Metabolism and Excretion of Therapeutic Peptides: Current Industry Practices, Perspectives, and Recommendations.

Therapeutic peptides (TPeps) have expanded from the initial endogenous peptides to complex modified peptides through medicinal chemistry efforts for almost a century. Different from small molecules and large proteins, the diverse submodalities of TPeps have distinct structures and carry different absorption, distribution, metabolism, and excretion (ADME) properties. There is no distinct regulatory guidance for the industry on conducting ADME studies (what, how, and when) for TPeps. Therefore, the Peptide ADME Working Group sponsored by the Translational and ADME Sciences Leadership Group of the International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) was formed with the goal to develop a white paper focusing on metabolism and excretion studies to support discovery and development of TPeps. In this paper, the key learnings from an IQ industry survey and U.S. Food and Drug Administration/European Medicines Agency submission documents of TPeps approved between 2011 and 2022 are outlined in detail. In addition, a comprehensive assessment of in vitro and in vivo metabolism and excretion studies, mitigation strategies for TPep metabolism, analytical tools to conduct studies, regulatory status, and Metabolites in Safety Testing considerations are provided. Finally, an industry recommendation on conducting metabolism and excretion studies is proposed for regulatory filing of TPeps. SIGNIFICANCE STATEMENT: This white paper presents current industry practices for metabolism and excretion studies of therapeutic peptides based on an industry survey, regulatory submission documents, and expert opinions from the participants in the Peptide Absorption, Distribution, Metabolism, and Excretion Working Group of the International Consortium for Innovation and Quality in Pharmaceutical Development. The group also provides recommendations on the Metabolites in Safety Testing considerations and metabolism and excretion studies for regulatory filing of therapeutic peptides.

Extraction, purification, bioactivity and pharmacological effects of capsaicin: a review.

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a well-known vanilloid, which is the main spicy component in chili peppers, showing several biological activities and the potential applications range from food flavorings to therapeutics. Traditional extraction of capsaicin by organic solvents was time-consuming, some new methods such as aqueous two-phase method and ionic liquid extraction method have been developed. During past few decades, an ample variety of biological effects of capsaicin have been evaluated. Capsaicin can be used in biofilms and antifouling coatings due to its antimicrobial activity, allowing it has a promising application in food packaging, food preservation, marine environment and dental therapy. Capsaicin also play a crucial role in metabolic disorders, including weight loss, pressure lowing and insulin reduction effects. In addition, capsaicin was identified effective on preventing human cancers, such as lung cancer, stomach cancer, colon cancer and breast cancer by inducing apoptosis and inhibiting cell proliferation of tumor cells. Previous research also suggest the positive effects of capsaicin on pain relief and cognitive impairment. Capsaicin, the agonist of transient receptor potential vanilloid type 1 (TRPV1), could selectively activate TRPV1, inducing Ca2+ influx and related signaling pathways. Recently, gut microbiota was also involved in some diseases therapeutics, but its influence on the effects of capsaicin still need to be deeply studied. In this review, different extraction and purification methods of capsaicin, its biological activities and pharmacological effects were systematically summarized, as well as the possible mechanisms were also deeply discussed. This article will give an updated and better understanding of capsaicin-related biological effects and provide theoretical basis for its further research and applications in human health and manufacture development.

Anomalous Capillary Rise under Nanoconfinement: A View of Molecular Kinetic Theory.

Understanding the capillary filling behaviors in nanopores is crucial for many science and engineering problems. Compared with the classical Bell-Cameron-Lucas-Washburn (BCLW) theory, anomalous coefficient is always observed because of the increasing role of surfaces. Here, a molecular kinetics approach is adopted to explain the mechanism of anomalous behaviors at the molecular level; a unified model taking account of the confined liquid properties (viscosity and density) and slip boundary condition is proposed to demonstrate the macroscopic consequences, and the model results are successfully validated against the published literature. The results show that (1) the effective viscosity induced by the interaction from the pore wall, as a function of wettability and the pore dimension (nanoslit height or nanotube diameter), may remarkably slow down the capillary filling process more than theoretically predicted. (2) The true slip, where water molecules directly slide on the walls, strongly depends on the wettability and will increase as the contact angle increases. In the hydrophilic nanopores, though, the magnitude may be comparable with the pore dimensions and promote the capillary filling compared with the classical BCLW model. (3) Compared with the other model, the proposed model can successfully predict the capillary filling for both faster or slower capillary filling process; meanwhile, it can capture the underlying physics behind these behaviors at the molecular level based on the effective viscosity and slippage. (4) The surface effects have different influence on the capillary filling in nanoslits and nanotubes, and the relative magnitude will change with the variation of wettability as well as the pore dimension.

Retrograde laparoscopic resection of left side of the liver: a safe and effective way.

OBJECTIVE: The safety and feasibility of retrograde laparoscopic resection of the left side of the liver. METHODS: Ninety-three laparoscopic left hepatic lobe cases were selected between August 2010 and August 2014 from our institution. A retrospective cohort study was performed between the antegrade partial hepatectomy group (47 cases; dissection from the first porta hepatis to the second) and the retrograde partial hepatectomy group (46 cases; dissection from the second porta hepatis to the first), to compare the length of time needed for resection, the amount of bleeding, post-operative time in the hospital, and the incidence of major complications, such as bile leakage, abdominal abscess, and post-hepatectomy hemorrhage. RESULTS: All of the cases had a successful laparoscopic partial hepatectomy without the need for an intraoperative blood transfusion. Patients were able to ambulate on post-operative day 1 and tolerated a liquid diet on post-operative day 1 or 2. There were no statistical differences of post-operative hospital length of stay or incidence of major complications between the two groups. Both duration of resection and the amount of bleeding were less in the retrograde group than of those in the antegrade group, due to the lower incidence of hepatic vein injury in the retrograde group. CONCLUSION: Occlusion of both the inflow and outflow hepatic vessels combined with retrograde hepatectomy from the second porta hepatis to the first, demonstrated less hemorrhage and lower incidence of hepatic veins injury during laparoscopic partial hepatectomy.

Metabolic capabilities of cytochrome P450 enzymes in Chinese liver microsomes compared with those in Caucasian liver microsomes.

AIM: The most common causes of variability in drug response include differences in drug metabolism, especially when the hepatic cytochrome P450 (CYP) enzymes are involved. The current study was conducted to assess the differences in CYP activities in human liver microsomes (HLM) of Chinese or Caucasian origin. METHODS: The metabolic capabilities of CYP enzymes in 30 Chinese liver microsomal samples were compared with those of 30 Caucasian samples utilizing enzyme kinetics. Phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion hydroxylation, amodiaquine N-desethylation, diclofenac 4'-hydroxylation (S)-mephenytoin 4'-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and midazolam 1'-hydroxylation/testosterone 6ß-hydroxylation were used as probes for activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Mann-Whitney U test was used to assess the differences. RESULTS: The samples of the two ethnic groups were not significantly different in cytochrome-b(5) concentrations but were significantly different in total CYP concentrations and NADPH-P450 reductase activity (P < 0.05). Significant ethnic differences in intrinsic clearance were observed for CYP1A2, CYP2C9, CYP2C19 and CYP2E1; the median values of the Chinese group were 54, 58, 26, and 35% of the corresponding values of the Caucasian group, respectively. These differences were associated with differences in Michaelis constant or maximum velocity. Despite negligible difference in intrinsic clearance, the Michaelis constant of CYP2B6 appeared to have a significant ethnic difference. No ethnic difference was observed for CYP2A6, CYP2C8, CYP2D6 and CYP3A. CONCLUSIONS: These data extend our knowledge on the ethnic differences in CYP enzymes and will have implications for drug discovery and drug therapy for patients from different ethnic origins.

The fate of 4-hydroxycarbazole metabolite: metabolism and carcinogenicity assessment of a ß-adrenergic receptor modulator containing carbazole structure.

LY377604 has a potential to form 4-hydroxycarbazole, which was reported in the literature as a mutagen. This safety concern led to our investigation of the metabolism and carcinogenicity of LY377604. In in vitro studies with LY377604, 4-hydroxycarbazole was detected in the presence of liver microsomes prepared from different species. When incubated with liver slices, only the conjugate of 4-hydroxycarbazole was detected. Subsequent in vivo radio-labelled studies were conducted to characterise the formation of 4-hydroxycarbazole from LY377604. Free 4-hydroxycarbazole was not detected in vivo, but the O-glucuronide conjugate was identified as a minor metabolite in urine samples, representing 0.2% and 0.9% of the radioactive dose in rats and monkeys. The low level of circulating 4-hydroxycarbazole glucuronide conjugate was also detected in plasma. LY377604 was negative in all genetic toxicology assays and was not associated with tumour induction in a 6-month carcinogenicity study using RasH2+/- mouse model. The exposure to free 4-hydroxycarbazole was not measurable after one dose and was about 0.1%-0.2% of the parent exposure at the end of the 6-month study. These data suggested that 4-hydroxycarbazole was formed as a minor metabolite in vivo, but it was primarily conjugated and excreted in urine as the glucuronide conjugate. The absence of tumours in the carcinogenicity study combined with the exposure data suggested that the low level of free 4-hydroxycarbazole did not represent a carcinogenic risk.

[Heavy Metal Ion Adsorption Properties and Stability of Amine-sulfur Modified Biochar in Aqueous Solution].

A novel biochar was prepared by modification with corn straw, ethylene triamine, and carbon disulfide, and its adsorption properties and stability with respect to heavy metal ions in single and mixed systems (Pb2+, Ni2+, and Cd2+) were investigated. Characterization analysis confirmed the successful modification of an amine-sulfur double group on the surface of the biochar, which had abundant functional groups with a large specific surface area. Adsorption experiments under the single system indicated that the adsorption equilibrium time was 4 h and the optimum dosages were 1, 0.8, and 1.2 g·L-1. The adsorption met the conditions of the quasi-second-order kinetic equation. Under the ternary system, the adsorption equilibrium time was reduced to 1.5 h, the optimum dosages were 0.4, 1.6, and 0.8 g·L-1, and the adsorption sequence was Pb2+ > Cd2+ > Ni2+. The total amount of adsorption was 0.67 mmol·g-1, which was higher than that of single heavy metal ions, indicating that amine-sulfur modified straw biochar (BC-SN) has an improved treatment effect on polluted water under the coexistence of three heavy metal ions. The Pb2+ and Cd2+ adsorbed by the biochar was stably bound in the form of heavy metal sulfide and a chelated amino group. In contrast, the adsorption of Ni2+ was via the mixed adsorption of various functional groups. When Pb2+ and Cd2+ compete for adsorption, the binding energy is higher and adsorption stability is more reliable.

Effect of Displacement Pressure on Oil-Water Relative Permeability for Extra-Low-Permeability Reservoirs.

The oil-water relative permeability is an important parameter to characterize the seepage law of fluid in extra-low-permeability reservoirs, and it is of vital significance for the prediction and evaluation of the production. The pore throat size of extra-low-permeability reservoirs is relatively small, and the threshold pressure gradient and capillary pressure cannot be negligible. In this study, the oil-water relative permeability experiments with three different displacement pressures were carried out on the same core from the extra-low-permeability reservoir of Chang 4+5 formation in Ordos basin by the unsteady experimental method. The results show that the relative permeability of oil increases, while the relative permeability of water remains unchanged considering the capillary pressure and oil threshold pressure gradient compared with the JBN method. As the displacement pressure enlarges, the relative permeability of oil and water both increases; the residual oil saturation decreases, therefore the range of the two-phase flow zone is improved. Moreover, the isotonic point of water-oil relative permeability curves moves to the upper right region, and the reference permeability improves as well with the increasing pressure.

Identification, synthesis and pharmacological activity of moxonidine metabolites.

The metabolism of moxonidine, 4-chloro-N-(4,5-dihydro-1H-imidazol-2-yl)-6-methoxy-2-methyl-5-pyrimidinamine, LY326869, in rats, mice, dogs, and humans has been examined. At least 17 metabolites were identified or tentatively identified in the different species by HPLC, LC/MS and LC/MS/MS. The identities of seven of the major metabolites have been verified by independent synthesis. The metabolites are generally derived from oxidation and conjugation pathways. Oxidation occurred at the imidazolidine ring as well as the methyl at the 2 position of the pyrimidine ring. All seven metabolites were examined in the spontaneously hypertensive rats (3 mg kg(-1), i.v.) for pressure and heart rate. Only one, 2-hydroxymethyl-4-chloro-5-(imidazolidin-2-ylidenimino)-6-methoxypyrimidine, exerted a short-lasting decrease in blood pressure, albeit attenuated in magnitude compared to moxonidine.

Investigation of clinical pharmacokinetic variability of an opioid antagonist through physiologically based absorption modeling.

Identifying the source of inter- and/or intrasubject variability in pharmacokinetics (PK) provides fundamental information in understanding the pharmacokinetics-pharmacodynamics relationship of a drug and project its efficacy and safety in clinical populations. This identification process can be challenging given that a large number of potential causes could lead to PK variability. Here we present an integrated approach of physiologically based absorption modeling to investigate the root cause of unexpectedly high PK variability of a Phase I clinical trial drug. LY2196044 exhibited high intersubject variability in the absorption phase of plasma concentration-time profiles in humans. This could not be explained by in vitro measurements of drug properties and excellent bioavailability with low variability observed in preclinical species. GastroPlus™ modeling suggested that the compound's optimal solubility and permeability characteristics would enable rapid and complete absorption in preclinical species and in humans. However, simulations of human plasma concentration-time profiles indicated that despite sufficient solubility and rapid dissolution of LY2196044 in humans, permeability and/or transit in the gastrointestinal (GI) tract may have been negatively affected. It was concluded that clinical PK variability was potentially due to the drug's antagonism on opioid receptors that affected its transit and absorption in the GI tract.

Combination of a Beta adrenoceptor modulator and a norepinephrine-serotonin uptake inhibitor for the treatment of obesity.

We report the novel combination of a selective beta adrenoceptor modulator and a norepinephrine-serotonin uptake inhibitor (sibutramine) with potential for the treatment of obesity. The synthesis and characterization of 6-[4-[2-[[(2S)-3-(9H-carbazol-4-yloxy)-2-hydroxypropyl]amino]-2-methylpropyl]phenoxy]pyridine-3-carboxamide (LY377604), a human ß3-adrenergic receptor agonist and ß1- and ß2-adrenergic receptor antagonist with no sympathomimetic activity at the ß1- and ß2-adrenergic receptors, is reported. Some in vivo data in both rats and humans is presented.

Comparative metabolic capabilities and inhibitory profiles of CYP2D6.1, CYP2D6.10, and CYP2D6.17.

Polymorphisms in the cytochrome P450 2D6 (CYP2D6) gene are a major cause of pharmacokinetic variability in human. Although the poor metabolizer phenotype is known to be caused by two null alleles leading to absence of functional CYP2D6 protein, the large variability among individuals with functional alleles remains mostly unexplained. Thus, the goal of this study was to examine the intrinsic enzymatic differences that exist among the several active CYP2D6 allelic variants. The relative catalytic activities (enzyme kinetics) of three functionally active human CYP2D6 allelic variants, CYP2D6.1, CYP2D6.10, and CYP2D6.17, were systematically investigated for their ability to metabolize a structurally diverse set of clinically important CYP2D6-metabolized drugs [atomoxetine, bufuralol, codeine, debrisoquine, dextromethorphan, (S)-fluoxetine, nortriptyline, and tramadol] and the effects of various CYP2D6-inhibitors [cocaine, (S)-fluoxetine, (S)-norfluoxetine, imipramine, quinidine, and thioridazine] on these three variants. The most significant difference observed was a consistent but substrate-dependent decease in the catalytic efficiencies of cDNA-expressed CYP2D6.10 and CYP2D6.17 compared with CYP2D6.1, yielding 1.32 to 27.9 and 7.33 to 80.4% of the efficiency of CYP2D6.1, respectively. The most important finding from this study is that there are mixed effects on the functionally reduced allelic variants in enzyme-substrate affinity or enzyme-inhibitor affinity, which is lower, higher, or comparable to that for CYP2D6.1. Considering the rather high frequencies of CYP2D6*10 and CYP2D6*17 alleles for Asians and African Americans, respectively, these data provide further insight into ethnic differences in CYP2D6-mediated drug metabolism. However, as with all in vitro to in vivo extrapolations, caution should be applied to the clinical consequences.

Clinical pharmacokinetics of alamifovir and its metabolites.

Alamifovir, a purine nucleotide analogue prodrug, and its hydrolyzed derivatives have shown preclinical efficacy activity against wild-type and lamivudine-resistant hepatitis B virus. Two studies were conducted to examine the single- and multiple-dose alamifovir pharmacokinetics after oral administration in healthy males. In study 1, subjects were given single doses (0.2 to 80 mg), with a subset receiving 20 mg in a fed state. Study 2 subjects were dosed with 2.5 to 15 mg twice daily for 15 days. Plasma samples were collected over 72 h in study 1 and over 24 h on days 1 and 15 in study 2. Concentrations of alamifovir and its major metabolites were determined using liquid chromatography/tandem mass spectrometry methods. The data were analyzed using a noncompartmental technique. Although alamifovir was rapidly absorbed, there was limited systemic exposure due to its rapid hydrolysis and formation of at least three metabolites, suggesting that alamifovir acts as a prodrug. The major metabolites detected were 602074 and 602076, with 602075 detectable only in higher-dose groups. Maximum 602074 plasma concentration was achieved at approximately 0.5 h (T(max)) and declined with a 1- to 2-h terminal half-life (t(1/2)). Maximum concentrations of 602076 (C(max)) averaged 10% of the 602074 C(max) and reached T(max) in 2.5 h with a 4-h t(1/2). Food appeared to decrease the extent of absorption of the compound. Multiple dosing resulted in minimal accumulation, and the concentrations following multiple doses could be predicted using the single-dose data. Alamifovir was well tolerated and the pharmacokinetics were characterized in these studies.

Quinidine does not affect the renal clearance of moxonidine.

AIMS: To test the hypothesis that the renal clearance of moxonidine decreases when dosed with quinidine. METHODS: A randomized, two-period study was conducted with six healthy, male subjects orally dosed with either 0.2 mg moxonidine alone or 1 h after 400 mg quinidine sulphate. Pharmacokinetic parameters were calculated using a noncompartmental analysis method. RESULTS: When coadministered, quinidine significantly increased moxonidine AUC and t1/2 by 11% and 15%, respectively, and decreased CL/F by 10% compared with the control dosing. CLR and Aeur were not significantly different. Clinically, both treatments were well tolerated. CONCLUSIONS: Quinidine does not affect the renal clearance of moxonidine. The decrease in apparent total clearance of moxonidine with quinidine coadministration was possibly due to metabolic inhibition, though not likely to be clinically significant.

Characterization of dark liver pigment observed in rats after subchronic dosing of the beta3-adrenergic receptor agonist LY368842.

Dark liver pigmentation was observed in F344 rats in a subchronic toxicology study after daily dosing of LY368842 glycolate. In addition, green-colored urine was observed in some animals. To identify the source of the pigment and its potential for toxic consequences, the liver pigment was isolated from the liver tissue of rats. The resulting material was a dark brown to black powder that was insoluble in water, organic solvents, or a tissue-solubilizing agent. Several techniques, such as chemical degradation, HPLC, tandem mass spectrometry (LC/MS/MS), (1)H NMR, and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), were employed to characterize the dark liver pigment. Following oxidative degradation of the isolated pigment, degradation products related to LY368842 were identified or tentatively identified using LC/MS/MS. Two degradation products had the same protonated molecular ion at m/z 505, which is 30 amu higher than that of LY368842. The major m/z 505 product has been identified as the indole-2,3-dione oxidative product based on (1)H NMR data and confirmed by an authentic standard. In addition, monohydroxylated product was also identified in the degradation mixture. These degradation products were consistent with the metabolites found in vivo in rats. MALDI-MS analyses of liver and urine pigment both identified a product with a protonated molecular ion at m/z 977, suggesting formation of indirubin-like and indigo-like pigments. The results obtained suggest that the oxidative metabolites of LY368842 played a key role in the formation of the liver and urine pigments.

Metabolism and disposition of the antihypertensive agent moxonidine in humans.

The metabolism and pharmacokinetics of moxonidine, a potent central-acting antihypertensive agent, were studied in four healthy subjects after a single oral administration of approximately 1 mg (approximately 60 muCi) of [(14)C(3)]moxonidine. Moxonidine was rapidly absorbed, with peak plasma concentration achieved between 0.5 to 2 h postdose. The maximal plasma concentration and the area under the curve of unchanged moxonidine are lower than those determined for radioactivity, indicating presence of circulating metabolite(s). The total recovery of radiocarbon over 120 h ranged from 99.6 to 105.2%, with 92.3 to 103.3% of the radioactivity excreted in the urine and only 1.9 to 7.3% of the dose excreted in the feces. Thus, renal elimination represented the principal route of excretion of radioactivity. Metabolites of moxonidine were identified in urine and plasma samples by high performance liquid chromatography and liquid chromatography-tandem mass spectrometry. Oxidation of moxonidine on the methyl group or on the imidazoline ring resulted in the formation of hydroxymethyl moxonidine, hydroxy moxonidine, dihydroxy moxonidine, and dehydrogenated moxonidine. Metabolite profiling results indicated that parent moxonidine was the most abundant component in the urine. The dehydrogenated moxonidine was the major urinary metabolite as well as the major circulating metabolite. Moxonidine also underwent phase II metabolism, generating a cysteine conjugate. In summary, moxonidine is well absorbed after oral administration. The major clearance pathway for moxonidine in humans is via renal elimination. Furthermore, seven metabolites were identified with three metabolites unique to humans.