Abstract
Aims
This study explores the impact of paediatric patient related factors and choice of formulation on the dissolution characteristics of nifedipine and lorazepam, 2 drug substances regularly applied in very young patients and in compounded formulations.
Methods
Dissolution experiments were designed to reflect clinical practice in a paediatric hospital, with respect to dosage forms, feeding regimens and methods of administration. Solubility studies addressed the influence of age and prandial state. Drug solubility and dissolution experiments were conducted in biorelevant media and adapted age‐specific (neonate and infant) media. Dissolution studies were performed with the mini‐paddle apparatus and the flow‐through cell apparatus.
Results
Dissolution of nifedipine formulations was not affected by age‐related changes of the fasted state simulated gastrointestinal fluids, and by disintegration of the formulation before administration. However, a significant difference in nifedipine's dissolution rate from commercial tablets and compounded capsules was observed. The dissolution of lorazepam tablets was affected by fasted‐ vs fed‐state media, but it was deemed less likely to be clinically relevant. The significant effect of fed‐state media on nifedipine's solubility was considered to have possible clinical relevance since very young patients are almost continuously in a fed state.
Conclusion
The in vitro results obtained from these studies reveal the potential of biorelevant solubility and dissolution studies reflecting clinical practice to predict drug performance in paediatric patients.
Keywords: biorelevant, dissolution, lorazepam, nifedipine, paediatric, solubility
1. What is already known about this subject
Using biorelevant dissolution, successful in vitro–in vivo relations/correlations have been established for oral immediate and modified release formulations in adults.
Evaluation of oral drug absorption is often only performed for fasted state conditions, which are not applicable to new‐borns and infants.
What this study adds
In vitro dissolution tests were conducted for 2 nifedipine formulations under settings considered to reflect clinical conditions. Both nifedipine conventional commercial tablets and compounded capsules displayed slow dissolution in fasted state conditions.
Our biorelevant solubility results suggest that there is a food effect on the solubility of nifedipine in gastrointestinal fluids in neonates and infants, which might be translated into an effect on in vivo drug absorption after administration of conventional formulations.
1. INTRODUCTION
During recent decades, the purpose of dissolution testing of drugs has expanded from pure quality control to prediction of in vivo drug performance and identifying potential bioavailability problems of pharmaceutical formulations. With development of biorelevant dissolution media (reflecting the main properties of gastrointestinal [GI] fluids), simulation of GI residence times, and simulation of GI hydrodynamics, successful in vitro–in vivo relations and correlations (IVIVRs/IVIVCs) have been established for oral immediate and modified release formulations in adults, using compendial dissolution apparatus.1, 2 Furthermore, in vitro dissolution data are used as input in physiologically based pharmacokinetic (PBPK) prediction models in which different factors influencing drug absorption are integrated. Both biorelevant dissolution and PBPK modelling are regularly applied in drug development by the pharmaceutical industry, i.e. to select and optimize formulations, or to predict solubilization and precipitation in the human GI tract under various conditions.3 Ideally, in vitro predictive methods, combined with in silico models, will replace in vivo experiments and clinical trials.
In recent years, advances towards the availability of suitable paediatric biorelevant dissolution tests have been made with the development of paediatric media.4 These media were based on the available literature on the composition of paediatric luminal fluids and the established adult media.5 Experimental dissolution parameters that mimic paediatric conditions have been proposed.6, 7 The search for alternatives to clinical trials is essential, especially in paediatrics, given the ethical and methodological difficulties involved in performing trials in paediatric patients.8 Predictive biopharmaceutical methods representing the in vivo drug dissolution in children would be of huge benefit for early formulation screening and assessing the influence of different administration strategies on drug performance, with the ultimate goal to reduce the amount of in vivo studies required and accelerating paediatric drug development.
Apart from the obvious differences between children and adults regarding the physiological development of the GI tract, the effect of pharmacotherapy in paediatric patients can be further influenced by a range of patient‐related factors, such as feeding regimens, the presence of feeding tubes, immobility, and the selected drug formulation. Pharmacists often have to rely on extemporaneous compounding and unlicensed manufacturing, or manipulation of adult dosage forms, in case licensed, age‐appropriate paediatric drug products are not available.
This study aims to explore the application of biopharmaceutical methods to study the impact of patient related factors on drug performance in paediatric patients, using 2 biopharmaceutical tools: drug substance solubility and drug product dissolution in biorelevant media adapted to reflect paediatric conditions. Dissolution experiments were designed to simulate the impact of formulation handling and dosage form manipulation. Dissolution parameters were set based on what is currently known about physiological conditions in the GI tract of children. To assess the age‐ and prandial state related changes in paediatric GI solubility, respective studies were performed in both adult biorelevant media and adapted paediatric media.
The compounds that were chosen to be studied were nifedipine and lorazepam, as they are both regularly applied in unlicensed formulations and in very young patients. Nifedipine is a biopharmaceutical classification system (BCS) class II drug substance, with an aqueous solubility of around 5–9 μg/mL9 and a logP value of 2.20.10 With pKa values of −0.9 and 13, it is not ionizable in the GI pH range. Therefore, under physiologically relevant conditions, nifedipine acts as a neutral molecule and its solubility is independent of the pH of the medium.11 Lorazepam is a compound with a slightly better aqueous solubility (80 μg/mL) compared to nifedipine, and a logP value of 2.39.12 With pKa values of 1.3 and 11.5,13 it can be partly ionized in the acidic conditions of the fasted stomach. Using the above stated solubility value, a dose number (D 0) ≤ 1 is calculated for dose strengths up to 20 mg, and thus lorazepam would be considered as a highly soluble, class I compound within the adult BCS.
2. METHODS
2.1. Materials
Pepsin from porcine gastric mucosa (powder, ≥400 units/mg protein), nifedipine drug substance (≥98% high‐performance liquid chromatography [HPLC] grade), lorazepam reference standard (≥98% HPLC grade) and Whatman GF/D (pore size 2.7 μm, 25‐mm diameter) and GF/F (pore size 0.7 μm, 25‐mm diameter) filters were purchased from Sigma–Aldrich (Dorset, UK). UHMW polyethylene 10‐μm full‐flow cannula filters were bought from Quality Lab Accessories LCC (Telford, USA). Egg‐lecithin (Lipoid E PCS) was purchased from Lipoid GmbH (Ludwigshafen, Germany). Sodium taurocholate (NaTc) was purchased from Prodotti Chimici e Alimentari S.p.A (Basaluzzo, AL, Italy). Cronus 13‐mm regenerated cellulose (RC) 0.45‐μm syringe filters were purchased from LabHut Ltd (Maisemore, UK). Aptamil 1 (Nutricia, Trowbridge, UK), SMA Wysoy Soya Infant Formula (SMA Nutrition, Gatwick, UK) and Ultra Heat Treated Standardised Whole Milk 3.6% fat (Sainsbury's, London, UK) were purchased from a local supermarket. Water was of Milli‐Q grade. All other reagents and chemicals were of analytical grade and were used as received, without further purification.
2.2. Instrumentation
Equipment utilized in the current study included a R114 Rotavapor (Buchi, Flawil, Switzerland), a SevenCompact pH/Ion S220 pH meter (Mettler‐Toledo AG, Schwerzenbach, Switzerland), Heraeus Fresco 17 and Heraeus Biofuge Primo R centrifuges (Thermo Scientific, Hanau, Germany), an Agilent Technologies 708‐DS (USP II) apparatus configured with Agilent TruAlign 200‐mL vessels and Agilent electropolished stainless steel mini‐paddles (Santa Clara, CA), a Sotax CE7 smart flow‐through cell (USP IV) apparatus connected to a CP 7 Piston Pump (Sotax, Switzerland). The Agilent 1100 HPLC system consisted of a G1311A Quaternary Pump, G1315A DAD detector, G1316A Column Compartment, G1322A Degasser, G1329A Autosampler, G1330A Autosampler Thermostat and ChemStation software (Agilent Technologies, Santa Clara, USA).
2.3. Drug products
Conventional commercial nifedipine retard 10‐mg tablets (Centrafarm, Etten‐Leur, the Netherlands), unlicensed GMP‐grade nifedipine 1‐ and 5‐mg capsules (Apotheek A15, Gorinchem, the Netherlands), commercial lorazepam 1‐mg IR tablets (Mylan, Bunschoten, The Netherlands) and unlicensed GMP‐grade lorazepam oral solution 1 mg/mL (Apotheek A15, Gorinchem, the Netherlands) were used. Nifedipine capsules were compounded from pure API into hard gelatine capsules using lactose as single excipient. Lorazepam oral solution 1 mg/mL contains glycerol 85%v/v (87%v/v), polyethylene glycol (PEG) 400 (10%v/v) and propylene glycol (3%v/v).14 All formulations used in this study were part of the formulary of the Sophia Children's Hospital, Rotterdam, The Netherlands.
Although no dosing advice is available for neonates, in clinical practice nifedipine is sometimes administered to patients below the age of 1 month, in dosages from 0.1 up to 1.0 mg/kg.15 For experiments simulating neonatal or infant conditions, nifedipine unlicensed 1‐mg capsules were used to reflect clinical practice.
Oral lorazepam is used off‐label to gradually taper‐off benzodiazepines that have been administered as continuous intravenous sedation at the paediatric intensive care unit. It is usually administered 4 times daily, in single dosages up to 0.5 mg (neonates), 1 mg (infants) and 3 mg (children).16 To allow for precise dosing and ease of administration, a 1 mg/mL oral solution was developed specifically for paediatric patients.14
2.4. Media used for solubility and dissolution studies
Simulated gastric fluid without pepsin (SGFsp) pH 1.2 and simulated intestinal fluid without pancreatin (SIFsp) pH 6.8 were used in dissolution studies.17 Freshly prepared adult and age‐specific (neonate and infant) biorelevant media were used in solubility and dissolution studies (Table 1).4, 5, 17, 18
Table 1.
| Fasted‐state simulated gastric fluid | Fed‐state simulated gastric fluid | ||||||
|---|---|---|---|---|---|---|---|
| Adult (FaSSGF) | Neonate (Pn‐FaSSGF) | Infant (pi‐FaSSGF) | Adult (FeSSGF) | Neonate—cow formula (Pnc‐FeSSGF) | Neonate—soy formula (Pns‐FeSSGF) | ||
| Sodium chloride (mM) | 34.2 | 34.2 | 34.2 | 237.02 | 100.35 | 94.79 | |
| Sodium taurocholate (μM) | 80 | 20 | 60 | ||||
| Lecithin (μM) | 20 | 5 | 15 | ||||
| Pepsin (mg/mL) | 0.1 | 0.015 | 0.025 | ||||
| Acetic acid (mM) | 17.12 | 7.25 | 7.25 | ||||
| Sodium acetate (mM) | 29.75 | 64.65 | 64.65 | ||||
| Milk:buffer | 1:1 | 1:1 | 1:1 | ||||
| HCl/NaOH qs | pH 1.6 | pH 1.6 | pH 1.6 | pH 5 | pH 5.7 | pH 5.7 | |
| pH | 1.6 | 1.6 | 1.6 | 5 | 5.7 | 5.7 | |
| Osmolarity (mOsm/kg) | 120.7 ± 2.5 | 120.7 ± 2.5 | 120.7 ± 2.5 | 400 | 340 | 240 | |
| Buffering capacity (mmol/L/ ∆pH) | ‐ | ‐ | ‐ | 25 | 15 | 15 | |
| Fasted‐state simulated intestinal fluid | Fed‐state simulated intestinal fluid | ||||||
|---|---|---|---|---|---|---|---|
| Adult (FaSSIF‐V2) | FaSSIF‐50% | FaSSIF‐150% | Adult (FeSSIF‐V2) | Neonate—breast fed (Pnb‐FeSSIF) | Neonate—cow formula (Pnc‐FeSSIF) | Infant—cow formula (pi‐Fessif) | |
| Sodium hydroxide (mM) | 34.8 | 34.8 | 34.8 | 81.65 | 81.65 | 81.65 | 81.65 |
| Sodium taurocholate (mM) | 3 | 1.5 | 4.5 | 10 | 2.5 | 2.5 | 7.5 |
| Lecithin (mM) | 0.2 | 0.1 | 0.3 | 2 | 0.5 | 0.5 | 1.5 |
| Sodium chloride (mM) | 68.62 | 68.62 | 68.62 | 125.5 | 95 | 111.73 | 107.35 |
| Maleic acid (mM) | 19.12 | 19.12 | 19.12 | 55.02 | 55.02 | 55.02 | 55.02 |
| Glyceryl monooleate (mM) | 5 | 5 | 6.65 | 5 | |||
| Sodium monooleate (mM) | 0.8 | 0.8 | 1.06 | 0.8 | |||
| HCl/NaOH qs | pH 6.5 | pH 6.5 | pH 6.5 | pH 5.8 | pH 5.8 | pH 5.8 | pH 5.8 |
| Osmolarity (mOsm/kg) | 180 ± 10 | 180 ± 10 | 180 ± 10 | 300 ± 10 | 330 ± 10 | 330 ± 10 | 390 ± 10 |
| Buffering capacity | 10 | 10 | 10 | 25 | 25 | 25 | 25 |
| (mmol/L/ ∆pH) | |||||||
FaSSGF = Fasted‐state Simulated Gastric Fluid; FeSSGF = Fed‐state Simulated Gastric Fluid; FaSSIF = Fasted‐state Simulated Intestinal FLuid; FeSSIF= Fed‐state Simulated Intestinal Fluid; P = paediatric; i = infant; n = neonate; c = cow milk formula; s = soy milk formula; b = breast fed.
2.5. In vitro dissolution studies
2.5.1. Experimental set‐up
Dissolution experiments were performed with the mini‐paddle apparatus and the flow‐through cell apparatus. The mini‐paddle apparatus is particularly suitable for working with reduced fluid volumes, to better mimic intraluminal fluid volumes in the GI tract of paediatric patients.19 The flow‐through cell apparatus (USP IV apparatus) offers the advantage of easily changing the medium and flow rate during an experiment, and maintaining sink conditions when operated in the open mode.20
The mini‐paddle apparatus was equipped with 200‐mL vessels and matching paddles, using a smaller volume compared to adult biorelevant studies.1 As intestinal motor activity matures throughout early infancy,21 the agitation rate of the paddle was set at 50 rotations/min. Samples (2 mL) were removed (with sample replacement) using a 5‐mL Fortuna Optima syringe fitted with stainless tubing and a cannula filter to facilitate representative sampling.
The flow‐through cell apparatus was equipped with large cells (22.6‐mm diameter), with a 5‐mm ruby bead at the bottom of the cell and small glass beads (1‐mm diameter) filling the cone of the cell. Test formulations were placed on a tablet holder. Two filters were placed on top of each cell: a GF/D and a GF/F filter (Glass Microfibre Filters 24 mm, Whatman). The open mode was used in all experiments. Samples were collected in glass cylinders or Erlenmeyer flasks, which were weighed to determine the volume of the sample.
All experiments, both in the mini‐paddle and the flow‐through cell apparatus, were conducted at 37°C. Sample collection for nifedipine took place at 5, 15, 30, 40, 50, 60, 75, 90 and then every 30 minutes up to 270 minutes, after the start of the experiment. Sample collection for lorazepam took place at 5, 15, 30, 45, 60, 75, 90 and 120 minutes after the start of the experiment. Before HPLC analysis, samples were filtered through a 0.45‐μm RC filter, discarding the first 10 drops (adsorption of the drugs onto the filters was checked and confirmed to be negligible). Calibration curves were prepared in corresponding media for each experiment on the day of the experiment. All experiments were performed in triplicate and in the case of nifedipine under protection from light.
2.5.2. Screening the impact of patient related variables
To reflect clinical practice in a paediatric hospital with respect to dosage forms, feeding regimens, and methods of administration, information from standard operating procedures of the Sophia Children's Hospital was retrieved. A clinically relevant design of the in vitro experiments was followed by considering: (i) the enteral feeding protocol; (ii) the protocol for administration of medicines through a feeding tube; and (iii) the local drug formulary of the Sophia Children's Hospital. The patient relevant parameters studied in the dissolution experiments were age, prandial state, method of administration, and formulation type.
Even though oral administration is the preferred route to feed paediatric patients, enteral feeding through a nasogastric tube is often indicated, due to an inability or unwillingness of eating or swallowing, anorexia, motility problems etc. This mode of administration has implications for the gastric‐emptying rate, which increases with enteral feeding compared to oral feeding.22 When possible, breast milk is the preferred type of food for children for a minimum duration of 4–6 months from the day of birth. Otherwise, patients are fed with formula milk, adjusted to their energy and protein requirements and potential fluid restriction. Table 2 displays the standard neonate and infant formulas administered to enterally fed patients. For normal birth‐weight neonates and infants, the feeding interval is gradually reduced from 8 times a day at birth to 4 times a day age 8 months. In certain conditions, such as gastroparesis, hyperemesis or recurrent aspiration, gastric feeding is not suitable and transpyloric feeding directly into the duodenum is required. Because the duodenum has no reservoir capacity like the stomach, transpyloric feeding is always administered as a continuous drip.
Table 2.
Neonate and infant nutrition
| PICU non ventilated | PICU ventilated | ||||||
|---|---|---|---|---|---|---|---|
| Weight (kg) | Age (months) | Standard | Caloric content per 100 mL | Standard + energy enriched | Caloric content per 100 mL | Standard + energy and protein enriched | Caloric content per 100 mL |
| 2–3.5 | 0–1 | Nenatal start | 78 kcal 2.5 g protein | Nenatal start 18% w/v | 87 kcal 2.7 g protein | Nenatal start 18% w/v + 0.5% NPF | 89 kcal 3.2 g eiwit |
| 3.5–8 | 0–6 | Nutrilon 1 | 66 kcal 1.3 g protein | Nutrilon 1 17% w/v | 82 kcal 1.6 g protein | Infatrini | 100 kcal 2.6 g protein |
| 8–9.5 | 7–9 | Nutrilon 2 | 68 kcal 1.4 g protein | Nutrilon 2 17% w/v | 79 kcal 1.6 g protein | Infatrini | 100 kcal 2.6 g protein |
| 9.5–10.5 | 10–12 | Nutrilon 3 |
70 kcal 1.5 g protein |
Nutrilon 3 17% w/v | 79 kcal 1.7 g protein | Infatrini | 100 kcal 2.6 g protein |
Nutrilon = Aptamil first milk; NPF = Nutrilon Nenatal Protein Fortifier; PICU = paediatric intensive care unit.
All products are manufactured by Danone (Paris, France).
Administration of solid dosage forms to paediatric patients is often not possible, which means that the formulation has to be manipulated before administration. In the Sophia Children's Hospital, as per protocol, immediate release capsules and tablets are dispersed in an oral syringe with a small amount of lukewarm water (1–20 mL).
2.5.3. Nifedipine solubility studies
To study the influence of age‐related changes in GI fluid composition on compound solubility, nifedipine solubility studies were performed according to methods described by Maharaj et al.4 In summary, for aqueous based media, an excess amount of nifedipine was added to 2 mL of medium, dwelled for 24 hours at a shaking water bath at 37°C, filtered through an 0.45‐μm RC filter and diluted with fresh medium prior to HPLC analysis. For the milk‐based media, a drug extraction step was required, which consisted of a centrifugation step, precipitation of proteins with methanol, a second centrifugation and filtration of the resulting supernatant through an 0.45‐μm RC filter. All solubility experiments were conducted in triplicate.
2.5.4. Nifedipine dissolution studies
An overview of the dissolution experiments is given in Table 3. Firstly, pH changes in the fasted state, resulting from passage through the stomach and small intestine, were simulated in the mini‐paddle apparatus using SGFsp and SIFsp (see section 2.4). The pH shift from the gastric to the intestinal conditions was achieved by the addition of an equal volume of double concentrated SIFsp (with additional NaOH), after a simulated gastric residence time of 45 minutes. Secondly, to compare the dissolution of the 2 nifedipine formulations that are regularly used in the Sophia Children's Hospital, commercial nifedipine 10‐mg tablets and compounded 5‐mg capsules, an experiment was performed in the flow‐through cell apparatus. To simulate administration to fasted children, adult biorelevant media were used (FaSSGF/FaSSIF), the gastric residence was set at 30 minutes and the flow rate was reduced from 5 mL/min to 4 mL/min after the medium switch in order to reflect the in vivo gastric and intestinal conditions (in terms of residence time and volume). The dose of the 10‐mg tablets was matched by using 2 × 5‐mg capsules per cell. Thirdly, a dissolution experiment in fasted state neonatal media (Pn‐FaSSGF/FaSSIF) was performed with the flow‐through cell apparatus, in order to reveal the effect of different hydrodynamics compared to the mini‐paddle apparatus.
Table 3.
Parameters used for the dissolution experiments in the mini‐paddle and the flow‐through‐cell apparatus (USP IV apparatus)
| Gastric conditions | Intestinal conditions | |||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Apparatus | API | Formulation | Agitation (rpm) | Medium | pH | Volume (mL) | Time (min) | Medium | pH | Volume (mL) | Time (min) | Total volume (mL) |
| Mini‐paddle | Nifedipine | Capsule 5 mg | 50 | SGFsp | 1.2 | 100 | 45 | SIFsp | 6.8 | 100 | 225 | 200 |
| Capsule 1 mg | 50 | Pn‐FaSSGF | 1.6 | 50 | 45 | FaSSIF | 6.5 | 150 | 225 | 200 | ||
| Capsule 1 mg | 50 | Pi‐FaSSGF | 1.6 | 75 | 30 | FaSSIF | 6.5 | 125 | 240 | 200 | ||
| Capsule 1 mg, dissolved in syringe | 50 | Pn‐FaSSGF | 1.6 | 50 | 45 | FaSSIF | 6.5 | 150 | 225 | 200 | ||
| Lorazepam | Oral solution 1 mL (1 mg/mL) | 75 | SGFsp | 1.2 | 100 | 45 | SIFsp | 6.8 | 100 | 75 | 200 | |
| Tablet 1 mg | 75 | SGFsp | 1.2 | 100 | 45 | SIFsp | 6.8 | 100 | 75 | 200 | ||
| Medium | pH | Flow (mL/min) | Time (min) | Medium | pH | Flow (mL/min) | Time (min) | Total volume (mL) | ||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Flow‐through cell | Nifedipine | Capsule 1 mg | Pi‐FaSSGF | 1.6 | 4 | 30 | FaSSIF | 6.5 | 3 | 240 | 840 | |
| Capsule 2 × 5 mg | FaSSGF | 1.6 | 5 | 30 | FaSSIF | 6.5 | 4 | 240 | 1110 | |||
| Tablets retard 10 mg | FaSSGF | 1.6 | 5 | 30 | FaSSIF | 6.5 | 4 | 240 | 1110 | |||
| Lorazepam | Tablet 1 mg | Pi‐FaSSGF | 1.6 | 4 | 30 | FaSSIF | 6.4 | 3 | 90 | 390 | ||
| Tablet 1 mg | Pnc‐FeSSGF | 5.7 | 5 | 60 | Pi‐FeSSIF | 5.8 | 5 | 60 | 600 | |||
| Tablet 1 mg | Pi‐FeSSIF | 5.8 | 5 | 120 | 600 |
API = active pharmaceutical ingredient; rpm = rotations/min.
The effects of age‐related differences in GI conditions on dissolution were simulated with the use of neonatal (Pn‐FaSSGF/FaSSIF) and infant (Pi‐FaSSGF/FaSSIF) fasting media in the mini‐paddle apparatus (see Table 3). To reflect the in vivo conditions in neonates, the gastric residence time was prolonged, and the gastric volume was decreased compared to infants.8 Thirdly, administration through a gastric feeding tube, where the capsule is dispersed in an oral syringe with 5 mL of warm water before administration, was also simulated in fasted state neonatal media (Pn‐FaSSGF/FaSSIF).
2.5.5. Lorazepam dissolution studies
Details of the design of the dissolution studies are presented in Table 3. Dissolution of the 1 mg tablets was studied in SGFsp and SIFsp and the mini‐paddle apparatus, with an increased rotational speed of 75 rotations/min to prevent coning of the tablet formulation. This experiment was repeated using the lorazepam 1‐mg/mL oral solution to compare the profiles.
Subsequently, 2 experiments were conducted with the flow‐through cell apparatus. The effect of prandial state on dissolution was explored using lorazepam 1‐mg tablets and infant fasted state and fed state simulated gastric and intestinal fluids. Pnc‐FeSSGF was considered appropriate to simulate infant fed‐state gastric fluid, as it contains milk formula that is given to infants up to the age of 6 months. Gastric residence time in the fed state was prolonged compared to the fasted state and the flow rates were set to reflect the prandial state and GI tract compartment. Administration directly into the duodenum was simulated using lorazepam 1‐mg tablets and infant fed state intestinal fluid (Pi‐FeSSIF).
2.6. Analytical quantification
For the quantitative analysis of nifedipine, HPLC combined with UV detection was used. The method was adapted from the method previously reported by Vertzoni et al.23, 24 Nifedipine was separated on an analytical C18 column (Thermo Hypersil GOLD, 5 μm, 250 × 4.6 mm) with UV detection at 238 nm, a column temperature of 30°C, mobile phase of a 60:40 mixture (v/v) of methanol and water (Milli‐Q), a flow rate of 1.0 mL/min, and injection volume of 50 μL. For the quantitative analysis of lorazepam, the HPLC‐UV method as reported by Share et al. was used.25 Lorazepam was separated using a Zorbax SB‐C18 analytical column (3.5 μm, 150 × 4.6 mm) with UV detection at 230 nm, a column temperature of 30°C, mobile phase of a 60:40 mixture (v/v) of methanol and water (Milli‐Q), a flow rate of 0.75 mL/min, and injection volume of 20 μl. Quantification of nifedipine and lorazepam was made based on calibration curves constructed from stock solutions in the corresponding medium (range 0.5–12 μg/mL). For milk‐ and formula‐based media, calibration curves were created in triplicate, and the same protein precipitation, centrifugation and filtration process was applied as described in section 2.5.
2.7. Statistical analysis
One‐way analysis of variance (ANOVA) with a posthoc Tukey's test was applied to identify statistically significant differences in solubility between adult and age‐specific media, using a significance level of p ≤ 0.05. Statistical analysis was performed in GraphPad Prism version 5.01 for Windows (GraphPad Software, San Diego, CA, USA).
3. RESULTS AND DISCUSSION
3.1. Paediatric GI solubility of nifedipine
Nifedipine is a substance with poor aqueous solubility (around 5–9 μg/mL).9 As shown in Figure 1, nifedipine solubility in adult FaSSGF (3.81 μg/mL) was different to solubility in Pi‐FaSSGF (7.04 μg/mL), but not to Pn‐FaSSGF (4.99 μg/mL). Although the relative difference between the solubility values for adult FaSSGF and Pi‐FaSSGF was large, all values were very similar to the range reported for aqueous solubility. This implies that the small amounts of bile salts and pepsin present in FaSSGF have a negligible effect on nifedipine solubility, and that age‐related changes in fasted state gastric fluid may be unlikely to significantly influence the absorption of nifedipine.
Figure 1.
Nifedipine 24‐hour solubility (mean ± standard deviation, n = 3) in adult and paediatric biorelevant gastrointestinal media. Statistically significant solubility differences compared to the adult media are denoted with * (P ≤ .05) or *** (P ≤ .001). FaSSGF = Fasted‐state Simulated Gastric Fluid, FeSSGF = Fed‐state Simulated Gastric Fluid, FaSSIF = Fasted‐state Simulated Intestinal Fluid, FeSSIF= Fed‐state Simulated Intestinal Fluid, P = paediatric, i = infant, n = neonate, c = cow milk formula, s = soy milk formula, b = breast fed
Compared to FaSSGF, solubility in FaSSIF was increased. Paediatric investigations examining luminal fluids within the fasted‐state proximal intestine are thus far limited, therefore the different FaSSIF media were developed to explore the impact of variations in bile salt concentrations.4 The nifedipine solubility values that were found in FaSSIF‐V2 (12.9 μg/mL), FaSSIF‐50% (9.3 μg/mL) and FaSSIF‐150% (15.1 μg/mL) reflected these variations.
In FeSSGF, nifedipine solubility was markedly increased compared to FaSSGF, indicating a substantial effect of prandial state on nifedipine solubilization in the gastric fluid. Additional changes in solubilization can be expected for energy and/or protein enriched nutrition.26
The biggest relative differences in solubility between adult and paediatric media were observed in FeSSIF. Nifedipine solubility in adult FeSSIF‐V2 (45.2 μg/mL) was higher than Pi‐FeSSIF (32.1 μg/mL), Pnc‐FeSSIF (18.3 μg/mL) and Pnb‐FeSSIF (18.5 μg/mL), reflecting the solubilizing effects of lipids and bile salts.
It should be noted that the solubility of a drug substance in a certain medium is a compound specific property. Nevertheless, the increased solubility in fed‐state media could have some implications for clinical practice. Both nifedipine formulations do not have any attributes that slow or alter the release of the drug substance. The slow dissolution rate of the drug substance itself is considered to provide the slow onset of action of the drug. In paediatric hypertension, nifedipine is administered to patients from the age of 1 month, who are effectively in an almost continuous fed prandial state. This means that, in these patients, a much more rapid dissolution from the nifedipine conventional tablets and capsules could be expected, possibly leading to a shortened time to maximum plasma concentration, an increased maximum plasma concentration and an altered drug exposure, compared to administration to fasted state patients. An increased maximum plasma concentration may lead to typical dihydropyridine adverse effects like headache and flushing. More serious adverse events reported in paediatric patients possibly caused by nifedipine included change in neurological status, severe hypotension, and oxygen desaturation.27 For this reason, the use of immediate release capsules, filled with a liquid solution of nifedipine, is not recommended in the Netherlands.15
3.2. Dissolution studies
3.2.1. Nifedipine
The nifedipine dissolution results in both the mini‐paddle apparatus and the flow‐through cell apparatus are presented in Figure 2 and Figure 3.
Figure 2.
Dissolution profiles of nifedipine (mean ± standard deviation, n = 3) under different conditions. A, Nifedipine 5‐mg compounded capsules in SGFsp (45 minutes)/SIFsp (225 minutes), mini‐paddle apparatus (50 rotations/min). B, Nifedipine 10‐mg commercial tablets (black circles) vs 2 × 5‐mg compounded capsules (grey squares) in FaSSGF (30 minutes, 5 mL/min)/FaSSIF (240 minutes, 4 mL/min), flow‐through cell apparatus. C, Nifedipine 1‐mg compounded capsules in Pi‐FaSSGF (30 minutes)/FaSSIF(240 minutes) in the mini‐paddle apparatus (50 rotations/min) and the flow‐through cell apparatus (4 mL/min and 3 mL/min; mean ± standard deviation, n = 3). Dotted lines represent the time of medium change. SGFsp = Simulated Gastric Fluid without pepsin; SIFsp = Simulated Intestinal Fluid without pancreatin; FaSSGF = Fasted‐state Simulated Gastric Fluid; FaSSIF = Fasted‐state Simulated Intestinal Fluid; pi‐ = paediatric infant
Figure 3.
Dissolution profiles of nifedipine (mean ± standard deviation, n = 3) under different conditions. A, Nifedipine 1‐mg compounded capsules in Pn‐FaSSGF (45 minutes)/FaSSIF‐V2 (225 minutes) (black circles) and Pi‐FaSSGF (30 minutes)/FaSSIF‐V2 (240 minutes) (grey squares), mini‐paddle apparatus (50 rotations/min). B, Nifedipine 1‐mg compounded capsules administered intact (black circles) vs capsule content dispersed in water before administration (grey squares), in Pn‐FaSSGF (45 minutes)/FaSSIF‐V2 (225 minutes), mini‐paddle apparatus (50 rotations/min). Dotted lines represent the time of medium change. FaSSGF = Fasted‐state Simulated Gastric Fluid; FaSSIF = Fasted‐state Simulated Intestinal Fluid
3.2.2. Experimental aspects
Impact of pH
Figure 2A shows the dissolution profile of a 5‐mg compounded capsule in SGFsp and SIFsp in the mini‐paddle apparatus. As nifedipine is not ionizable in the GI pH range, no apparent effect on nifedipine's dissolution from the pH switch was observed. A plateau was reached after around 180 minutes, with just over 20% of nifedipine dissolved. After the media switch, a rise in the amount dissolved was observed, resulting from an increased volume of dissolution medium. The large variability in the early phase of the experiment was caused by a variable capsule rupture time.
Impact of formulation
The dissolution profiles of the commercial 10‐mg nifedipine tablets and the compounded nifedipine 5‐mg capsules in FaSSGF/FaSSIF‐V2 (simulating children) in the flow‐through cell apparatus (displayed in Figure 2B) show a similar dissolution/release pattern, but the dissolution extent and rate from the capsules were slightly higher. As mentioned before, both formulations are presented as slow‐release formulations, even though they are not formulated as such. The dissolution profiles confirm this assumption under fasted conditions; however, from our results, the capsules and tablets cannot be considered interchangeable, as they previously were.28
Impact of hydrodynamics
Figure 2C shows the dissolution profiles of nifedipine 1‐mg compounded capsules in Pi‐FaSSGF/FaSSIF in the mini‐paddle and the flow‐through cell apparatus. The extent of nifedipine dissolution was affected by the type of in vitro dissolution apparatus used. Dissolution of nifedipine 1‐mg capsules in the mini‐paddle apparatus reached a plateau of 40% after around 2 hours, due to the lack of sink conditions, whereas in the flow‐through cell apparatus a continuous dissolution of nifedipine was observed with 70% dissolved at 270 minutes. Due to the continuous flow of fresh medium, sink conditions were achieved when the system operated in the open‐loop configuration. In this way, the dissolution rate reflects the behaviour of the formulation and not the solubility of the substance, as in the closed systems.20
3.2.3. Patient‐related aspects
Impact of age
Figure 3A shows the dissolution profiles of 1‐mg compounded capsules in Pn‐FaSSGF/FaSSIF‐V2 and Pi‐FaSSGF/FaSSIF‐V2 in the mini‐paddle apparatus. Between the dissolution profiles, a small effect of age was observed due to the difference in gastric emptying time/media switch, but an overall similar extent of dissolution was seen at the end of the experiment. This was an expected result as dissolution conditions with regard to fluid composition only moderately differed in the gastric phase. The gastric release was again variable and low in both experiments, as a result of a variable capsule rupture time. Also, the bile salt content, an important constituent to nifedipine solubility, is much lower in (paediatric) FaSSGF compared to FaSSIF‐V2.4
Impact of method of administration
Figure 3B shows the dissolution profiles of nifedipine compounded capsules administered intact vs capsule content dispersed in water before administration in Pn‐FaSSGF (45 minutes) and FaSSIF‐V2 in the mini‐paddle apparatus. The amount released was slightly higher in the gastric phase in the case that the capsule content had been mixed with water before administration compared to the direct administration of the capsule, and profiles in the intestinal phase were similar (Figure 2B). Since absorption mainly takes place from the small intestine and onwards, this suggests that a large change in the rate and extent of absorption from the different mode of administration of the capsule is unlikely.29
3.3. Lorazepam
The lorazepam dissolution results under paediatric biorelevant conditions in both the mini‐paddle apparatus and the flow‐through cell apparatus are presented in Figure 4.
Figure 4.
Dissolution profiles of lorazepam (mean ± standard deviation, n = 3) under different conditions. A, 1‐mg commercial tablet (black circles) vs 1 mL compounded oral solution 1 mg/mL (grey squares), SGFsp (45 minutes)/SIFsp (75 minutes), mini‐paddle apparatus (75 rotations/min). B, 1‐mg commercial tablets, fasted‐state pi‐FaSSGF (30 minutes, 4 mL/min)/FaSSIF‐V2 (90 minutes, 3 mL/min) vs fed‐state Pn‐FeSSGF (60 minutes, 5 mL/min)/Pi‐FeSSIF (60 minutes, 5 mL/min), flow‐through cell apparatus. C, 1‐mg commercial tablets, regular administration Pn‐FeSSGF (60 minutes, 5 mL/min)/i‐FeSSIF (60 minutes, 5 mL/min) vs duodenal administration Pi‐FeSSIF (120 minutes, 5 mL/min), flow‐through cell apparatus. Dotted lines represent the time of medium change. FaSSGF = Fasted‐state Simulated Gastric Fluid; FeSSGF = Fed‐state Simulated Gastric Fluid; FaSSIF = Fasted‐state Simulated Intestinal Fluid; FeSSIF= Fed‐state Simulated Intestinal Fluid
3.3.1. Experimental aspects
Impact of pH and formulation
Performance of the lorazepam oral solution and tablet formulation was assessed in SGFsp and SIFsp with the mini‐paddle apparatus (Figure 4A). During the simulated gastric residence time (45 minutes), almost all lorazepam was dissolved from the tablet, reaching similar concentrations as compared to the lorazepam liquid after only 15 minutes.
3.3.2. Patient‐related aspects
Impact of prandial state
Results from the flow‐through cell apparatus dissolution studies depicted in Figure 4B show the dissolution profiles of lorazepam 1‐mg tablets in Pi‐FaSSGF/FaSSIF‐V2 and Pnc‐FeSSGF/Pi‐FeSSIF. A slower dissolution rate was observed in fasted state conditions, leading to a lower amount dissolved after 2 hours (81.3 ± 8.4%) compared to fed state conditions (95.3 ± 3.0%). This could be predictive of a slower rate of in vivo drug absorption. Nevertheless, this is a relatively small difference, considering that lorazepam is likely to be absorbed almost completely, as is known from adult data.30
Impact of method of administration
Sometimes a patient does not tolerate gastric feeding and transpyloric feeding directly into the duodenum is required. When necessary, medication is also administered through the duodenal feeding tube and solid dosage forms are crushed or dispersed. When direct administration of the lorazepam tablet to the duodenum was simulated in Pi‐FeSSIF in the flow‐through cell apparatus, the dissolution profile was similar to the 1 obtained in Pnc‐FeSSGF/Pi‐FeSSIF (Figure 4C). These results suggest that administration through a duodenal feeding tube will not impact the in vivo dissolution. A change in tmax is still possible, however, as lorazepam reaches the site of absorption, namely the upper intestine, more quickly than when it is administered orally or via a gastric feeding tube.
4. GENERAL DISCUSSION
This study has yielded information on the application of nifedipine and lorazepam products in paediatric pharmacotherapy based on biopharmaceutical in vitro investigations.
As mentioned in section 3.1, markedly increased or accelerated absorption of nifedipine could lead to adverse effects such as severe hypotension, and must be avoided. In the solubility studies, we observed a significant food effect on the solubility of nifedipine in paediatric media, which is in agreement with previously reported clinical data in adults.31 This expected impact of food is also applicable in paediatric patients, and needs to be taken into account when administering nifedipine to paediatric patients. Clinicians should be aware that they cannot rely on the slow onset of action associated with nifedipine when conventional capsules or tablets are administered to patients in the fed state. Reassuring results came from the experiment dispersing the nifedipine capsule in water before administration, a commonly applied administration technique, revealing no significant differences in dissolution and thus implying no altered bioavailability in comparison to administration of the intact capsule.
In our experiments, dissolution of the lorazepam tablets was affected to a small extend by the different experimental set‐ups, namely the simulated prandial state and administration site. As there are no indications that lorazepam is a substrate to GI drug transporters, and absorption is almost complete in adults,30 it is also unlikely that excipients from the oral liquid will alter the lorazepam absorption compared to the tablets. When administering lorazepam to paediatric intensive care unit patients to prevent iatrogenic withdrawal syndrome, precise dosing is required.16 For this reason, and for ease of administration through a feeding tube, a liquid formulation would be the dosage form of choice. The fast and complete dissolution of the tablets gives reassurance about the interchangeability of liquid vs immediate release lorazepam tablets, and are supplemented with negligible effects of prandial state and administration site on the in vitro performance of the lorazepam formulations.
Drug substance solubility, together with the highest dose, upper GI lumen fluid volume, and volume used for administration are key factors defining the solubility classification of a drug within the BCS. The adult BCS is widely used in support of waivers of in vivo bioequivalence studies for immediate release solid oral formulations containing BCS class I and III (high solubility) substances, but it has been recognized that BCS classifications do not necessarily translate to paediatric populations.32 When the dose number (D 0) of nifedipine is calculated using the age adjusted initial gastric volumes (V 0) proposed by Shawahna33 and the saturated solubility values (C s) measured for Pnc‐FeSSGF, dosages up to 0.22 mg/kg would result in a D 0 < 1, and thus be considered BCS I. This example illustrates 1 of the challenges of developing a relevant paediatric BCS.
Ideally, the results obtained from in vitro dissolution experiments would be integrated into more complex in silico prediction models, which are able to include other factors influencing absorption, like gastric emptying. This PBPK modelling and simulation is already commonly used in formulation development/bridging for adult medicines and provides a promising tool for paediatric in vivo drug performance prediction, provided we gain a better understanding of the developmental changes of the GI tract in the paediatric population.3 Aside from the factors influencing in vivo dissolution, specific research is still required on the factors influencing permeability, mainly the ontogeny of metabolizing enzymes and drug transporters, to better predict oral drug absorption in this population.34 Ultimately, the developed biopharmaceutical tools could be validated using paediatric pharmacokinetic data, when available and possible to be shared by the pharmaceutical industry. The validated biopharmaceutical tools can then be used to study off‐patent paediatric drugs that would otherwise be neglected.
There is still a knowledge gap concerning GI physiology in paediatric patients. With the development of the paediatric biorelevant media, extrapolations from adult values had to be made for some aspects when availability of paediatric data was limited.4 Future adaptations of the media compositions are therefore likely when clinical investigations yield more accurate paediatric values. Other aspects that would gain from future clinical information and would be further updated in the design of in vivo predictive dissolution tests for the paediatric population would relate to the fluid volumes available at the GI lumen, and the motility patterns and hydrodynamics.
5. CONCLUSION
The in vitro results obtained from the experiments in this study, designed to reflect clinical practice in a paediatric hospital, suggest that biorelevant solubility and dissolution studies could assist in the understanding of drug performance in paediatric patients. The dissolution setups aiming to simulate physiological conditions to address numerous different administration scenarios, which would not be feasible or ethical in pharmacokinetic studies in children.
COMPETING INTERESTS
There are no competing interests to declare.
ACKNOWLEDGEMENT
This work was financially supported by the Erasmus Trustfonds.
Van der Vossen AC, Hanff LM, Vulto AG, Fotaki N. Potential prediction of formulation performance in paediatric patients using biopharmaceutical tools and simulation of clinically relevant administration scenarios of nifedipine and lorazepam. Br J Clin Pharmacol. 2019;85:1728–1739. 10.1111/bcp.13956
Present Address: Anna C. Van der Vossen, Department of Pharmacy, Maasstadziekenhuis, Rotterdam, The Netherlands.
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