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ANESTHETIC PHARMACOLOGY

A Novel Molecule with Peripheral Opioid Properties: The Effects on Hypercarbic and Hypoxic Ventilation at Steady-State Compared with Morphine and Placebo

Åsa Österlund Modalen, MD, PhD^*, Hans Quiding, PhD, Joana Frey, MD^*, Lars Westman, MD, PhD^*, and Sten Lindahl, MD, PhD^*

*Department of Anesthesiology and Intensive Care, Danderyds Hospital and Karolinska Institute, Stockholm; and Experimental Medicine, AstraZeneca R&D, Södertälje, Sweden

Address correspondence and reprint requests to Åsa Österlund Modalen, MD, PhD, Danderyds Hospital, S-182 88 Danderyd, Sweden. Address e-mail to asamodalen{at}hotmail.com.

	Abstract

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Frakefamide (FF), is a new peripherally acting µ-opioid receptor agonist. The aim of this double-blind, randomized, double-dummy, four-way, crossover study was to investigate FF effects on hypercarbic and hypoxic ventilation at steady-state after a 6-h infusion. We compared the effect with 2 clinical doses of morphine (M-small and M-large) and placebo in 12 healthy men. The subjects received 1.22 mg/kg of FF, 0.44 mg/kg of M-large, and 0.11 mg/kg of M-small. Sodium chloride 9 mg/mL was used as placebo. Ventilation was studied by pneumotachography and in-line capnography. There were no ventilatory effects caused by FF or placebo. As expected, large doses of morphine influenced both hypercarbic and hypoxic ventilatory responses. We conclude that there were no signs of central respiratory depression caused by FF after 6 h of constant infusion, which supports a peripheral action of the compound. However, morphine caused a dose-dependent central depression during the hypercarbic ventilatory response and a mild depression of hypoxic ventilatory response.

	Introduction

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Opioids, such as morphine and pethidine, are often used to treat acute and chronic pain (1) but are associated with various adverse effects, such as sedation, nausea and vomiting, constipation, and respiratory depression (2). The mechanism behind opioid-induced respiratory depression is not entirely understood, although µ- and -opioid agonists depress the brainstem respiratory center (3). Opioid receptors are also found in peripheral nerves, and drugs acting on these receptors, but not on central receptors, could be useful for analgesic treatment without serious side effects (4). Frakefamide (FF), a fluorinated tetrapeptide, H-Tyr-(D)Ala-(pF)Phe-Phe-NH2 hydrochloride, is a new peripherally acting µ-opioid receptor agonist, and it has been shown to be a potent analgesic in rats (5) and humans (6,7). In rats, the intrinsic activity is similar to morphine and the potency eightfold higher (5). FF was designed to not penetrate the blood-brain barrier (5,8,9). In line with this, FF was recently shown in the first part of our study not to impair resting ventilation in humans (10).

The purpose of the current, second part, of the study was to further characterize the respiratory effects of FF during hypercarbic and hypoxic respiratory challenges. For comparison, two clinical doses of morphine (M-small and M-large) and placebo were used. The study was a double-blind, randomized, double-dummy, four-way, crossover design.

	Methods

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The Ethics Committee of Human Research at the Karolinska Hospital and Institute, Stockholm, Sweden, approved the study protocol, and all volunteers gave their written informed consent before inclusion.

Twelve healthy male volunteers, 18–45 yr old, body weight between 65–95 kg, and body mass index 30, participated in study sessions that started in the mornings. Appropriate toxicology was not available to allow inclusion of women with childbearing potential. All subjects had to refrain from alcohol and medication for 48 h before the study. They were instructed to eat a standardized breakfast at home. During the study, food and drink were not allowed until measurements were completed. All volunteers were nonsmokers and healthy as judged by medical history, physical examination, and electrocardiogram (ECG). They were informed about the breathing equipment and allowed to test it. Adverse events reported spontaneously by the subjects or observed by the research team were recorded.

The subjects were studied in a double-blind, double-dummy, randomized, placebo-controlled, four-way, crossover design with at least 7 days between the investigations. A computer-generated randomization list was prepared at Biostatistics (AstraZeneca R&D, Sweden). Treatment order was only made available to those responsible for the labeling and bioanalysis of the study drug.

Venous catheters were placed in right and left cubital fossa veins. The cannula on the left side was used for continuous administration of the study drug by an infusion pump and on the right for infusion of crystalloid solution (5 mL/kg).

The subjects rested in a comfortable bed with a 15-degree head-up tilt and were not allowed to leave the bed for 6 h after the start of drug infusion.

The subjects received FF 1 nmol^–1 · kg^–1 · min^–1 during the first 15 min, 3 nmol^–1 · kg^–1 · min^–1 the next 15 min, and thereafter 6 nmol^–1 · kg^–1 · min^–1 for up to 6 h, with a total dose of 1.22 mg/kg. The stepwise start of infusion was designed to minimize myalgia, a side effect of FF. Administration of clinically small and large doses of morphine (M-small and M-large) were 8.73 and 34.78 nmol^–1 · kg^–1 · min^–1, respectively, for the first 15 min, and thereafter, 0.44 and 1.77 nmol^–1 · kg^–1 · min^–1 for the remaining time up to 6 h. M-small corresponds to a dose of 0.05 mg/kg for 15 min and 0.06 mg/kg for 345 min, and M-large corresponds to 0.20 mg/kg for 15 min followed by 0.24 mg/kg for 345 min. Sodium chloride 9 mg/mL was used as placebo.

Before and at 110, 350, and 470 min after study drug administration, 12-lead ECG (Siemens Sicard 460; Siemens, Elema, Germany) was recorded. Systolic and diastolic blood pressures were measured noninvasively.

Resting ventilation was recorded with the subjects breathing air through a transparent face mask (Gibeck, Gibeck-Dryden, Germany) positioned over the nose and mouth. The subjects were allowed an initial resting period of approximately 20–30 min to get adjusted to the apparatus. An in-line infrared capnometer (Hewlett-Packard 14360 A) and a pneumotachograph (Fleisch #2, linear for flows up to 2.4 L/s) were placed in the circuit. The dead space of the system was 70 mL measured by water displacement. Inspiratory and expiratory resistances of the measuring apparatus were 2 cm H₂O · L^–1 · s^–1. Minute ventilation (e) and respiratory rate were measured by integration of the flow signal from the pneumotachograph. Flow, tidal volume, and in-line end-tidal CO₂ (ETco₂) were recorded on an ink-jet recorder (Mingograf 800; Siemens) and stored in a computer. Inspired and expired concentrations of O₂ were continuously recorded by a Datex Ultima S device (Datex, Helsinki, Finland), which also measured arterial oxygen saturation by finger pulse oximetry (Spo₂). Ventilation volumes are corrected for body temperature and saturated vapor pressure. The Hewlett-Packard capnometer was calibrated with certified gases before each experiment. Calibration of the pneumotachograph was performed before and after each experiment using a high precision syringe (model 553; Hans Rudolph Inc.). Ventilatory measurements were performed both at resting ventilation and during hypercapnic and hypoxic challenges before study drug infusion and at 15 and 45, 80 and 110, 330 and 350, and 440 and 470 min (respectively for hypercapnic and hypoxic challenges) after the start of administration of the study drug. Measurements at 6 h (330 and 350 min) of infusion were targeted for evaluations.

Hypercarbic ventilatory responses (HCVR) were measured with a steady-state technique after adding 5% CO₂ to inspired air during 5- to 6-min periods. A steady-state was reached after 3 min. Ventilatory measurements were then performed over 2 min. HCVR was defined according to the expression: HCVR (L · min^–1 · mm Hg^–1) = e/ ETco₂.

Hypoxic ventilatory responses (HVR) were measured after adding 5% oxygen/nitrogen gas to inspired air to obtain a stable level of 80% Spo₂. Steady-state was reached after approximately 5 min. After another 1–2 min with stable hypoxia, ventilatory measurements were performed over 2 min. Hence, acute HVR were measured. The isocapnic test condition was achieved by adding CO₂ to inspired air to keep ETco₂ constant during hyperventilation. HVR was defined according to the expression: HVR (L · min^–1 · %^–1) = e/ Spo₂.

An analysis of covariance model was used for the statistical analysis. Independent variables in the model were subjects, period, and treatment. To control for possible one-period carryover effects, corresponding indicator variables were also included in the model. Estimates (least square means) with corresponding confidence intervals from the analysis are presented. The validity of the underlying assumptions about the model was assessed using model-checking tools such as residual plots. All analyses were performed using SAS, version 8.2. To obtain a power of 90% (two-sided paired t-test at the 5% significance level), a sample size of 12 subjects was required.

	Results

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There were only minor variations of arterial blood pressure and heart rate during the study period, with no differences between groups. At resting ventilation there were no differences in peripheral O₂ saturation between groups.

After administration of FF, all subjects experienced a transient pain described as myalgia. The muscle symptoms were minor to severe (one subject) and started within 15 min after the administration of FF and vanished after 15–20 min. The same muscle symptoms were noted in two subjects after M-small and in five subjects after M-large administration. Rhinitis was observed in six subjects in the FF group. Nausea was the most common adverse event in both M-small (three subjects) and M-large (nine subjects) groups. Pruritus was noted in one subject in M-small and in five subjects in M-large.

Before the infusion of FF, e increased from 10.6 L/min to 19.4 L/min during CO₂ stimulation (Table 1). The ETco₂ increased from 38.3 to 48.0 mm Hg.

At 6-h measurements, during resting conditions before HCVR testing, ETco₂ was increased by 12% in M-large and by 4% in M-small groups compared with the control situation. There was no increase in ETco₂ in the FF and placebo groups (Table 1).

At 6-h measurements, there were statistically significant reductions in HCVR in both M-small and M-large groups (Fig. 1). FF and placebo groups maintained HCVRs comparable to the control situation.

View larger version (18K): [in this window] [in a new window]   Figure 1. Median hypercarbic ventilatory responses (HCVR) after a 6-h infusion of frakefamide (FF), morphine 0.11 mg/kg (M-small), morphine 0.44 mg/kg (M-large), and placebo in 12 healthy volunteers in a crossover study. Slopes of the HCVR are presented. *P < 0.05; **P < 0.001 represents pairwise differences in HCVR. Pairwise differences in HCVR at 330 min after the start of infusion (differences of least squares mean values and corresponding 95% confidence limits).

Before infusions of FF, e increased from 10.8 L/min to 16.3 L/min during hypoxia (Table 2). e was lower during M-small and M-large administration as compared with placebo and FF. Parallel with this, ETco₂ was increased (Table 2). Compared with the influence on HCVR presented above, effects of the drugs on HVR were small and did not reach statistical significance (Fig. 2). Hence, FF did not impair HVR (Fig. 2).

View larger version (17K): [in this window] [in a new window]   Figure 2. Median hypoxic ventilatory responses (HVR) after a 6-h infusion of frakefamide (FF), morphine 0.11 mg/kg (M-small), morphine 0.44 mg/kg (M-large), and placebo in 12 healthy volunteers in a crossover study. Slopes of the HVR are presented. *P < 0.05 represents pairwise differences in HVR. Pairwise differences in HVR at 360 min after the start of infusion (differences of least squares mean values and corresponding 95% confidence limits).

	Discussion

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The main result in the current, last part of the study was that FF did not impair the responses to hypercarbic or hypoxic ventilatory challenges. However, morphine caused depression of HCVR, as expected. In the FF group, all subjects described a mild to moderate pain that lasted up to 30 minutes after the start of infusion.

The rationale behind the design of our study using six-hour measurements for comparisons was to ensure a steady-state and near complete distribution of the drug in peripheral tissues to assess the peripheral effect of this novel compound. The initial myalgia caused by the drug composition also invalidates respiratory measurements too close to the initiation of the infusion. By using the ventilatory measurements at steady-state six hours after the start of the infusion, the pain was regarded to have no influence on respiratory functions.

A major long-term goal for the pharmacology of analgesics has always been to diminish side effects, in particular, those that jeopardize control of breathing. Bouillon et al. (11) demonstrated that if there are rapid changes of opioid concentrations in the medulla oblongata, patients cannot maintain ventilation. Thus, bolus doses are more likely to cause irregular breathing and apnea. In our study, with the use of an IV infusion during six hours, it is probable that there were no sudden changes in brainstem opioid concentrations, and hence, we did not record problems with irregular breathing or apnea. FF with analgesic properties and seemingly peripheral activation only seems to be a step forward in the search for a potent opioid analgesic without respiratory depression.

Because opioid receptors are present on peripheral sensory nerves and function by preventing nociception (12), the search for a substance solely acting on peripheral opioid receptors has been a pharmacological undertaking for many years. FF is a large molecule (molar mass = 600.1 g) and a selective µ agonist with restricted passage into the central nervous system (5,8,9). In humans, it produces a dose-related analgesia with a rapid onset and short duration of analgesia (6,7). In the present study, we investigated doses 33% larger than those previously used in human studies. These doses of FF had no effects on hypercarbic and hypoxic ventilatory challenges. This demonstrates a lack of FF effects on central regulation of breathing, which is an indirect indication that FF does not penetrate the blood-brain barrier.

HVR were virtually similar in all studies. Acute hypoxia preferably stimulates ventilation by an action on peripheral chemoreceptors (13). Studies on opioids and the influence on hypoxic ventilatory sensitivity are difficult to interpret and compare because of different testing protocols. Berkenbosch et al. (14) studied influences of morphine on ventilatory responses to isocapnic hypoxia in anesthetized cats and found that morphine did not depress ventilation. Gross et al. (15), however, observed a 20% reduction of hypoxic sensitivity during alfentanil infusion; a similar, but longer lasting, depression of the ventilatory response to hypoxia was noted after intrathecal rather than IV administration of morphine (16). These authors also suggested that the respiratory depression during hypoxia is caused by a direct action of opioids on the central nervous system and not via peripheral action. Similarly, we found a reduction of e during M-small and M-large administration with increased ETco₂s (Table 2). In agreement with the findings of Berkenbosch et al.’s (14) findings in cats, however, there was no depression of HVR in the M-large (0.44 mg/kg) group in our study. There was, however, a significant depression during hypercarbia. Overall, the drugs used in this study did not impair the HVR. One explanation could be that the hypoxic stimulus used (Spo₂ 80%) was insufficient to elicit an adequate HR, or another explanation could be that there is a sex difference in HVR after morphine infusion (17). It seems as if HVR is a vital compensatory physiological defense mechanism that compensates, in a complex manner, for various drug effects. This study was, however, not designed to explain mechanisms for O₂ sensing.

We conclude that both HCVR and HVR were maintained during a six-hour infusion of the new compound FF, which is designed not to penetrate the blood-brain barrier. FF has a potent analgesic action and could be a major advance in the search for a clinically useful opioid with no effects on the central nervous system.

We are grateful for the excellent technical contributions from Anette Ebberyd and Ringvor Hägglöv, Department of Anesthesiology and Intensive Care, Karolinska Hospital, and for support and statistical evaluation from AstraZeneca R&D, Södertälje.

	Footnotes

 
Accepted for publication July 11, 2005.

	References

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