Introduction

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Morphine, a potent narcotic analgesic, produces a variety of pharmacological responses by interacting with the opioid receptors in the nervous system. It is used widely for preoperative and anxiolytic therapy in pediatric patients, for the management of postoperative pain, and for moderate-to-severe pain in cancer patients because of its general availability, the choice of different formulations and routes of delivery, and the well characterized pharmacological properties. At least one-third of newly diagnosed cancer patients and about two-thirds of patients with an advanced disease experience pain either as chronic pain or as breakthrough pain episodes or both (Foley, 1995). The World Health Organization recommended in 1986 that advanced cancer pain should be treated in accordance with the analgesic ladder (World Health Organization, 1986).

Morphine is most commonly administered via the oral route, either as an oral solution, as an immediate release or controlled release oral tablet, or capsule preparation and is readily absorbed in the small intestine. Due to considerable intestinal metabolism and extensive hepatic first pass effect the oral bioavailability has been reported to be as low as 20% (Bourget et al., 1995) and 32% (Westerling et al., 1995). The main metabolites of morphine are morphine-6-glucuronide (M-6-G), which is an active analgesic agent, and morphine-3-glucuronide (M-3-G), which is inactive (Osborne et al., 1990; Westerling et al., 1995; Faura et al., 1996). Oral morphine therapy results in a range of side effects (e.g., respiratory depression, constipation, nausea, and vomiting) in the majority of patients (Twycross, 1994) and even patients with generally well controlled (chronic) pain will experience several 30-60-min periods of excruciating "breakthrough pain" every day, triggered by manipulations of the patient or appearing spontaneously (Cleary, 1997). Breakthrough pain is normally treated by oral opioid medication such as a morphine solution or oral immediate release tablets, but the maximum plasma concentration may not be reached for 0.8 h, resulting in slow onset of analgesia.

Analgesic agents such as fentanyl, oxycodone, and butorphanol can be effectively and rapidly absorbed from the nasal cavity (due to their relative high lipophilicity) without the help of absorption promoters and thereby provide rapid onset of analgesia (Shyu et al., 1993; Takala et al., 1997). However, in humans morphine is only absorbed to a low degree when given by the nasal route and mainly when reaching the small intestine after clearance from the nasal cavity (Behl, 2000)

As shown by ourselves and other groups, the nasal absorption of small polar molecules and polypeptides can be greatly improved if administered in combination with an absorption-promoting agent such as chitosan (Illum et al., 1994, 1996, 2000; Illum, 1998a; Roon et al., 1999). Hence, when M-6-G (log P = 0.76), which is more hydrophilic than morphine (log P = 0.89), was formulated with a 0.5% chitosan solution the bioavailability in sheep after nasal administration was 31% relative to an intravenous injection (Illum et al., 1996).

Chitosan is a linear polysaccharide comprised of two monosaccharides: N-acetyl-D-glucosamine and D-glucosamine linked together by glucosidic bonds. Chitosan is produced by alkaline hydrolysis (deacetylation) of chitin obtained from crustacean shells and forms positively charged salts when dissolved in inorganic or organic acids. Chitosan is available in a wide range of molecular weights and degrees of deacetylation. The chitosan most commonly chosen for nasal delivery of drugs is the glutamate salt with a mean molecular weight of around 200 kDa and a degree of deacetylation of 80 to 90%. Chitosan is bioadhesive and able to interact strongly with the nasal mucus layer and with the nasal epithelial cells. The clearance of chitosan formulations from the nasal cavity of sheep and humans has been shown to be significantly slower than that of simple aqueous solutions (Soane et al., 1999, 2001). Hence, nasal chitosan drug formulations provide longer time for drug transport across the nasal membrane, before the formulation is cleared by the mucociliary clearance mechanism. Furthermore, chitosan has also been shown in Caco-2 cell culture studies to open transiently the tight junctions between cells, which enables hydrophilic drugs to pass through the membrane by the paracellular route (Dodane et al., 1999).

The purpose of the present work was to study the nasal absorption of morphine in an animal model and in humans and to develop a suitable nasal morphine formulation that could provide rapid and efficient absorption of the morphine across the nasal membrane. Various formulations, expected to enhance the nasal absorption of morphine, were tested in sheep (to include bioadhesive starch microspheres, and chitosan solution and powder formulations). Selected formulations were subsequently administered to human volunteers and the pharmacokinetic profile and tolerability of the formulations were evaluated.

	Experimental Procedures

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Materials

Morphine hydrochloride BP was purchased from MacFarlane Smith Ltd. (Edinburgh, Scotland, UK). Morphine sulfate (10 mg/ml) in a sterile saline solution was obtained from Martindale Pharmaceuticals (Essex, UK). Chitosan glutamate (Sea Cure G + 210) and chitosan hydrochloride (Sea Cure Cl 113) were obtained from Pronova (Drammen, Norway). The chitosan was supplied spray dried and had the form of microspheres. Crosslinked Eldexomer starch microspheres (SMS) were supplied by Perstorp Pharma (Perstorp, Sweden) and the L--lysophosphatidylcholine (LPC) by Sigma-Aldrich (Poole, Dorset, UK). All other materials were of pharmaceutical grade or at least analytical grade.

Prototype devices from Bespak Ltd. (King's Lynn, UK) were used to administer the powder formulations to the nasal cavity in the human clinical trial. These single dose devices contained a polypropylene capsule loaded with the correct dose of powder formulation. The capsule was pierced by priming the device and the dose delivered by the volunteer breathing in rapidly through one nostril.

The dosing devices used in the clinical trial for administration of a single dose of a liquid formulation were supplied by Pfeiffer GmbH (Radolfzell, Germany). The dose was contained in a glass vial, which was assembled into the dosing unit. Each device was calibrated to deliver a dose of 125 µl, for which a loading of 145 µl was required. The dose was released as a spray into the nasal cavity by pressing the plunger on the device.

Formulation Preparation

Formulations Used in Sheep Studies. A summary of the morphine formulations administered to the sheep is given in Table 1. The morphine solution for intravenous injection (formulation 1) was prepared by dissolving 40 mg of morphine hydrochloride in 50 ml of sterile isotonic saline and filtering through a sterile (0.2-µm) membrane filter (Satorius, Gottingen, Germany). The osmolality of this solution was 0.292 Osmol/kg.

Formulations Used in Human Studies. A summary of the morphine formulations administered to human volunteers is given in Table 2.

Analytical Methods

In Vitro Morphine Assay. The morphine hydrochloride analysis was performed by reverse phase HPLC with ultraviolet detection by using a method slightly modified from the assay method described by Svensson et al. (1982). The limit of detection of the assay was 10 µg/ml.

Plasma Morphine Levels in Sheep. The plasma morphine levels were measured in the sheep plasma samples by a solid phase quantitative radioimmunoassay, by using a commercial Coat-A-Count serum morphine kit (Diagnostics Product Corporation, Abingdon, Oxfordshire, UK). The RIA-CALC program was used for calculating the plasma morphine concentrations in nanomoles per milliliter, by using a morphine calibration curve. All measurements were performed using plasma samples. Validation of the assay showed the intraday and interday variation to be within the acceptable range. The coefficient of variation was less than 15% for all the quality control samples (low, medium, and high). The limit of detection was found to be 2.8 nM. The cross-reactivity of the method with M-6-G and M-3-G metabolites was reported as negligible. All samples were analyzed at least in duplicate.

Plasma Morphine Levels in Human Volunteers. The plasma samples were analyzed by HPLC for morphine, morphine-6-glucuronide, and morphine-3-glucuronide by Hafslund Nycomed Pharma (Linz, Austria). The extraction method used was a modification of the method described by Murphey et al. (1993) and the HPLC conditions used based on the method described by Todd et al. (1982). The method was shown to be linear over the chosen concentration ranges and stability of the analytes was demonstrated in the injection solution. A series of quality control samples were included in each extraction and accuracy and precision were demonstrated to deviate by less than 20% for morphine. Pharmacokinetic analysis was performed using the program TOPFIT version 2.0 according to noncompartmental methods.

Sheep Studies

The sheep nasal model was chosen for the initial studies because it has been shown in various studies and by various groups that this model is very predictive of results in humans (Illum, 1996). Twenty male, cross-bred Texel and Suffolk sheep of 49.1 ± 12.1 kg (mean ± S.D.) were used in the study and divided into five groups of four animals. The sheep were housed indoors for the duration of the study and fed ad libitum on a nut concentrate and hay. The animals were not fasted before the experiment. On the first day of the study, an indwelling Secalon cannula fitted with a flow switch was placed approximately 15 cm into one of the external jugular veins of each animal. The cannulae were kept patent by flushing with heparinized (25 IU/ml) 0.9% saline solution. On the second day, the sheep were sedated for about 3 min with an intravenous dose of 100 mg/ml ketamine (Vetalar; Fort Dodge Animal Health, Ltd., Southhampton, UK) at 2.25 mg/kg during dosing to prevent sneezing. The solution formulations were instilled nasally from a 1-ml syringe (0.01 ml/kg) attached to a blueline umbilical cannula inserted approximately 8 cm into the nasal cavity. The dose was divided equally between the two nostrils. The powder formulations were administered nasally using a blueline siliconized oral/nasal tracheal tube containing the preweighed dose, inserted approximately 8 cm into the nasal cavity, by means of a simple one-way spray bellows. The intravenous administration was given as a slow injection (0.125 ml/kg over 1 min) via the indwelling jugular vein cannula. The cannula was flushed with 10 ml of sterile normal saline. Blood samples of 4.0 ml were collected from the cannulated jugular vein of the sheep at 20, 15, and 5 min before morphine administration and at 5, 10, 15, 20, 30, 45, 60, 90, 120, 150, 180, 240, 300, and 360 min after dosing. For the intravenous administration an extra blood sample was collected at 2 min after administration. The blood samples were gently mixed in 4 ml of heparinized tubes (60 IU of lithium heparin; Sarstedt, Leicester, UK) and kept on crushed ice until plasma separation. The plasma samples were stored at 20°C awaiting analysis. The cannulae were removed upon completion of the study and the sheep returned to their normal housing. The animal studies were performed under an approved Home Office Animal Project License in accordance with the Animals (Scientific Procedures) Act 1986.

Human Volunteer Trial

The study was conducted as a three-way crossover design in 12 healthy volunteers (male and female) between 18 and 36 years of age. The volunteers were fasted from 10:00 PM the night before each dose administration. A light breakfast was allowed 2 h postdose. Lunch and evening meals were provided at 5 and 10 h postdose, respectively. A cannula was inserted into a vein in the lower arm for blood sampling at the start of each study day. The volunteers received the three morphine formulations (A, B, or C) in a randomized order according to a Latin square design. There was a 1-week washout period between the administration of the various doses. Before recruitment into the trial, volunteers were given detailed information about the study and signed a consent form. They then underwent a medical screening procedure, including a physical examination, medical history, clinical laboratory tests, and ECG recording, according to the protocol. Only volunteers complying with the inclusion and exclusion criteria were used in the study. No volunteer with a history of intravenous drug abuse or abuse of opioids was included in the study. The clinical protocol was approved by an Ethics Committee and the study carried out at Medeval Ltd. (Skelton House, Manchester Science Park, Manchester, UK) in accordance with the Declaration of Helsinki.

The nasal solution and powder formulations were administered by a trained nurse or clinician to the volunteers according to written instructions. The volunteers received the content of a capsule in each nostril (a nominal 10 mg of morphine hydrochloride) for the powder formulation and 125 µl in each nostril (10 mg of morphine hydrochloride) for the solution formulation. Ten milligrams of morphine sulfate was infused over a period of 30 min via an indwelling intravenous catheter in a forearm vein that was not used for blood sampling. The infusions were prepared by adding 18 ml of sterile normal saline to 2 ml of the morphine sulfate commercial preparation. After priming the giving set, the infusion pumps were set to infuse 20 ml/h for 30 min giving a total of 10 mg of morphine sulfate.

The formulations to be tested in the human studies were selected from the sheep studies on the basis of an evaluation of potential toxicological problems that might be encountered in the clinic for some of the formulations.

The residual doses left in the nasal devices were analyzed by HPLC for morphine content and the exact doses delivered to each volunteer calculated by subtracting the residual morphine dose from the original dose in the device. All pharmacokinetic results were adjusted to account for the dose given. The mean residual doses constituted less than 20% of the total dose.

Blood samples (8 ml) were taken at 15 min (before dosing) and at 5 min, 15 min, 30 min, 32 min, 35 min, 40 min, 45 min, 1 h, 1 h 15 min, 1 h 30 min, 2 h, 3 h, 4 h, 6 h, 8 h, and 12 h postdose for the intravenous administration of morphine or 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 1 h 30 min, 2 h, 2 h 30 min, 3 h, 4 h, 6 h, 8 h, and 12 h postdose for the nasal doses. The total volume of blood sampled during the whole study was approximately 490 ml from each volunteer. The samples were collected into heparinized tubes and maintained on ice until centrifugation. The samples were centrifuged within 15 min of collection on a refrigerated centrifuge at 4°C at 2000g for 10 min. The resultant plasma was divided into two samples of 2.5 and 1.5 ml and stored at 20°C until analysis.

At specified times after dosing the volunteers were asked to complete a form describing the taste and tolerability of the drug formulation in the nasal cavity on a scale from 0 to 10. A questionnaire was used to record the central effects of the morphine such as the degree of drowsiness and nausea on a similar scale from 0 to 10. For each time point, the total score for all volunteers and the number of volunteers recording a score greater than zero are recorded. The maximum score for each type of intolerability or central effect is 120 and the maximum total intolerability score is 600.

Blood pressure, respiratory rate, and heart rate were monitored before dosing and at specific times afterward. Volunteers were closely monitored for effects on the central nervous system for the duration of the study, especially in the first 2 h after dose administration.

Statistical Analysis

Statistical analysis of data obtained from the sheep/human studies was performed using GraphPad Instat software (GraphPad Software, San Diego, CA). Throughout, the level of statistical significance was chosen as p < 0.05. For comparison of intravenous and/or nasal sheep data, a one-way analysis of variance (ANOVA) with Tukey-Kramer multiple comparisons post test was used. The post test was performed only if findings of the ANOVA were significant. Analysis of human nasal and intravenous data was by one-way ANOVA with Tukey-Kramer multiple comparison post test as appropriate. Comparison of data from the two nasal groups was performed using unpaired (two-tailed) t tests.

	Results

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Sheep Studies. The pharmacokinetic values for the nasal absorption of morphine in sheep are shown in Table 3 and the plasma profiles for the nasal formulations for the first 120 min after dosing are given in Fig. 1. The absorption of morphine across the nasal membrane from a nasal morphine hydrochloride solution formulation given as a control (formulation 2) was limited with a C_max of 151 nM and an F% in the order of 10%. The T_max of 20 min indicated relatively slow rate of nasal absorption of morphine from the control formulation. When 0.5% chitosan was coadministered with morphine in a solution formulation (formulation 3) the nasal absorption was increased with a C_max of 657 nM and a bioavailability of 26.6%. The rate of absorption was also improved with T_max at about 14 min. Chitosan formulated into microspheres and administered with the morphine (formulation 4) further improved nasal morphine absorption. The C_max was found to be 1010 nM, the T_max about 8 min, and the bioavailability 54.6%, representing more than a 4-fold increase in absorption compared with the morphine control solution formulation. Still further improvement in nasal morphine absorption was observed after dosing a powder formulation comprising starch microspheres, LPC, and morphine (formulation 5); values of C_max, T_max, and F% of 1875 nM, 10 min, and 75%, respectively, were recorded.

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Morphine plasma concentration after nasal administration of morphine formulations in sheep. Mor Sol, morphine solution; Mor Chi Sol, morphine solution containing chitosan; Mor Chi PWD, morphine-chitosan powder; Mor SMS LPC, starch microspheres with lysophosphatidylcholine and morphine as a freeze-dried powder.

Human Phase I Clinical Trial. The pharmacokinetic values for the nasal absorption of morphine in human volunteers are shown in Table 4 and the plasma profiles for the nasal and intravenous formulations are given in Fig. 2. After slow intravenous administration of 10 mg of morphine sulfate the mean plasma concentration of morphine (C_max) was 336 ± 68 nM, 30 min after the start of dose administration. The plasma half-life of morphine was 1.67 ± 0.26 h. After nasal administration of a solution formulation containing 0.5% chitosan and morphine hydrochloride (formulation B, nominal dose 10 mg of morphine per volunteer) peak plasma concentrations of morphine were rapidly attained (C_max of 98 ± 57 nM, T_max of 16 ± 7 min). The shape of the plasma morphine profile was similar to that obtained after the slow intravenous injection (Fig. 2). The plasma half-life (t_1/2) obtained after the nasal solution formulation (2.98 ± 2.39 h) was not significantly different (p > 0.05) from that after slow intravenous morphine administration. The mean bioavailability of the nasal chitosan-morphine formulation was 56 ± 27%. For the nasal powder formulation comprising chitosan and morphine hydrochloride (formulation A, nominal dose 10 mg of morphine per volunteer) the results were not significantly different (p > 0.05) from those of the chitosan-based solution formulation and the shape of the plasma morphine profile obtained was similar. Values of C_max, T_max, t_1/2, and F% were 92 ± 36 nM, 21 ± 7 min, 2.72 ± 2.17 h, and 56 ± 20%, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Morphine plasma concentration in human volunteers after intravenous administration of morphine and after nasal administration of morphine as chitosan solution and powder formulations. Mor Chi Sol, morphine solution containing chitosan; Mor Chi PWD, morphine-chitosan powder; IN, intranasal.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Total central effects scores in human volunteers after nasal administration of a morphine solution formulation with chitosan, a powder formulation containing morphine and chitosan and an intravenous administration of morphine. IN MOR CHI PWD, intranasal morphine-chitosan powder; IN MOR CHI Sol, intranasal morphine-chitosan solution; i.v. MOR, intravenous morphine.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Plasma concentration of morphine metabolites M-3-G and M-6-G after intravenous administration of morphine (A) and nasal administration of a morphine-chitosan solution formulation (B).

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Relative amount of morphine metabolites M-6-G (A) and M-3-G (B) after intravenous administration of morphine, nasal administration of a morphine-chitosan solution formulation, and oral administration of morphine. The oral data taken from Osborne et al. (1990).

	Discussion

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In the management of breakthrough pain it is of importance to use a drug and a route of administration that will provide a time-action profile characterized by rapid onset and early peak effect and duration commensurate with the span of most breakthrough pain situations. Hence, a pure µ-opioid agonist such as morphine, with relatively short plasma half-life, administered nasally with an adequate delivery system would be a suitable choice.

Very few studies on the nasal delivery of morphine to humans have been published. Chast et al. (1992) administered 20 mg of morphine acetate to six postoperative patients by the nasal and oral routes and reported a peak plasma concentration 15 min after nasal and 30 min after oral administration. The plasma profile after nasal administration was very similar to that seen after parenteral administration. However, in the article, the bioavailability was not disclosed, although pharmacokinetic data were given. Recently, data on the nasal delivery of morphine sulfate to humans as a simple solution were presented by Behl (2000). The nasal administration of morphine provided a plasma profile very similar to that found after oral administration, most likely due to an expected limited nasal absorption of the hydrophilic drug followed by a more extensive oral absorption after clearance from the nasal cavity in humans.

Studies in Sheep. Because of its polar nature, morphine is not easily transported across the nasal membrane with a bioavailability of only 10.5% in the sheep model (Fig. 1; Table 3). Due to the special nature of the sheep stomach (rumen) the absorption profile obtained in the sheep model can be credited to purely nasal absorption. The absorption found herein is much lower than that reported by Kondo et al. (1995) (60%) in a rat model and in rabbits by Chast et al. (1992) (86%). This is most likely due to the use of anesthesia in the rat and rabbit models during administration of the nasal formulations. Anesthesia is known to give rise to a decrease in mucociliary clearance rate and has been shown to enhance the nasal absorption of drugs (Illum, 1996; Mayor and Illum, 1997). The sheep model used in these experiments only involved mild sedation for about 3 min during dosing and the model has been shown to be predictive of absorption in humans (Illum, 1996).

Studies in Volunteers. The human volunteers were given morphine doses of 10 mg in three different formulations: a nasal solution formulation and a powder formulation both containing chitosan and morphine hydrochloride and an intravenous infusion of morphine sulfate over 30 min, in a crossover design. The nasal solution and powder formulations resulted in substantially identical morphine plasma profiles with rapid and high peak plasma concentrations, which were similar in shape to the profile obtained for intravenous administration (Fig. 2). The T_max for the nasal powder formulation was slightly longer (21 min) than for the solution formulation (15 min), which would be expected for a mucoadhesive powder formulation. The reason why in humans the nasal powder formulation did not increase the absorption of the morphine to a higher degree than the chitosan solution formulation could partly be due to the similarities that exist between the physicochemical characteristics of the two chitosan formulations. The devices used for the solution and powder formulations in sheep and humans were different but both active in action. Different devices were necessary due to the different morphology of the nasal cavity of sheep and humans (Illum, 1996). However, it has been shown by our own group that both for solution and powder formulations the clearance times obtained in sheep and humans are very comparable when administered by such methods (Soane et al., 1999, 2001). Because the clearance of formulations from the nasal cavity is dependent upon the site of deposition, the comparability between the two species and the delivery device are evident. The increased clearance time of both the solution and powder chitosan formulations is reflected in the apparent prolonged half-life of the two nasal formulations compared with intravenous administration.

	Conclusion

Top Abstract Introduction Experimental Procedures Results Discussion Conclusion References

It can be concluded from these studies that it is possible with a nasal morphine formulation containing chitosan to obtain a rapid and therapeutically relevant peak plasma level of morphine. The plasma profiles after nasal administration were similar to those obtained after intravenous administration of morphine and a bioavailability of about 60% can be obtained. The pharmacokinetic data from the sheep and human studies was subjected to statistical analysis. Pilot studies in cancer patients have shown the efficacy of the nasal morphine formulation as a means of improving the treatment of breakthrough pain. The nasal morphine formulation containing chitosan has been shown to be well tolerated and well accepted by both volunteer subjects and cancer patients.