authors revised the article critically for important intellectual content and gave final approval of the version to be published.

Appendix S1 Selection of study population: full inclusion and exclusion criteria

Click here for additional data file.

TARGETS
G protein‐coupled receptors 2	Enzymes 3
μ receptor	Cyclooxygenase
5‐HT receptor

These Tables list key protein targets and ligands in this article that are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY 1, and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 2, 3.