Strategic Approaches to Optimizing Peptide ADME Properties

Development of peptide drugs is challenging but also quite rewarding. Five blockbuster peptide drugs are currently on the market, and six new peptides received first marketing approval as new molecular entities in 2012. Although peptides only represent 2% of the drug market, the market is growing twice as quickly and might soon occupy a larger niche. Natural peptides typically have poor absorption, distribution, metabolism, and excretion (ADME) properties with rapid clearance, short half-life, low permeability, and sometimes low solubility. Strategies have been developed to improve peptide drugability through enhancing permeability, reducing proteolysis and renal clearance, and prolonging half-life. In vivo, in vitro, and in silico tools are available to evaluate ADME properties of peptides, and structural modification strategies are in place to improve peptide developability.KEY WORDS: ADME, peptides, pharmacokinetics, proteolysis, renal clearance     

Protein and Peptide Drug Delivery: Oral Approaches

Till recent, injections remained the most common means for administering therapeutic proteins and peptides because of their poor oral bioavailability. However, oral route would be preferred to any other route because of its high levels of patient acceptance and long term compliance, which increases the therapeutic value of the drug. Designing and formulating a polypeptide drug delivery through the gastro intestinal tract has been a persistent challenge because of their unfavorable physicochemical properties, which includes enzymatic degradation, poor membrane permeability and large molecular size. The main challenge is to improve the oral bioavailability from less than 1% to at least 30-50%. Consequently, efforts have intensified over the past few decades, where every oral dosage form used for the conventional small molecule drugs has been used to explore oral protein and peptide delivery. Various strategies currently under investigation include chemical modification, formulation vehicles and use of enzyme inhibitors, absorption enhancers and mucoadhesive polymers. This review summarizes different pharmaceutical approaches which overcome various physiological barriers that help to improve oral bioavailability that ultimately achieve formulation goals for oral delivery.     

Chemical Approaches to Improve the Oral Bioavailability of Peptidergic Molecules

This review discusses both tools and strategies that may be employed as approaches towards the pursuit of orally active compounds from peptidergic molecules. Besides providing a review of these subjects, this paper provides an example of how these were utilized in a research programme at SmithKline Beecham involving the development of orally active GPIIb/IIIa antagonists. The tools for studying oral drug absorption in-vitro include variants of the Ussing chamber which utilize either intestinal tissues or cultured epithelial cells that permit the measurement of intestinal permeability. Example absorption studies that are described are mannitol, cephalexin, the growth hormone-releasing peptide SK&F 110679 and two GPIIb/ IIIa antagonist peptides SK&F 106760 and SK&F 107260. With the exception of cephalexin, these compounds cross the intestine by passive paracellular diffusion. Cephalexin, on the other hand, crosses the intestine via the oligopeptide transporter. Structure-transport studies are reviewed for this transporter. The tools for studying oral drug absorption in-vivo involve animals bearing in-dwelling intestinal or portal vein catheters. A study of the segmental absorption of SK&F 106760 is provided. The review concludes with two chemical strategies that may be taken towards the enhancement of oral bioavailability of peptidergic molecules. The first strategy involves the chemical modification of peptides which enhance intestinal permeability, specifically the modification of amide bonds. The second strategy involves the design of compounds bearing nonpeptide templates, which are more amenable to the discovery of compounds with oral activity, from peptide pharmacophore models. An example is given regarding the discovery of SB 208651, a potent orally active GPIIb/IIIa antagonist, designed from the peptides SK&F 106760 and SK&F 107260.     

药剂学实验教学改进刍议

药剂学是一门实践性、交叉性学科。药剂学实验是培养学生独立思考、动手实验、分析解决问题、创新能力的重要手段。通过讨论传统药剂学实验教材,在教学模式、考核方式等方面提出自己的见解。     

Molecular Approaches to Improve the Insecticidal Activity of Bacillus thuringiensis Cry Toxins

Bacillus thuringiensis (Bt) is a gram-positive spore-forming soil bacterium that is distributed worldwide. Originally recognized as a pathogen of the silkworm, several strains were found on epizootic events in insect pests. In the 1960s, Bt began to be successfully used to control insect pests in agriculture, particularly because of its specificity, which reflects directly on their lack of cytotoxicity to human health, non-target organisms and the environment. Since the introduction of transgenic plants expressing Bt genes in the mid-1980s, numerous methodologies have been used to search for and improve toxins derived from native Bt strains. These improvements directly influence the increase in productivity and the decreased use of chemical insecticides on Bt-crops. Recently, DNA shuffling and in silico evaluations are emerging as promising tools for the development and exploration of mutant Bt toxins with enhanced activity against target insect pests. In this report, we describe natural and in vitro evolution of Cry toxins, as well as their relevance in the mechanism of action for insect control. Moreover, the use of DNA shuffling to improve two Bt toxins will be discussed together with in silico analyses of the generated mutations to evaluate their potential effect on protein structure and cytotoxicity.     

提高有机化学教学质量的方法探讨

从注重教学环节、采用现代化多媒体教学手段、加强课外辅导答疑、及时听取反馈意见、创新考试方式及注重能力评价等方面,探讨提高有机化学教学质量的方法。     

探索提高药物临床试验机构的核心竞争力

目的:为药物临床试验机构提高新药Ⅰ～Ⅳ期临床试验的质量,保障受试者权益,增强核心竞争力提供参考。方法:将《药物临床试验质量管理规范》(GCP)的指导原则和《检测和校准实验室能力的通用要求》(ISO/IEC17025)质量管理标准优势互补,贯穿于药物临床试验质控的全过程。结果与结论:将GCP的原则和ISO/IEC17025质量管理标准结合,建立既符合国际规范,又符合我国国情的质量管理及督察体制,有利于全面提高药物临床试验研究质量,增强我国药物临床试验机构的核心竞争力。     

Systematic Approaches for Incorporating Control Spots and Data Quality Information to Improve Normalization of cDNA Microarray Data

Background: Normalization and data quality control are two important aspects in microarray data analysis. Proper normalization and data quality control ensure that intensity ratios provide meaningful and accurate measurement of relative gene expression values. Control spots such as spikes and housekeeping genes with known concentrations in two channels are often used for calibrating experimental parameters. They provide valuable information about experimental variation which can be utilized for better normalization. They are also needed for proper normalization in cases that the most of the spots tend to change in one direction. In addition, it is desirable to include information on spot quality. Such information is available in a typical microarray data set, but is not fully utilized by existing normalization methods.

Results: We propose two extensions of the two-way semi-linear model (TW-SLM) for appropriately combining control genes and spot quality information in normalization. The first extension (TW-SLMC) is designed to systematically incorporate control spots in a semi-parametric model to calibrate estimated normalization curves so that the relative fold changes of gene expressions are accurately estimated. Extrapolation is not required in this approach. The second extension (TW-SLMQ) is proposed to incorporate spot quality measure into normalization. This approach down-weights spots with lower quality scores in normalization. These two extensions can be used simultaneously for normalizing a data set. Two microarray data sets are used to demonstrate the proposed methods. Availability: An R based computing package is developed for the proposed methods and available from the corresponding authors.

Contact: Deli Wang: deliwang@uab.edu or Jian Huang: jian-huang@uiowa.edu.     

Prodrug and Analog Approaches to Improving the Intestinal Absorption of a Cyclic Peptide,GPIIb/IIIa Receptor Antagonist

Purpose. The purpose of this study was to test whether structural modifications improve the intestinal absorption of DMP 728 (cyclo(D-Abu-NMeArg-Gly-Asp-Amb)), a GPIIb/IIIa receptor antagonist. Methods. In vitro permeabilities of prodrugs and analogs of DMP 728 across excised rat intestinal segments were determined. Results. n-Butyl and n-octyl esters of DMP 728 were relatively stable during in vitro permeation of rat intestine. Intestinal permeation rates of these compounds were no greater than that of DMP 728, even though the octyl ester was much more lipophilic. A pivaloyloxymethyl ester, which was hydrolyzed to DMP 728 during intestinal permeation, also did not improve permeability. In another approach, analogs with an additional methyl substituent on various amide nitrogens were evaluated. Cyclo(D-Val-NMeArg-Gly-Asp-NMeAmb), cyclo(D-Abu-diN-MeLys-Gly-Asp-Amb), and cyclo(NMeGly-NMeArg-Gly-Asp-Amb) each had about 2-fold greater permeability than DMP 728. Two other analogs with improved permeability were linear Ac-D-Abu-NMeArg-Gly-Asp-Amb and a DMP 728 derivative in which the Asp was rearranged. An analog in which the charged amino acids were replaced by neutral amino acids had permeability similar to DMP 728. Conclusions. Within this series of peptides, hydrogen bonding tendency and structural constraint influenced intestinal permeation, but not always in ways consistent with the literature, whereas charge and lipophilicity were not shown to influence intestinal permeability. The failure of these approaches to improve permeation more significantly could be due to the influence of secretory transport.     

在制药化工原理教学中培养学生的工程素质

制药化工原理是制药工程专业课程体系的主要核心课程之一，也是学校重点建设的课程。结合教学实践，阐述在制药化工原理教学中如何培养学生的工程素质。理论上应突出工程方法，使学生逐步树立工程观点；例题与习题应理论联系实际，结合工程实例培养学生解决工程问题的能力；通过实践教学，加强实验环节，提高学生的工程实践能力。     

Analytical Tools and Approaches for Metabolite Identification in Early Drug Discovery

Determination of the chemical structures of metabolites is a critical part of the early pharmaceutical discovery process. Understanding the structures of metabolites is useful both for optimizing the metabolic stability of a drug as well as rationalizing the drug safety profile. This review describes the current state of the art in this endeavor. The likely outcome of metabolism is first predicted by comparison to the literature. Then metabolites are synthesized in a variety of in vitro systems. The various approaches to LC/UV/MS are applied to learn information about these metabolites and structure hypotheses are made. Structures are confirmed by synthesis or NMR. The special topic of reactive metabolite structure determination is briefly addressed.     

Developability Assessments of Monoclonal Antibody Candidates to Minimize Aggregation During Large-Scale Ultrafiltration and Diafiltration (UF-DF) Processing

Therapeutic proteins are subjected to a variety of stresses during manufacturing, storage or administration, that often lead to undesired protein aggregation and particle formation. Ultrafiltration-diafiltration (UF-DF) processing of monoclonal antibodies (mAbs) is one such manufacturing step that has been shown to result in such physical degradation. In this work, we explore the use of different analytical techniques and lab-scale setups as methodologies to predict and rank-order the aggregation potential of four different mAbs during large-scale UF-DF processing. In the first part of the study, a suite of biophysical techniques was applied to assess differences in their inherent bulk protein properties including conformational and colloidal stability in a PBS buffer. Additionally, the inherent interfacial properties of these mAbs in PBS were measured using a Langmuir trough technique. In the next part of the study, several different scale-down lab models were evaluated including a lab bench-scale UF-DF setup, mechanical stress (shaking/stirring) studies in vials, and application of interfacial dilatational stress using a Langmuir trough to assess protein particle formation in different UF-DF processing buffers. Taken together, our results demonstrate the ability of a Langmuir-trough methodology to accurately predict the mAb instability profile observed during large scale UF-DF processing.     

Surface Modification of Liposomes Using Polymer-Wheat Germ Agglutinin Conjugates to Improve the Absorption of Peptide Drugs by Pulmonary Administration

In this study, we investigated the feasibility of a system based on liposomal surface modification with a novel mucoadhesive polymer–lectin conjugate for the pulmonary delivery of therapeutic peptides and proteins. We covalently attached wheat germ agglutinin (WGA), a ligand that specifically interacts with alveolar epithelial cells, to carbopol (CP), a mucoadhesive polymer, using the carbodiimide method and then evaluated the efficacy and potential toxicity of CP–WGA surface-modified liposomes in vivo and in vitro. In association studies, CP–WGA modification enhanced the interaction with A549 lung epithelial cells compared with unmodified or CP-modified liposomes. This increased association was dependent on temperature and the surface concentration of free WGA. These results suggested synergy of WGA and CP, and retention of the biological cell binding activity of WGA, leading to improved liposome-cell interactions. Moreover, improvement of liposomal bioadhesion to lung epithelia significantly enhanced and prolonged the therapeutic efficacy of calcitonin, a model peptide drug, without any evidence of toxicity, following administration of calcitonin-loaded CP–WGA- modified liposomes. Hence, surface modification of liposomes with CP–WGA has potential for effective pulmonary administration of peptides.     

Mucoadhesive Polymers in Peroral Peptide Drug Delivery. VI. Carbomer and Chitosan Improve the Intestinal Absorption of the Peptide Drug Buserelin In Vivo

Purpose. To evaluate the effect of the crosslinked poly(acrylate) carbomer 934P (C934P) and its freeze-dried neutralized sodium salt (FNaC934P) as well as chitosan hydrochloride on the intestinal absorption of the peptide drug buserelin. Methods. Buserelin was applied intraduodenally in control buffer, 0.5% (w/v) C934P, 0.5% (w/v) FNaC934P, 1.5% (w/v) chitosan hydrochloride or FNaC934P/chitosan hydrochloride (1:1 (v/v)) mixture in rats. Results. All polymer preparation showed a statistically significant improvement of buserelin absorption compared to the control solution. The absolute bioavailabilities for the different polymer preparations were: control, 0.1%; 0.5% FNaC934P, 0.6%; 0.5% C934P, 2.0%; chitosan hydrochloride, 5.1% and FNaC934P/chitosan hydrochloride (1:1 (v/v)) mixture, 1.0%. The higher bioavailability with chitosan hydrochloride compared to C934P and FNaC934P indicates that for buserelin the intestinal transmucosal transport enhancing effect of the polymer plays a more dominant role than the protection against proteases such as -chymotrypsin. Conclusions. The mucoadhesive polymers carbomer 934P and chitosan hydrochloride are able to enhance the intestinal absorption of buserelin in vivo in rats, and may therefore be promising excipients in peroral delivery systems for peptide drugs.     

Approaches to Drug Stability

Pharmaceutical Research -     

Approaches to antiviral chemotherapy

Most of the drugs used today in the treatment of viral infections in man are purine-pyrimidine antimetabolites that interfere with viral replication. Work at Southern Research Institute has identified a number of compounds of this type with promising antiviral activity in both cell culture and rodent test systems. By far the most active and selective agents are carbocyclic nucleoside analogs in which the oxygen of the furanose ring is replaced by a methylene group. The effects of this change on the metabolism and antiviral activity of these compounds is discussed below.