N-terminal acylation of somatostatin analog with long chain fatty acids enhances its stability and anti-proliferative activity in human breast adenocarcinoma cells

The anti-proliferative activity of the somatostatin analog RC-160 is limited by its short serum half life. To circumvent this limitation, fatty acids of chain lengths ranging from 4 to 18 were individually conjugated to the N-terminal residue of RC-160. The lipophilized derivatives of RC-160 were synthesized, purified and characterized. The anti-proliferative activity of lipophilized-RC-160 on the human breast carcinoma cell line MCF-7, was evaluated in vitro. The long chain lipopeptides like pamitoyl-RC-160 exhibited significantly higher anti-proliferative activity on MCF-7 cells (p<0.001), relative to RC-160. The affinity of RC-160 towards somatostatin receptors remained unaltered by pamitoylation. However, the observed increase in bioactivity was manifested within an optimum range of chain length of the lipoppetide. Increasing the peptide hydrophobicity beyond this range reduced the bioactivity of lipophilized-RC-160. Accordingly, stearoyl-RC-160, manifested lower anti-neoplastic activity and receptor affinity relative to pamitoyl-RC-160 and RC-160 itself. The signaling pathways underlying the antineoplastic activity of these lipopeptides were found to be similar to RC-160. Pamitoyl-RC-160 displayed enhanced inhibition of protein tyrosine kinase activity and intracellular cAMP levels in MCF-7 cells, relative to butanoyl-RC-160 or RC-160 itself. Pamitoyl-RC-160 also displayed greater resistance towards trypsin and serum degradation than RC-160. Lipophilization of RC-160 with long chain fatty acids like pamitic acid improves its stability and anti-proliferative activity, thereby improving the scope of enhancing its therapeutic index. However, the optimization of peptide hydrophobicity seems to be a crucial factor governing the efficacy of bioactive lipopeptides.     

Lipophilization of somatostatin analog RC-160 with long chain fatty acid improves its antiproliferative and antiangiogenic activity in vitro

The therapeutic potential of the somatostatin analogue RC-160 having antiproliferative activity, is limited by its short serum half life. To overcome this limitation, fatty acids namely butanoic acid and myristic acid were conjugated to the N-terminal residue of RC-160. The lipophilized derivatives of RC-160 were synthesized, purified by reverse phase HPLC and characterized by ES-mass spectroscopy. The antiproliferative activity of lipophilized derivatives of RC-160 on the growth of MIA-PaCa2 (human pancreatic carcinoma), DU145 (human prostate carcinoma), ECV304 (human umbilical chord endothelioma), as well as their antiangiogenic activity was evaluated in vitro. The relative stability of myristoyl-RC-160 towards degradation by proteases and serum was also determined. Myristoyl-RC-160 exhibited significantly higher antiproliferative efficacy than RC-160, on the above cell lines (P<0.01). Receptor binding assays, demonstrated that the affinity of RC-160 towards somatostatin receptors remains unaltered by myristoylation. Unlike RC-160, the myristoylated derivative was found to have significantly greater resistance to protease and serum degradation (P<0.01). Myristoyl-RC-160 exhibited significantly greater antiproliferative activity on ECV304, than RC-160 (P<0.01). Myristoyl RC-160 could also inhibit capillary tube formation more efficiently than RC-160 in a dose dependent manner, suggesting that it possessed enhanced antiangiogenic activity in vitro (P<0.001). Lipophilization of RC-160 with long chain fatty acids like myristic acid endows it with improved antiproliferative and antiangiogenic activity, stability and therapeutic index. British Journal of Pharmacology (2000) 109, 101 - 109     

N-Acyl-(α,γ Diaminobutyric Acid)n Hydrazide as an Efficient Gene Transfer Vector in Mammalian Cells in Culture

Purpose. This study investigates the structure/activity relationship of a series of N-acyl-peptides (lipopeptides) for the transfection of mammalian cells. Methods. Lipopeptides comprising 1 to 3 basic amino-acids and a single fatty acid chain were synthesized. Transfecting complexes between lipopeptide, plasmid DNA and dioleoyl phosphatidylethanolamine were prepared and applied on cells in culture. Transfection efficiency was evaluated by measuring -galactosidase activity 48 h post-transfection. Lipopeptide-DNA binding was also investigated by physical means and molecular modelling. Results. Besides the length of the fatty acid chain, the nature of the basic amino-acid and the C-terminal group were crucial parameters for high transfection efficiency. The N-acyl-(diaminobutyric acid)n derivatives were the most potent transfecting agents among those tested and induced a -galactosidase activity 2 to 20 times higher than the N-acyl-lysine, -ornithine or -diaminopropionic acid derivatives. Furthermore, a hydrazide C-terminal modification greatly enhanced transfection efficiency for all compounds tested. The reason why , -diaminobutyric acid hydrazide-based lipopeptides were the most potent in transfection is not fully understood but could be related to their high DNA binding. Conclusions. Poly- or oligo-diaminobutyric acid containing or not a hydrazide C-terminus could advantageously be used in peptide-based gene delivery systems.     

Effect of somatostatin analogs on gastric acid secretion in dogs and rats

The effects of several superactive analogs of somatostatin on gastric acid response to various exogenous and endogenous stimulants were investigated in conscious dogs and rats with gastric fistulae (GF). The inhibition was compared to that induced by somatostatin-14 (S-S-14) at two dose levels. Several octapeptide analogs of somatostatin including (RC-160) and (RC-121), which were superactive in tests on suppression of GH levels, were 4-5 times more potent than S-S-14 in inhibiting desglugastrin-stimulated gastric acid secretion in GF dogs. The analog RC-160 also reduced the rise in serum gastrin levels and gastric acid secretion induced by sham feeding (SF) in dogs with gastric and esophageal fistulae (EF), but did not decrease food consumption. Gastric acid secretion induced by histamine (80μg/kg/h) in dogs was not affected by 1-5μg/kg/h of analog RC-121 or by 5μg/kg/h of S-S-14. Analogs RC-160, RC-121, and RC-98-I and others also powerfully inhibited desglugastrin-induced gastric acid secretion in GF rats. Hexapeptide cyclo(-Pro-Phe-D-Trp-Lys-Thr-Phe) was only about half as potent as S-S-14 in dogs but its activity was higher in rats. The results indicate that octapeptide analogs which are superactive in GH-inhibition tests are also more potent than S-S-14 in suppressing gastric acid secretion. These findings may be of clinical value.     

An update on treatment strategies for acromegaly

Background: Acromegaly is an insidious disease due to growth hormone (GH) hypersecretion from a pituitary adenoma, and is associated with multiple comorbidities and risk of premature mortality. Objectives: To review the therapeutic goals and options for acromegaly. Methods: Literature review. Results/conclusion: Surgery is the mainstay of therapy, but a role for primary medical therapy using somatostatin analogs is described as well. Somatostatin analogs are the mainstay for medical therapy, largely in an adjuvant setting. The GH receptor antagonist is also used and may be considered in addition to the somatostatin analogs, or as second line therapy. Based on these multiple modalities of therapy, it should be possible to achieve biochemical control in almost all patients.     

Current and future medical treatments for patients with acromegaly

Introduction: Acromegaly is a relatively rare condition of growth hormone (GH) excess associated with significant morbidity and, when left untreated, high mortality. Therapy for acromegaly is targeted at decreasing GH and insulin-like growth hormone 1 levels, ameliorating patients' symptoms and decreasing any local compressive effects of the pituitary adenoma. The therapeutic options for acromegaly include surgery, medical therapies (such as dopamine agonists, somatostatin receptor ligands and the GH receptor antagonist pegvisomant) and radiotherapy. However, despite all these treatments option, approximately 50% of patients are not adequately controlled.

Areas covered: In this paper, the authors discuss: 1) efficacy and safety of current medical therapy 2) the efficacy and safety of the new multireceptor-targeted somatostatin ligand pasireotide 3) medical treatments currently under clinical investigation (oral octreotide, ITF2984, ATL1103), and 4) preliminary data on the use of new injectable and transdermal/transmucosal formulations of octreotide.

Expert opinion: This expert opinion supports the need for new therapeutic agents and modalities for patients with acromegaly.     

Selective uptake of the somatostatin analog RC-160 across the blood-brain tumor barrier of mice with KHT sarcomas.

The development of somatostatin analogs with anti-tumor effects has raised hopes for their use in various cancers and tumors of the central nervous system. However, for many therapeutic agents, access to normal brain is retarded by the blood-brain barrier (BBB) and to tumor tissues by a blood-brain tumor barrier (BBTB). We examined the ability of RC-160, a somatostatin analog with known anti-tumor activity, to cross the normal BBB and the BBTB in mice with brain sarcomas. In comparison with the normal BBB, the BBTB was about 10 times more permeable to the vascular marker albumin (radioactively labeled with 99mTc), but the BBTB still represents a substantial barrier. By contrast, the entry rate of RC-160, radioactively labeled with 125I, into brain sarcomas was 60 times higher than into normal brain tissue; more than 1% of the RC-160 injected i.v. was taken up by each gram of brain tumor. These results show that a brain tumor can selectively accumulate the potentially therapeutic agent RC-160.     

Fat Emulsions Based on Structured Lipids (1,3-specific triglycerides): An Investigation of the in Vivo Fate

Purpose. Structured lipids (1,3-specific triglycerides) are new chemical entities made by enzymatic transesterification of the fatty acids in the 1,3 positions of the triglyceride. The purpose of this study is to characterize structured lipids with either short chain fatty acids or medium chain fatty acids in the 1,3 positions with regard to their hydrophobicity, and investigate the in vivo fate in order to evaluate the potential of structured lipids as core material in fat emulsions used as parenteral drug delivery system. Methods. The lipids were characterized by employing reversed phase high performance liquid chromatography. The biodistribution of radio-actively labeled emulsions was studied in rats. Results. By employing high performance liquid chromatography a rank order of the hydrophobicities of the lipids could be given, with the triglycerides containing long chain fatty acids being the most hydrophobic and the structured lipid with short chain fatty acids in the 1,3 positions the least. When formulated as fat emulsions, the emulsion based on structured lipids with short fatty acids in the 1,3 positions was removed slower from the general blood circulation compared to emulsions based on lipids with long chain fatty acids in the 1,3 positions. Conclusions. The type of core material influences the in vivo circulation time of fat emulsions.     

山奈酚对白假丝酵母生物被膜抑制作用的研究

目的 研究山奈酚抗白假丝酵母生物被膜的作用及其可能机制。方法 测定山奈酚对白假丝酵母处于形成过程中的生物被膜和成熟生物被膜代谢活性的影响;测定山奈酚对生物被膜基质产生水平的影响;显微镜下观察山奈酚对菌丝形成的抑制作用;水-烃两相分离法测定山奈酚对白假丝酵母细胞表面疏水性的影响;实时定量RT-PCR法测定山奈酚对生物被膜形成相关基因表达的影响。结果 山奈酚抑制白假丝酵母生物被膜形成,且呈剂量依赖性,同时具有抗成熟生物被膜作用,显著降低生物被膜基质含量;与对照组相比,山奈酚明显抑制白假丝酵母菌丝形成并降低其细胞表面疏水性;经山奈酚处理的白假丝酵母生物被膜形成相关基因BCR1、NRG1和TUP1的表达升高,同时HWP1、EFG1、CPH1、ALS1、ALS3和CSH1的表达下降。结论 山奈酚具有抗白假丝酵母生物被膜活性,其机制与抑制菌丝形成及降低其细胞表面疏水性相关。     

Pharmacokinetic-Pharmacodynamic Modeling of Ipamorelin,a Growth Hormone Releasing Peptide,in Human Volunteers

Purpose. To examine the pharmacokinetics (PK) and pharmacodynamics (PD) of ipamorelin, a growth hormone (GH) releasing peptide, in healthy volunteers. Methods. A trial was conducted with a dose escalation design comprising 5 different infusion rates (4.21, 14.02, 42.13, 84.27 and 140.45 nmol/kg over 15 minutes) with eight healthy male subjects at each dose level. Concentrations of ipamorelin and growth hormone were measured. Results. The PK parameters showed dose-proportionality, with a short terminal half-life of 2 hours, a clearance of 0.078 L/h/kg and a volume of distribution at steady-state of 0.22 L/kg. The time course of GH stimulation by ipamorelin showed a single episode of GH release with a peak at 0.67 hours and an exponential decline to negligible GH concentration at all doses. The ipamorelin–GH concentration relationship was characterized using an indirect response model and population fitting. The model employed a zero-order GH release rate over a finite duration of time to describe the episodic release of GH. Ipamorelin induces the release of GH at all dose levels with the concentration (SC₅₀) required for half-maximal GH stimulation of 214 nmol/L and a maximal GH production rate of 694 mlU/L/h. The inter-individual variability of the PD parameters was larger than that of the PK parameters. Conclusions. The proposed PK/PD model provides a useful characterization of ipamorelin disposition and GH responses across a range of doses.     

The place of medical treatment of acromegaly: current status and perspectives

Introduction: Acromegaly is characterized by elevated growth hormone (GH) and insulin-like growth factor-I (IGF-I) levels and by progressive somatic disfigurement and systemic manifestations, which lead to a mortality rate higher than the general population. Therefore, diagnosis and properly treatment should be performed as soon as possible.

Areas covered: This article focuses on the state of the art of acromegaly medical treatment. Somatostatin analogs, dopamine agonists and GH receptor antagonist were reviewed. Somatostatin analogs, the first-choice pharmacotherapy, can be used as primary or pre-operative treatment or as secondary therapy after failed surgery. Dopamine agonists have been used in patients with slightly elevated hormone levels and/or mixed GH/prolactin adenomas. Pegvisomant is indicated for resistant to somatostatin analogs/dopamine agonists. Combined treatment is also an option for resistant cases. Other non-conventional therapies and perspectives of treatment were also been discussed.

Expert opinion: The control of disease activity in acromegaly is of paramount importance. Medical treatment is an important option for cases in which surgery was unsuccessful or not indicated. Despite the achievements in medical treatment, somatotropic tumor aggressiveness and/or resistance to the drugs currently available remain a concern. Therefore, novel therapy targets based on molecular pathogenesis of GH-secreting tumors are currently in development, aiming at fulfilling this important gap.     

Boosting Antioxidants by Lipophilization: A Strategy to Increase Cell Uptake and Target Mitochondria

Purpose

To explore the possibility to boost phenolic antioxidants through their structural modification by lipophilization and check the influence of such covalent modification on cellular uptake and mitochondria targeting.

Methods

Rosmarinic acid was lipophilized by various aliphatic chain lengths (butyl, octyl, decyl, dodecyl, hexadecyl, and octadecyl) to give rosmarinate alkyl esters which were then evaluated for their ability (i) to reduce the level of reactive oxygen species (ROS) using 2′,7′-dichlorodihydrofluorescein diacetate probe, (ii) to cross fibroblast cell membranes using confocal microscopy, and (iii) to target mitochondria using MitoTracker® Red CMXRos.

Results

Increasing the chain length led to an improvement of the antioxidant activity until a threshold is reached for medium chain (10 carbon atoms) and beyond which lengthening resulted in a decrease of activity. This nonlinear phenomenon—also known as the cut-off effect—is discussed here in connection to the previously similar results observed in emulsified, liposomal, and cellular systems. Moreover, butyl, octyl, and decyl rosmarinates passed through the membranes in less than 15 min, whereas longer esters did not cross membranes and formed extracellular aggregates. Besides cell uptake, alkyl chain length also determined the subcellular localization of esters: mitochondria for medium chains esters, cytosol for short chains and extracellular media for longer chains.

Conclusion

The localization of antioxidants within mitochondria, the major site and target of ROS, conferred an advantage to medium chain rosmarinates compared to both short and long chains. In conjunction with changes in cellular uptake, this result may explain the observed decrease of antioxidant activity when lengthening the lipid chain of esters. This brings a proof-of-concept that grafting medium chain allows the design of mitochondriotropic antioxidants.     

Use of the Intestinal Bile Acid Transporter for the Uptake of Cholic Acid Conjugates with HIV-1 Protease Inhibitory Activity

Purpose. To investigate the ability of the human intestinal bile acid transporter to transport cholic acid conjugates with potential HIV-1 protease inhibitory activity. Methods. Cholic acid was conjugated at the 24 position of the sterol nucleus with various amino acids and amino acid analogs. The CaCo-2 cell line was used as a model to investigate the interaction of these bile acid conjugates with the human intestinal bile acid transporter. Interaction between the carrier and the conjugates was quantified by inhibition of taurocholic acid transport and confirmed by transport of radiolabelled conjugates in this cell line. Results. The highest interaction with the transporter, as quantified by inhibition of taurocholic acid transport, occurred when a single negative charge was present around the 24 to 29 region of the sterol nucleus. A second negative charge or a positive charge significantly reduced the interaction. Transport of radiolabelled cholyl-L-Lys--tBOC ester and cholyl-D-Asp--benzyl ester was inhibited by taurocholic acid. Of all tested compounds, only cholyl-D-Asp--benzyl ester showed modest HIV-1 protease inhibitory activity with an IC₅₀ of 125 M. Conclusions. Cholic acid-amino acid conjugates with appropriate stereochemistry are recognized and transported by the human bile acid transporter and show modest HIV- 1 protease inhibitory activity. Transport of these conjugates by the bile acid carrier is influenced by charge and hydrophobicity around the 24 position of the sterol nucleus.     

Low Potential Ocular Irritation of Arginine-Based Gemini Surfactants and Their Mixtures with Nonionic and Zwitterionic Surfactants

Purpose. The aim of this study was to find new biocompatible surfactants and mixtures with low ocular irritant action for application in pharmaceutical formulations and to establish a relationship between their structure and their potential ocular irritant activity. Methods. An alternative method to the Draize in vivo test, based on the adverse effects of surfactants on the cytoplasmic membrane of red blood cell, was used to evaluate the potential ocular irritation of the surfactants. Results. It was found that the hemolytic activity of arginine-based gemini surfactants increased with the aliphatic alkyl chain lengths of the hydrophobic tail. The addition of the surfactant with an alkyl chain length of 10 carbon atoms to cocoamidopropilbetaina (TB), decylglucoside (APG), and N-lauroyl-arginine ethyl ester (LAE) increases the hemolytic activity moderately for the mixtures with TB and LAE (1.1- and 1.5-fold, respectively) and strongly for APG (fivefold). Conclusions. The new arginine-based gemini surfactants constitute a suitable alternative to commercial surfactants because of their natural origins, which make them biocompatible and renewable products. Based on their hemolytic activity as an alternative to the Draize test, these new arginine-based gemini surfactants and their mixtures can be classified as mild irritants. This fact constitutes an advantage, especially for pharmaceutical and cosmetic applications.     

Fundamental Relationships Between the Composition of Pluronic Block Copolymers and Their Hypersensitization Effect in MDR Cancer Cells

Purpose. Previous studies have demonstrated that Pluronic block copolymers hypersensitize multiple drug resistant (MDR) cancer cells, drastically increasing the cytotoxic effects of anthracyclines and other anticancer cytotoxics in these cells. This work evaluates the dose dependent effects of these polymers on (i) doxorubicin (Dox) cytotoxicity and (ii) cellular accumulation of P-glycoprotein probe, rhodamine 123 (R123) in MDR cancer cells. Methods. Dox cytotoxicity and R123 accumulation studies are performed on monolayers of drug-sensitive (KB, MCF-7, Aux-Bl) and MDR (KBv, MCF-7/ADR, CH^rC5) cells. Results. Both tests reveal strong effects of Pluronic copolymers observed at concentrations below the critical micelle concentration (CMC) and suggest that these effects are due to the copolymer single chains ('unimers'). Using block copolymers with various lengths of hydrophobic propylene oxide (PO) and hydrophilic ethylene oxide (EO) segments these studies suggest that the potency of Pluronic unimers in MDR cells increases with elevation of the hydrophobicity of their molecule. Optimization of Pluronic composition in R123 accumulation and Dox cytotoxicity studies reveals that Pluronic copolymers with intermediate lengths of PO chains and relatively short EO segments have the highest net efficacy in MDR cells. Conclusions. The relationship between the structure of Pluronic block copolymers and their biological response modifying effects in MDR cells is useful for determining formulations with maximal efficacy with respect to MDR tumors.     

Halogenated benzoate derivatives of altholactone with improved anti-fungal activity

Altholactone exhibited the anti-fungal activity with a high MIC value of 128 μg ml^?1 against Cryptococcus neoformans and Saccharomyces cerevisiae. Fifteen ester derivatives of altholactone 1–15 were modified by esterification and their structures were confirmed by spectroscopic methods. Most of the ester derivatives exhibited stronger anti-fungal activities than that of the precursor altholactone. 3-Bromo- and 2,4-dichlorobenzoates (7 and 15) exhibited the lowest minimal inhibitory concentration (MIC) values against C. neoformans at 16 μg ml^?1_, while the 4-bromo-, 4-iodo-, and 1-bromo-3-chlorobenzoates (11–13) displayed potent activity against S. cerevisiae with MIC values of 1 μg ml^?1. In conclusion, this analysis indicates that the anti-fungal activity of altholactone is enhanced by addition of halogenated benzoyl group to the 3-OH group.     

Biodistribution of Long-Circulating PEG-Grafted Nanocapsules in Mice: Effects of PEG Chain Length and Density

Purpose: To study the pharmacokinetics and biodistribution of novel polyethyleneglycol (PEG) surface-modified poly(rac-lactide) (PLA) nanocapsules (NCs) and to investigate the influence of PEG chain length and content. Methods: The biodistribution and plasma clearance in mice of different NC formulations were studied with [³H]-PLA. PLA-PEG copolymers were used in NC preparations at different chain lengths (5 kDa and 20 kDa) and PEG contents (10% and 30% w/w of total polymer). In vitro and in vivo stability were also checked. Results: Limited [³H]-PLA degradation was observed after incubation in mouse plasma for 1 h, probably because of to the large surface area and thin polymer wall. After injection into mice, NCs prepared with PLA-PEG copolymers showed an altered distribution compared to poloxamer-coated PLA NCs. An increased concentration in plasma was also observed for PLA-PEG NCs, even after 24 h. A dramatic difference in the pharmacokinetic parameters of PLA-PEG 45-20 30% NCs compared to poloxamer-coated NCs indicates that covalent attachment, longer PEG chain lengths, and higher densities are necessary to produce an increased half-life of NCs in vivo. Conclusions: Covalently attached PEG on the surface of NCs substantially can reduce their clearance from the blood compartment and alter their biodistribution.     

Synthesis of piscidinol A derivatives and their ability to inhibit HIV-1 protease

Four types of piscidinol A derivatives were synthesized and evaluated their ability to inhibit HIV-1 protease to understand their structure-activity relationships. Of these tirucallane-type triterpene derivatives, an A-seco derivative (1b) moderately inhibited human immunodeficiency virus (HIV) protease (IC₅₀ 38.2 μM). The 2,2-dimethyl succinic acid (DMS) acylated tirucallane derivatives (4b, 6a, and 7b, 50 < IC₅₀ < 100 μM) were more inhibitory against HIV-1 PR than the others (PA, 2a, 4a, 4c–4d, 5a, 6b–6d, and 7a, IC₅₀ > 100 μM). These findings indicated that the 2,3-seco-2,3-dioic acid (1b) and DMS-acylated tirucallane-type derivatives preferably inhibited HIV viral protease.     

New polyacetylenes glycoside from Eclipta prostrate with DGAT inhibitory activity

One new polyacetylene glycoside eprostrata Ⅰ (1), together with seven known compounds (2–8), were isolated from Eclipta prostrata. Their structures were elucidated on the basis of spectroscopic and physico-chemical analyses. All the isolates were evaluated inhibitory activity on DGAT in an in vitro assay. Compounds 1–8 were found to exhibit inhibitory activity of DGAT1 with IC₅₀ values ranging from 74.4 ± 1.3 to 101.1 ± 1.1 μM.     

Development of Activity Assays for High-Volume Evaluation of Human Immunodeficiency Virus (HIV) Protease Inhibitors in Rat Serum: Results with Ditekiren

We showed previously that a commercially available synthetic tetradecapeptide, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, produces authentic angiotensin I (Ang I) upon incubation with the HIV-1 protease (S. K. Sharma et al., Anal. Biochem. 198:363, 1991). Therefore, we developed an Ang-I based activity assay for HIV protease inhibitors based on the technology developed earlier (M. J. Ruwart et al., Pharm. Res. 7:407, 1990; S. K. Sharma et al., Anal. Biochem. 186:24, 1990) for tracking renin inhibitors in rat sera. Ditekiren was either extracted from sera with ethyl acetate or assayed after the interfering substances in sera were precipitated with acetonitrile. Purified recombinant HIV-1 protease was added to extracted rat serum and the enzymatic reaction was initiated in the presence of the tetradecapeptide substrate. The inhibition of Ang I production was measured by a commercially available RIA kit. The cleanup methodology also enabled a commercially available Proteinase Scintillation Proximity Assay (SPA, Amersham) to quantify ditekiren in rat serum through the addition of recombinant HIV-1 protease and cleavage of substrate from SPA beads. Results were confirmed by HPLC or by the renin assay for ditekiren, which inhibits both aspartyl proteases. These technologies should prove useful for assessing serum levels of HIV protease inhibitors in rat.