Characterization of Membrane-Type Dissolution Profiles of Clinically Available Orally Inhaled Products Using a Weibull Fit and a Mechanistic Model.

Dissolution rate impacts the absorption rate of poorly soluble inhaled drugs. In vitro dissolution tests that can capture the impact of changes in critical quality attributes of the drug product on in vivo dissolution are important for the development of products containing poorly soluble drugs, as well as modified release formulations. In this study, an extended mathematical model allowing for dissolution of polydisperse powders and subsequent diffusion of dissolved drug across a membrane is described. In vitro dissolution profiles of budesonide, fluticasone propionate, and beclomethasone dipropionate delivered from three commercial drug products were determined using a membrane-type Transwell dissolution test, which consists of a donor and an acceptor compartment separated by a membrane. Subsequently, the profiles were analyzed using the developed mechanistic model and a semi-empirical model based on the Weibull distribution. The two mathematical models provided the same rank order of the performance of the three drug products in terms of dissolution rates, but the rates were significantly different. The faster rate extracted from the mechanistic model is expected to reflect the true dissolution rate of the drug; the Weibull model provides an effective and slower rate that represents not only drug dissolution but also diffusion across the Transwell membrane. In conclusion, the developed extended model provides superior understanding of the dissolution mechanisms in membrane-type (Transwell) dissolution tests.

Characterisation of pharmacokinetics, safety and tolerability in a first-in-human study for AZD8154, a novel inhaled selective PI3Kγδ dual inhibitor targeting airway inflammatory disease.

AIMS: This 3-part, randomised, phase 1 first-in-human study (NCT03436316) investigated the safety, tolerability and pharmacokinetics (PK) of AZD8154, a dual phosphoinositide 3-kinase (PI3K) γδ inhibitor developed as a novel inhaled anti-inflammatory treatment for respiratory disease. METHODS: Healthy men, and women of nonchildbearing potential, were enrolled to receive single and multiple ascending inhaled doses of AZD8154 in parts 1 and 3 of the study, respectively, while part 2 characterised the systemic PK after a single intravenous (IV) dose. In part 1, participants received 0.1-7.7 mg AZD8154 in 6 cohorts. In part 2, participants were given 0.15 mg AZD8154 as an IV infusion. In part 3, AZD8154 was given in 3 cohorts of 0.6, 1.8 and 3.1 mg, with a single dose on Day 1 followed by repeated once-daily doses on Days 4-12. RESULTS: In total, 78 volunteers were randomised. All single inhaled, single IV and multiple inhaled doses were shown to be well tolerated without any safety concerns. A population PK model, using nonlinear mixed-effect modelling, was developed to describe the PK of AZD8154. The terminal mean half-life of AZD8154 was 18.0-32.0 hours. The geometric mean of the absolute pulmonary bioavailability of AZD8154 via the inhaled route was 94.1%. CONCLUSION: AZD8154 demonstrated an acceptable safety profile, with no reports of serious adverse events and no clinically significant drug-associated safety concerns reported in healthy volunteers. AZD8154 demonstrated prolonged lung retention and a half-life supporting once-daily dosing.

Discovery of AZD8154, a Dual PI3Kγδ Inhibitor for the Treatment of Asthma.

Starting from our previously described PI3Kγ inhibitors, we describe the exploration of structure-activity relationships that led to the discovery of highly potent dual PI3Kγδ inhibitors. We explored changes in two positions of the molecules, including macrocyclization, but ultimately identified a simpler series with the desired potency profile that had suitable physicochemical properties for inhalation. We were able to demonstrate efficacy in a rat ovalbumin challenge model of allergic asthma and in cells derived from asthmatic patients. The optimized compound, AZD8154, has a long duration of action in the lung and low systemic exposure coupled with high selectivity against off-targets.

Model for the Analysis of Membrane-Type Dissolution Tests for Inhaled Drugs.

Impactor-type dose deposition is a common prerequisite for dissolution testing of inhaled medicines, and drug release typically takes place through a membrane. The purpose of this work is to develop a mechanistic model for such combined dissolution and release processes, focusing on a drug that initially is present in solid form. Our starting points are the Noyes-Whitney (or Nernst-Brunner) equation and Fick's law. A detailed mechanistic analysis of the drug release process is provided, and approximate closed-form expressions for the amount of the drug that remains in solid form and the amount of the drug that has been released are derived. Comparisons with numerical data demonstrated the accuracy of the approximate expressions. Comparisons with experimental release data from literature demonstrated that the model can be used to establish rate-controlling release mechanisms. In conclusion, the model constitutes a valuable tool for the analysis of in vitro dissolution data for inhaled drugs.

Ranking in Vitro Dissolution of Inhaled Micronized Drug Powders including a Candidate Drug with Two Different Particle Sizes.

Pulmonary dissolution of poorly soluble drug substances (DSs) may limit the drug absorption rate and consequently influence clinical performance. Dissolution rate is thus an important quality attribute, and its influence on in vivo drug release must be characterized, understood, and controlled early in the development process. The aim of this study is to establish an in vitro dissolution method with the capability to capture therapeutically relevant differences in the dissolution rate between drug batches and drug compounds. A method was developed by which a biorelevant aerosol fraction was captured on a filter using a sedimentation technique in a modified Andersen cascade impactor to avoid particle agglomeration. Subsequently, the filters were transferred to a commercial Transwell system where dissolution in 3 mL of phosphate buffer at pH 6.8 with 0.5% sodium dodecyl sulfate (SDS) occurred at sink conditions. Dissolved DS was quantified over time using UPLC-UV. Dissolution data was obtained on a series of micronized and aerosolized lipophilic DSs, budesonide, fluticasone furoate (FF), fluticasone propionate (FP), and AZD5423. The latter is a lipophilic AstraZeneca development compound available in two different mass median diameters (MMD), 1.3 (AZD54231.3) and 3.1 µm (AZD54233.1). Dissolution data were evaluated using a Weibull fit and expressed as t63, the time to dissolution of 63% of the initial dose. The following rank-order of t63 was obtained (mean t63 and MMD in brackets), budesonide (10 min, 2.1 µm) = AZD54231.3 (10 min, 1.3 µm) < AZD54233.1 (19 min, 3.1 µm) < FP (38 min, 2.4 µm) < FF (63 min, 2.5 µm). The method could differentiate between different drug compounds with different solubility but similar particle size distribution, as well as between the same drug compound with different particle size distributions. Furthermore, a relation between the in vitro dissolution rate ( t63) and mean pulmonary absorption time in man (literature data) was observed, indicating clinical relevance. It is thus concluded, that the method may be useful for the characterization and ranking of DSs and drug products in early development, as well as being a potential tool for the control of dissolution as a potential quality attribute.

Bile salt enhancers for inhalation: Correlation between in vitro and in vivo lung effects.

The lungs have potential as a means of systemic drug delivery of macromolecules. Systemic delivery requires crossing of the air-blood barrier, however with molecular size-dependent limitations in lung absorption of large molecules. Systemic availability after inhalation can be improved by absorption enhancers, such as bile salts. Enhancers may potentially interfere with the different constituents of the lungs, e.g. the lung surfactant lining the alveoli or the lung epithelium. We used two in vitro models to investigate the potential effects of bile salts on lung surfactant function (with the constrained drop surfactometer) and on the epithelium in the proximal airways (with the MucilAir™ cell system), respectively. In addition, we measured direct effects on respiration in mice inhaling bile salt aerosols. The bile salts inhibited lung surfactant function at different dose levels, however they did not affect the integrity of ciliated cells at the tested doses. Furthermore, the bile salt aerosols induced changes in the breathing pattern of mice indicative of pulmonary irritation. The bile salts were ranked according to potency in vitro for surfactant function disruption and in vivo for induction of pulmonary irritation. The ranking was the same, suggesting a correlation between the interference with lung surfactant and the respiratory response.

An imidazole based H-Phe-Phe-NH2 peptidomimetic with anti-allodynic effect in spared nerve injury mice.

The dipeptide amide H-Phe-Phe-NH2 (1) that previously was identified as a ligand for the substance P 1-7 (SP1-7) binding site exerts intriguing results in animal models of neuropathic pain after central but not after peripheral administration. The dipeptide 1 is derived from stepwise modifications of the anti-nociceptive heptapeptide SP1-7 and the tetrapeptide endomorphin-2 that is also binding to the SP1-7 site. We herein report a strong anti-allodynic effect of a new H-Phe-Phe-NH2 peptidomimetic (4) comprising an imidazole ring as a bioisosteric element, in the spare nerve injury (SNI) mice model after peripheral administration. Peptidomimetic 4 was stable in plasma, displayed a fair membrane permeability and a favorable neurotoxic profile. Moreover, the effective dose (ED50) of 4 was superior as compared to gabapentin and morphine that are used in clinic.

Impact of N-methylation of the substance P 1-7 amide on anti-allodynic effect in mice after peripheral administration.

Substance P 1-7 (SP1-7, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7) is the major bioactive metabolite formed after proteolytic degradation of the tachykinin substance P (SP). This heptapeptide often opposes the effects of the mother peptide. Hence, SP1-7 is having anti-inflammatory, anti-nociceptive and anti-hyperalgesic effects in experimental models. Despite all encouraging properties of SP1-7 its exact mode of action has not yet been elucidated which has hampered further development of this heptapeptide in drug discovery. Contrary to SP that mediates its biological activity via the NK-1 receptor, the N-terminal fragment SP1-7 acts through an unknown target that is distinct from all known opioid and tachykinin receptors. The SP1-7 amide 1 (Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-NH2) was previously shown to be superior to the endogenous SP1-7 in all experimental pain models where the two compounds were compared. Herein, we report that N-methylation scan of the backbone of the SP1-7 amide (1) results in peptides that are significantly less prone to undergo proteolysis in plasma from both mouse and human. However, with the two exceptions of the [MeLys3]SP1-7 amide (3) and the [MeGln5]SP1-7 amide (4), the peptides with a methyl group attached to the backbone are devoid of significant anti-allodynic effects after peripheral administration in the spared nerve injury (SNI) mouse model of neuropathic pain. It is suggested that the N-methylation does not allow these peptides to form the accurate bioactive conformations or interactions required for efficient binding to the macromolecular target. The importance of intact N-terminal Arg1 and C-terminal Phe7, anticipated to serve as address and message residues, respectively, for achieving the anti-allodynic effect is emphasized. Notably, the three heptapeptides: the SP1-7 amide (1), the [MeLys3]SP1-7 amide (3) amide and the [MeGln5]SP1-7 amide (4) are all considerably more effective in the SNI mouse model than gabapentin that is widely used in the clinic for treatment of neuropathic pain.

Importance of N- and C-terminal residues of substance P 1-7 for alleviating allodynia in mice after peripheral administration.

The heptapeptide SP1-7 (1, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7) is the major bioactive metabolite formed after proteolytic processing of the neuropeptide substance P (SP, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-Phe8-Gly9-Leu10-Met11-NH2). The heptapeptide 1 frequently exhibits opposite effects to those induced by SP, such as exerting antinociception, or attenuating thermal hyperalgesia and mechanical allodynia. The heptapeptide SP1-7 amide (2, Arg1-Pro2-Lys3-Pro4-Gln5-Gln6-Phe7-NH2) is often more efficacious than 1 in experimental pain models. We have now assessed the anti-allodynic outcome after systemic administration of 2 and a series of Ala-substituted and truncated analogues of 2, in the spared nerve injury (SNI) mice model and the results obtained were correlated with in vitro plasma stability and permeability measurements. It is herein demonstrated that an intact Arg1 in SP1-7 amide analogues is fundamental for retaining a potent in vivo effect, while Lys3 of 2 is less important. A displacement with Ala1 or truncation rendered the peptide analogues either inactive or with a significantly attenuated in vivo activity. Thus, the pentapeptide SP3-7 amide (7, t1/2=11.1 min) proven to be the major metabolite of 2, demonstrated an in vivo effect itself although considerably less significant than 2 in the SNI model. Intraperitoneal administration of 2 in a low dose furnished the most powerful anti-allodynic effect in the SNI model of all the analogous evaluated, despite a fast proteolysis of 2 in plasma (t1/2=6.4 min). It is concluded that not only the C-terminal residue, that we previously demonstrated, but also the N-terminal with its basic side chain, are important for achieving effective pain relief. This information is of value for the further design process aimed at identifying more drug-like SP1-7 amide related peptidomimetics with pronounced anti-allodynic effects.

Azacyclic FTY720 Analogues That Limit Nutrient Transporter Expression but Lack S1P Receptor Activity and Negative Chronotropic Effects Offer a Novel and Effective Strategy to Kill Cancer Cells in Vivo.

FTY720 sequesters lymphocytes in secondary lymphoid organs through effects on sphingosine-1-phosphate (S1P) receptors. However, at higher doses than are required for immunosuppression, FTY720 also functions as an anticancer agent in multiple animal models. Our published work indicates that the anticancer effects of FTY720 do not depend on actions at S1P receptors but instead stem from FTY720s ability to restrict access to extracellular nutrients by down-regulating nutrient transporter proteins. This result was significant because S1P receptor activation is responsible for FTY720s dose-limiting toxicity, bradycardia, that prevents its use in cancer patients. Here, we describe diastereomeric and enantiomeric 3- and 4-C-aryl 2-hydroxymethyl pyrrolidines that are more active than the previously known analogues. Of importance is that these compounds fail to activate S1P1 or S1P3 receptors in vivo but retain inhibitory effects on nutrient transporter proteins and anticancer activity in solid tumor xenograft models. Our studies reaffirm that the anticancer activity of FTY720 does not depend upon S1P receptor activation and uphold the promise of using S1P receptor-inactive azacyclic FTY720 analogues in human cancer patients.

Exploration and pharmacokinetic profiling of phenylalanine based carbamates as novel substance p 1-7 analogues.

The bioactive metabolite of Substance P, the heptapeptide SP1-7 (H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH), has been shown to attenuate signs of hyperalgesia in diabetic mice, which indicate a possible use of compounds targeting the SP1-7 binding site as analgesics for neuropathic pain. Aiming at the development of drug-like SP1-7 peptidomimetics we have previously reported on the discovery of H-Phe-Phe-NH2 as a high affinity lead compound. Unfortunately, the pharmacophore of this compound was accompanied by a poor pharmacokinetic (PK) profile. Herein, further lead optimization of H-Phe-Phe-NH2 by substituting the N-terminal phenylalanine for a benzylcarbamate group giving a new type of SP1-7 analogues with good binding affinities is reported. Extensive in vitro as well as in vivo PK characterization is presented for this compound. Evaluation of different C-terminal functional groups, i.e., hydroxamic acid, acyl sulfonamide, acyl cyanamide, acyl hydrazine, and oxadiazole, suggested hydroxamic acid as a bioisosteric replacement for the original primary amide.

N-terminal truncations of substance P 1-7 amide affect its action on spinal cord injury-induced mechanical allodynia in rats.

Central neuropathic pain can arise from injury of the spinal cord and can become chronic. Treatment is difficult and, because complete pain relief is currently very hard to achieve, there is a need for new, more effective treatment options. In this study we used an animal model of spinal cord injury to evaluate the potency of a bioactive fragment of substance P (SP), i.e. SP1-7, in alleviating signs of allodynia and acute pain. SP1-7 is known from earlier studies to possess antinociceptive properties. We also studied the effects of intraperitoneal injection of an amidated analog of this heptapeptide and of its truncated analogs, all of which had high affinity to the SP1-7 binding site, to evaluate the importance of the removed amino acids for the biodistribution and stability of the peptides. Most of the examined compounds alleviated mechanical allodynia without any signs of sedation or motor impairment in the rats. In contrast, the response threshold to acute nociceptive stimulation was not affected by any of the compounds tested. Most of the amino acids in the heptapeptide structure were essential for retaining the biological effect after peripheral injection. These observations suggest that the heptapeptide and its N-terminal truncated hexa- and pentapeptide analogs could be of interest for further development of analgesics in the management of mechanical allodynia.

Design, Synthesis, and Anti-leukemic Activity of Stereochemically Defined Constrained Analogs of FTY720 (Gilenya).

FTY720 functions as an immunosuppressant due to its effect on sphingosine-1-phosphate receptors. At doses well above those needed for immunosuppression, FTY720 also has anti-neoplastic actions. Our published work suggests that at least some of FTY720's anti-cancer activity is independent of its effects on S1P receptors and due instead to its ability to induce nutrient transporter down-regulation. Compounds that trigger nutrient transporter loss but lack FTY720's S1P receptor-related, dose-limiting toxicity have the potential to be effective and selective anti-tumor agents. In this study, a series of enantiomerically pure and stereochemically diverse O-substituted benzyl ethers of pyrrolidines was generated and tested for the ability to kill human leukemia cells. The stereochemistry of the hydroxymethyl was found to be a key determinant of compound activity. Moreover, phosphorylation of this group was not required for anti-leukemic activity.

Aminocarbonylation of 4-iodo-1H-imidazoles with an amino acid amide nucleophile: synthesis of constrained H-Phe-Phe-NH2 analogues.

A simple and an expedient process to prepare 5-aryl-1-benzyl-1H-imidazole-4-carboxamides by the aminocarbonylation of 5-aryl-4-iodo-1H-imidazoles using ex situ generation of CO from Mo(CO)6 with an amino acid amide nucleophile is reported. Furthermore, a microwave-assisted protocol for the direct C-5 arylation of 1-benzyl-1H-imidazole and a regioselective C-4 iodination method to acquire starting material for our aminocarbonylation are presented. The method can be used to prepare imidazole based peptidomimetics, herein exemplified by the synthesis of constrained H-Phe-Phe-NH2 analogues.

Constrained H-Phe-Phe-NH2 analogues with high affinity to the substance P 1-7 binding site and with improved metabolic stability and cell permeability.

We recently reported the discovery of H-Phe-Phe-NH2 as a small and high affinity ligand for the substance P 1-7 (SP(1-7), H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) specific binding site and its intriguing ability to reduce neuropathic pain. With the overall aim to develop stable and orally bioavailable SP(1-7) mimetics, the dipeptide was chosen as a lead compound. Herein the structure-activity relationship (SAR) of a set of modified H-Phe-Phe-NH2 analogues is presented together with their potential active uptake by PEPT1 transporter, intestinal permeability, and metabolic stability. Local constraints via peptide backbone methylation or preparation of cyclized analogues based on pyrrolidine were evaluated and were shown to significantly improve the in vitro pharmacokinetic properties. The SAR was rationalized by deriving a plausible binding pose for the high affinity ligands. Rigidification using a 3-phenylpyrrolidine moiety in the C-terminal of H-Phe-Phe-NH2 resulted in high affinity and improved intrinsic clearance and intestinal epithelial permeability.

The dipeptide Phe-Phe amide attenuates signs of hyperalgesia, allodynia and nociception in diabetic mice using a mechanism involving the sigma receptor system.

BACKGROUND: Previous studies have demonstrated that intrathecal administration of the substance P amino-terminal metabolite substance P1-7 (SP1-7) and its C-terminal amidated congener induced antihyperalgesic effects in diabetic mice. In this study, we studied a small synthetic dipeptide related to SP1-7 and endomorphin-2, i.e. Phe-Phe amide, using the tail-flick test and von Frey filament test in diabetic and non-diabetic mice. RESULTS: Intrathecal treatment with the dipeptide increased the tail-flick latency in both diabetic and non-diabetic mice. This effect of Phe-Phe amide was significantly greater in diabetic mice than non-diabetic mice. The Phe-Phe amide-induced antinociceptive effect in both diabetic and non-diabetic mice was reversed by the σ1 receptor agonist (+)-pentazocine. Moreover, Phe-Phe amide attenuated mechanical allodynia in diabetic mice, which was reversible by (+)-pentazocine. The expression of spinal σ1 receptor mRNA and protein did not differ between diabetic mice and non-diabetic mice. On the other hand, the expression of phosphorylated extracellular signal-regulated protein kinase 1 (ERK1) and ERK2 proteins was enhanced in diabetic mice. (+)-Pentazocine caused phosphorylation of ERK1 and ERK2 proteins in non-diabetic mice, but not in diabetic mice. CONCLUSIONS: These results suggest that the spinal σ1 receptor system might contribute to diabetic mechanical allodynia and thermal hyperalgesia, which could be potently attenuated by Phe-Phe amide.

Microwave-assisted synthesis of Weinreb and MAP aryl amides via Pd-catalyzed Heck aminocarbonylation using Mo(CO)6 or W(CO)6.

A simple and expedient process for the Heck aminocarbonylative synthesis of Weinreb and MAP amide acylating agents, from aryl halides, is reported. This methodology utilizes solid sources of CO making it readily accessible to chemists working in small-scale laboratory applications.

The effect of substance P1-7 amide on nociceptive threshold in diabetic mice.

We previously demonstrated that intrathecal treatment with substance P metabolite substance P(1-7) induced anti-hyperalgesia in diabetic mice. In the present study, we have used a synthetic analog of this peptide, the substance P(1-7) amide, showing higher binding affinity than the native heptapeptide, for studies of the tail-flick response in diabetic and non-diabetic mice. Intrathecal injection of substance P(1-7) amide produced prolongation of the tail-flick latency in both diabetic and non-diabetic mice, an effect that was more pronounced in diabetic mice than non-diabetic mice. Moreover, the observed antinociceptive potency of the substance P(1-7) amide was higher in both diabetic and non-diabetic mice in comparison with the native substance P(1-7). The antinociceptive effect of substance P(1-7) amide was reversed by naloxone but not by the selective opioid receptor antagonist ß-funaltrexamine, naltrindole or nor-binaltorphimine, selective for the µ-, δ- or κ-opioid receptor, respectively. In addition, the antinociceptive effect induced by substance P(1-7) amide was partly reversed by the σ(1) receptor agonist (+)-pentazocine, suggesting a possible involvement of the σ(1) receptor for the action of this peptide. These results suggest that the actions of substance P(1-7) amide mimic the effects of the native substance P fragment but with higher potency and that the mechanisms for its action may involve the σ(1) receptor system.

Discovery of dipeptides with high affinity to the specific binding site for substance P1-7.

Substance P 1-7 (SP(1-7), H-Arg-Pro-Lys-Pro-Gln-Gln-Phe-OH) is the major bioactive metabolite of substance P. The interest in this heptapeptide originates from the observation that it modulates, and in certain cases opposes the effects of the parent peptide, e.g., the nociceptive effect. The mu-opioid receptor agonist endomorphin-2 (EM-2, H-Tyr-Pro-Phe-Phe-NH(2)) has been found to also interact with the specific binding site of SP(1-7) with only a 10-fold lower affinity compared to the native peptide. Considering the smaller size of EM-2 compared to the target heptapeptide, it was selected as a lead compound in the development of low-molecular-weight ligands to the SP(1-7) binding site. An alanine scan and truncation study led to the unexpected discovery of the dipeptide H-Phe-Phe-NH(2) (K(i) = 1.5 nM), having equal affinity as the endogenous heptapeptide SP(1-7.) Moreover, the studies show that the C-terminal phenylalanine amide is crucial for the affinity of the dipeptide.

The C-terminal amidated analogue of the substance P (SP) fragment SP(1-7) attenuates the expression of naloxone-precipitated withdrawal in morphine dependent rats.

We previously demonstrated that intracerebroventricular (i.c.v.) administration of the substance P (SP) aminoterminal fragment SP(1-7) attenuates the expression of morphine withdrawal in the male rat. In this study we have used a synthetic analogue of this peptide, i.e. the SP(1-7) amide showing higher binding potency than the native heptapeptide, in a similar experimental set-up. Thus, Wistar male rats were made tolerant to morphine by daily injections of the opiate during 8 days. Following peptide administration (i.c.v.) and a subsequent naloxone challenge a variety of physical syndromes of withdrawal were recorded. We observed that the SP(1-7) amide potently and dose-dependently reduced several signs of reaction to morphine withdrawal. Interestingly, the effect of the peptide amide was significantly attenuated by the addition of the sigma agonist (+)-SKF-10047. We conclude that the SP(1-7) amide mimics the effect of the native SP fragment and that the mechanisms for its action involve a sigma receptor site.