N-terminal 4-imidazolidinone prodrugs of Leu-enkephalin: synthesis, chemical and enzymatic stability studies

Four N-terminal 4-imidazolidinone prodrugs of Leu-enkephalin are prepared and characterized. Their enzymatic and chemical stability are assessed using high-performance liquid chromatography. The prodrug derivatives are shown to degrade stoichiometrically to Leu-enkephalin in phosphate buffer [t1/2 (0.05 M phosphate buffer without KCl): acetone prodrug (II) 930 min; cyclopentanone prodrug (III): 216 min; cyclohexanone prodrug (IV): 432 min; 4-methylcyclohexanone prodrug (V): 792 min]. Furthermore, the prodrugs are shown to afford global stabilization of the Leu-enkephalin molecule towards the enzymes, aminopeptidase N and angiotensin converting enzyme, primarily responsible for degradation of Leu-enkephalin at the blood-brain barrier and in plasma. Therefore, the 4-imidazolidinones, being metabolic stable and bioreversible, may be suitable prodrug candidates for delivery of Leu-enkephalin to important target areas such as the brain, if given intravenously.     

Acute and chronic effects of gallium chloride (GaCl3) on tilapia (Oreochromis mossambicus) larvae

PURPOSE: The synthesis of chemically stable triazene prodrugs capable of hydrolysing under physiological conditions to liberate cytotoxic monomethyltriazene alkylating agents. METHODS: A series of 3-aminoacyl-1-aryl-3-methyltriazenes was synthesised through reaction of 1-aryl-3-methyltriazenes with N-BOC protected amino acids using the DCC method of activation, followed by deprotection of the amino function using HCl in nitromethane. Half-lives for the hydrolysis of these compounds to the corresponding monomethyltriazenes at 37 degrees C in isotonic phosphate buffer and in 80% human plasma containing 20% phosphate buffer were determined by HPLC. RESULTS: The aminoacyltriazene prodrugs hydrolyse in isotonic phosphate buffer with t1/2 values ranging from 26 to 619 minutes. In human plasma, several decompose at the same rate as in phosphate buffer whereas those containing more lipophilic groups decompose more slowly. A beta-alanyl derivative was found to be more stable in phosphate buffer (t1/2 = 180 minutes) than in plasma (t1/2 = 53 minutes). An N-acetylated alpha-alanyl derivative was found to be chemically stable in phosphate buffer (t1/2 = 10 hours) but liberated the cytotoxic drug in t1/2 = 41 minutes in plasma, demonstrating its ability to act as a substrate for plasma enzymes. CONCLUSIONS: Aminoacyltriazenes are prodrugs of the antitumour monomethyltriazenes hydrolysing in human plasma with a range of reactivities. The acylation of the alpha-amino group seems to be an effective and simple means of reducing the chemical reactivity of the alpha-aminoacyl derivatives while retaining a rapid rate of enzymatic hydrolysis.     

New carriers for representative peptides and peptide drugs

3,5-Disubstituted tetrahydro-2H-1,3,5-thiadiazine-2-thione (THTT) derivatives; 4a-g were prepared and found to be a promising prodrug approach for peptide drugs. The pH profile for their degradation in aqueous buffer solutions was determined using HPLC technique and accounted for, in terms of specific base-catalyzed reactions. All of the compounds however, showed high acid-stability. Enzymatic (human serum) hydrolysis of the different derivatives offered an advantageous range of t1/2's, the property that permits controlling onset and duration of actions of drugs.     

Randomized comparison of etoposide pharmacokinetics after oral etoposide phosphate and oral etoposide

Etoposide phosphate is a water-soluble prodrug of etoposide. The plasma pharmacokinetics of etoposide following oral administration of etoposide phosphate or oral etoposide were compared. Seventeen patients with solid tumours were enrolled to receive oral etoposide phosphate 125 mg m(-2) on days 1-5 every 3 weeks, with escalation to 175 mg m(-2) from course 3 when possible. Patients were randomized to receive oral etoposide phosphate or oral etoposide on day 1 of course 1 and the alternative compound on day 1 of course 2. Fifteen patients received two or more courses and were evaluable for pharmacokinetic comparisons. The median AUC(inf) (area under the concentration vs time curve from zero to infinity) of etoposide was 77.7 mg l(-1) h after etoposide phosphate (95% CI 61.3-100.5) and 62.0 mg l(-1) h after oral etoposide (95% CI 52.2-76.9). The difference in favour of etoposide phosphate was borderline significant: median 9.9 mg l(-1) h (95% CI 0.1-32.8 mg l(-1) h; P = 0.05). However, the inter-patient variability of etoposide AUC(inf) was not improved (coefficients of variation 42.3% and 48.4%). Etoposide phosphate was undetectable in plasma after oral administration. Toxicities of oral etoposide phosphate were not different from those known for etoposide. In conclusion, oral etoposide phosphate does not offer a clinically relevant benefit over oral etoposide.     

Pharmacokinetics and tissue distribution of (+/-)-3'-azido-2',3'-dideoxy-5'-O-(2-bromomyristoyl)thymidine, a prodrug of 3'-azido-2',3'-dideoxythymidine (AZT) in mice

The in-vivo biodistribution and pharmacokinetics in mice of 3'-azido-2',3'-dideoxythymidine (1, AZT), 2-bromomyristic acid (2) and their common prodrug, (+/-)-3'-azido-2',3'-dideoxy-5'-O-(2-bromomyristoyl)thymidine (3) are reported. The objectives of the work were to enhance the anti-human immunodeficiency virus and anti-fungal effects of 1 and 2 by improving their delivery to the brain and liver. The pharmacokinetics of AZT (beta t1/2 (elimination, or beta-phase, half-life) = 112.5 min; AUC (area under the plot of concentration against time) = 29.1 +/- 2.9 micromol g(-1) min; CL (blood clearance) = 10.5 +/- 1.1 mL min(-1) kg(-1)) and its ester prodrug (3, beta t1/2 = 428.5 min; AUC = 17.3 +/- 4.7 micromol g(-1) min; CL = 17.6 +/- 4.8 mL min(-1) kg(-1) were compared after intravenous injection of equimolar doses (0.3 mmol kg(-1)) via the tail vein of Balb/c mice (25-30 g). The prodrug was rapidly converted to AZT in-vivo, but plasma levels of AZT (peak concentration 0.17 micromol g(-1)) and AUC (12.3 micromol min g(-1)) were lower than observed after AZT administration (peak concentration 0.36 micromol g(-1); AUC 29.1 micromol min g(-1). The prodrug also accumulated rapidly in the liver immediately after injection, resulting in higher concentrations of AZT than observed after administration of AZT itself (respective peak concentrations 1.11 and 0.81 micromol g(-1); respective AUCs 42.5 and 12.7 micromol min g(-1)). Compared with doses of AZT itself, 3 also led to significantly higher brain concentration of AZT (25.7 compared with 9.8 nmol g(-1)) and AUCs (2.8 compared with 1.4 micromol min g(-1)). At the doses used in this study the antifungal agent 2-bromomyristic acid was measurable in plasma and brain within only 2 min of injection. Hepatic concentrations of 2-bromomyristic acid were higher for at least 2 h after dosing with 3 than after dosing with the acid itself. In summary, comparative biodistribution studies of AZT and its prodrug showed that the prodrug led to higher concentrations of AZT in the brain and liver. Although the prodrug did not result in measurably different concentrations of 2-bromomyristic acid in the blood and brain, it did lead to levels in the liver which were higher than those achieved by dosing with the acid itself.     

Synthesis and evaluation of morpholinoalkyl ester prodrugs of indomethacin and naproxen

Morpholinoalkyl esters (HCl salts) of naproxen 1 and indomethacin 3 were synthesized and evaluated in vitro and in vivo for their potential use as prodrugs for oral delivery. Prodrugs were freely soluble in simulated gastric fluid (SGF) and pH 7.4 phosphate buffer and showed a minimum of a 2000-fold increase in solubility over the parent drugs. All prodrugs were more lipophilic than parent drugs as indicated by n-octanol/pH 7.4 buffer partition coefficients but less lipophilic in terms of n-octanol/SGF partition coefficients. Potentiometrically determined pKa's for prodrugs were in the range of 6.89 to 8.62 at 25 degrees C. All prodrugs were quantitatively hydrolyzed to their respective parent drugs by enzymatic and/or by chemical means. An increase in carbon chain length rendered the prodrugs more stable at pH 7.4 but less stable in SGF. The esters were generally found to be hydrolyzed rapidly in rat plasma at 37 degrees C, the half-lives being in the range of 1.2-31.0 min. Based on in vitro results, prodrugs 2c and 4c were chosen to evaluate solid-state stability, in vivo bioavailability, and ulcerogenicity. At elevated temperatures, the solid-state decomposition of 2c and 4c followed biphasic kinetics, with rapid decomposition occurring initially. The prodrugs were 30-36% more bioavailable orally than the parent drugs following a single equimolar solution dose in rats. Prodrugs 2c and 4c were significantly less irritating to gastric mucosa than parent drugs following single-dose and chronic oral administration in rats.     

Stereoselective hydrolysis of O-isovaleryl propranolol and its influence on the clearance of propranolol after oral administration

The stereoselective hydrolysis of O-isovaleryl propranolol (isovaleryl-PL) was studied using phosphate and Tris-HCl buffers (pH 7.4), dog plasma, and liver preparations. The 10000g supernatant, microsomes, and cytosol were prepared from the liver homogenate. The hydrolysis rate of isovaleryl-PL was accelerated in the order Tris buffer < plasma = phosphate buffer < 10000g supernatant of liver = liver cytosol < liver microsomes. The high plasma protein binding of the prodrug brought about the extremely slow hydrolysis rate of isovaleryl-PL in plasma. No difference was observed in the hydrolysis rate between the isomers of isovaleryl-PL in buffers. The hydrolysis rate was 2-3 times faster with the (R)-isomer than with the (S)-isomer using racemate in dog plasma and liver preparations. The hydrolysis of each enantiomer was inhibited by the other enantiomer. For hydrolysis in microsomes the Km values of (R)- and (S)-isomers were same, and the Vmax of the (R)-isomer was 3 times greater than that of the (S)-isomer. These data suggested the mutual interaction of (R)- and (S)-isomers during the hydrolysis process and the rapid hydrolysis of isovaleryl-PL in liver after absorption. The AUC of PL enantiomers after oral administration of racemic isovaleryl-PL was about 2 times higher compared to 2 mg/kg equivalent molar dose of racemic PL in beagle dogs, and the corresponding plasma levels were not stereoselective from both PL and prodrug. The amount of (R)-PL absorbed after administration of a 5 mg/kg dose of racemic PL was 2-fold greater than (S)-PL, because of the stereoselective oxidation and glucronidation of (S)-PL.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and antitumor evaluation of bis[(pivaloyloxy)methyl] 2'-deoxy-5-fluorouridine 5'-monophosphate (FdUMP): a strategy to introduce nucleotides into cells

The bis[(pivaloyloxy)methyl] [PIV2] derivative of 2'-deoxy-5- fluorouridine 5'-monophosphate (FdUMP) was synthesized as a potential membrane-permeable prodrug of FdUMP. The compound was designed to enter cells by passive diffusion and to revert to FdUMP after removal of the PIV groups by hydrolytic enzymes. The most convenient preparation of PIV2FdUMP was by condensation of 2'-deoxy-5-fluorouridine (FUdR) with PIV2 phosphate in the presence of triphenylphosphine and diethyl azodicarboxylate (the Mitsunobo reagent). PIV2FdUMP was stable in the pH range 1.0-4.0 (t1/2 > 100 h). It was also fairly stable at pH 7.4 (t1/2 = 40.2 h). In 0.05 M NaOH solution, however, it was rapidly degraded (t1/2 < 2 min). In the presence of hog liver carboxylate esterases, PIV2FdUMP was converted quantitatively to the mono-[(pivaloyloxy)methyl] [PIV1] analogue PIV1FdUMP. After a 24 h incubation, only trace amounts of FdUMP (1-3%) were observed, indicating that PIV1FdUMP is a poor substrate for carboxylate esterases. In mouse plasma, PIV2FdUMP was rapidly metabolized, first to PIV1FdUMP and then to FdUMP. With continued incubation, FUdR was formed, presumably due to further catabolism of FdUMP by plasma phosphatases or 5'-nucleotidases. Since PIV1FdUMP is a poor substrate for carboxylate esterase, the cleavage of the second PIV group is most likely mediated by plasma phosphodiesterases. The rate of degradation of PIV2FdUMP in the presence of acid and alkaline phosphatase, 5'-nucleotidase, or spleen phosphodiesterase was the same as that in buffer controls, indicating that the compound is not a substrate for these nucleotide catabolizing enzymes. The concentration of PIV2FdUMP and its 3'-O-acetyl ester (PIV2 3'-O-Ac-FdUMP) required to inhibit the growth of Chinese hamster ovary (CHO) cells in vitro to less than 50 cells per colony was 5 x 10(-6) M, the same as that required for 5-fluorouracil (FU). Both nucleotide prodrugs showed the same growth-inhibitory potency against a mutant CHO cell line that was 20-fold resistant to FU (CHO/FU). Administered intraperitoneally at optimal dosage for 5 consecutive days, PIV2FdUMP and PIV2 3'-O-Ac-FdUMP were as effective as FU at prolonging the life spans of mice bearing intraperitoneally implanted P388 leukemia. Both prodrugs retained full therapeutic activity against a P388 subline resistant to FU. Collectively, these data indicate that PIV2FdUMP and PIV2 3'-O-Ac-FdUMP are effective membrane-permeable prodrugs of FdUMP.     

Enhancement of the oral bioavailability of phenytoin by N-acetylation and absorptive characteristics

To improve the absorbability of phenytoin (DPH), a prodrug, N-acetyl-DPH (EDPH), was synthesized, and the absorptive characteristics and pharmacokinetics of the prodrug were evaluated in rats. EDPH was rapidly hydrolyzed to DPH in the intestinal fluid and the mucosa (rate constant, 0.055 and 0.169 min(-1), respectively). The plasma concentrations of DPH after intravenous dosing of EDPH declined in a biexponential manner, although two different elimination patterns were observed in these rats. When dosed orally (25 mg/kg, DPH equivalent), the plasma levels of DPH converted from the prodrug were significantly higher and more sustained than those after DPH alone, giving bioavailability 11.4 (rapid decay) and 9.1 times (slow decay) as high, respectively, as that after DPH alone. The concentrations of DPH distributed into the mucosa of the duodenum and jejunum 1 and 5 h after oral dosing of EDPH were significantly higher than those after DPH alone. The prodrug and DPH converted from the prodrug dissolved 2-4 fold more than DPH alone in bile salt solution and bile salt-oleic acid mixed micelles, indicating the increased solubility of the prodrug in the intestinal fluid. It is concluded from the data that such high solubility of EDPH enhanced the intestinal absorption of the prodrug, part of which would be absorbed in the amide form, and thus gave the high bioavailability.     

Enzyme immunoassay for specific analysis of pancreatic stone proteins in human pancreatic juice

In order to study the concentration of pancreatic stone protein (PSP) in human pancreatic juice, we investigated the influence of the insoluble form of PSP-S1 converted from PSP-S2-5 on PSP determination and the assay method for PSP-S1 precipitate after solubilizing PSP-S1. When bovine trypsin was added to pancreatic juice, PSP-S1 was converted from PSP-S2-5 and precipitated about 45-85% after 1 h. The precipitated PSP-S1 was dissolved in 0.1 M sodium acetate buffer, pH 4.0, and the concentration was measured by the enzyme immunoassay, with similar reactivity to PSP-S1 and PSP-S2-5. The proposed method can offer accurate and specific analysis of the PSP level in pancreatic juice. The results of the fractionation of pancreatic juice and duodenal juice on Mono S cation-exchange chromatography suggested that the major component of PSP was PSP-S2-5 in pancreatic juice and PSP-S1 in duodenal juice.     

Resolution of the diastereomers of a large synthetic peptide by capillary electrophoresis using nonionic surfactants

A capillary electrophoretic method was developed for the determination of the diastereomeric composition of the bradykinin B2 antagonist, SB-238592-DB. The nonionic surfactant Brij 35 was demonstrated to be suitable for the baseline resolution of the three diastereoisomers of SB-238592-DB in the presence of a low pH phosphate buffer and small volume percentages of acetonitrile.     

The character of inhibition of the metabolism of 1,2-benzopyrone (coumarin) by grapefruit juice in human

OBJECTIVE: Constituents of grapefruit juice are known to interfere with mammalian cytochrome P450 isozymes such as intestinal CYP3A4 and hepatic CYP2A6, lowering the biotransformation of drugs and increasing their bioavailability. The aim of this study was to investigate whether the presence of naringin is demanded for the inhibition of the coumarin 7-hydroxylase in man or other compounds are responsible for it. METHODS: In cross-over studies, doses of 10 mg coumarin, together with combinations of grapefruit juice, water and naringin, were given orally to one healthy male volunteer, We investigated increasing amounts of grapefruit juice, keeping the volume of liquid constant at 1 L; increasing doses of naringin given in water; increasing amounts of juice, keeping the dose of naringin constant; or increasing doses of naringin, keeping the amount of juice constant. Urine samples were collected up to 24 h after dosing and 7-hydroxycoumarin was quantified fluorimetrically in urine hydrolysates after HPLC separation to determine the excretion rates. RESULTS: While increasing amounts of grapefruit juice delay the excretion of 7-hydroxycoumarin by 2 h, increasing doses of naringin in water up to twofold (i.e. naringin content of 2 L grapefruit juice) do not cause any alteration in the time course of excretion. Experiments with increasing amounts of juice, keeping the dose of naringin constant, indicate that the inhibitory potency of small amounts of grapefruit juice can be amplified by naringin. The same is true when the ratio between juice constituents and naringin is enhanced up to threefold by adding naringin. CONCLUSION: As naringin alone is ineffective, the inhibitory effect of grapefruit juice on the metabolism of coumarin is caused by at least one compound other than naringin. The persistency of the primary inhibitor not identified yet can obviously be modulated by the naring(en)in-system.     

The effect of synthetic surfactant Exosurf on gene transfer in mouse lung in vivo

Gene transfer in the lung holds promise for the treatment of diseases such as pulmonary fibrosis, cystic fibrosis and asthma. Pulmonary surfactant has been reported to enhance expression from endobronchial, adenovirus-mediated gene transfer in experimental animals. This study examines the effect of exogenous synthetic surfactant (Exosurf) on gene expression from naked plasmid DNA administered endobronchially to adult mice. Transfection efficiency was evaluated by quantifying the expression of chloramphenicol acetyltransferase (CAT) and luciferase (Luc) genes in the lung. Endobronchial administration of either CAT or Luc expression plasmid DNA resulted in detectable concentrations of each reporter protein. CAT expression from plasmid DNA was monitored after endobronchial administration with the maximal expression observed at 3-5 days after administration and decreasing for 5 days thereafter. When DNA was delivered in a 50% suspension of Exosurf, the expression of either CAT or Luc was significantly reduced by 89.6 +/- 1.4% and 82.7 +/- 10.5%, respectively. The decrease in Luc expression was closely correlated (r = 0.99, P < 0.001) to log concentration of surfactant in the plasmid buffer solution (IC50 = 8.6%). CAT expression was not altered when surfactant was administered either 2 h before or after plasmid DNA instillation. Examination of the components of Exosurf revealed that two compounds, DPPC and tyloxapol, showed inhibitory effects on CAT expression. However, the inhibition caused by Exosurf appeared greater than that of either component. Our results suggest that the lung surfactant is a barrier to transfection of the endobronchial airway and may be partly responsible for the low expression of exogenous DNA in vivo in the bronchial tree.     

Effects of combretastatin on murine tumours monitored by 31P MRS, 1H MRS and 1H MRI

PURPOSE: Combretastatins have tubulin-binding activity and are being investigated for their toxicity against tumour vasculature. We report the use of 31P and 'H magnetic resonance (MR) spectroscopy and 1H MR imaging for monitoring the effects of combretastatin A-4 prodrug (100mg/kg, i.p.) on energy metabolism and necrosis, respectively, in the C3H murine mammary tumour. MATERIALS AND METHODS: The tumours (volume ca. 200mm3) were grown in the hind foot of mice. MR examinations were performed without anaesthesia within a 7.1 Tesla magnet. 31P MRS (TR = 6 s) was performed before treatment and at 1-, 2-, 3-, and 24-h after injection of drug or saline via an i.p. line. 1H MRS (PRESS; 24microl voxel; TR = 2 s; TE = 135 ms) and both T1-weighted (TR = 0.2 s; TE = 0.02 s) and T2-weighted (TR = 2 s; TE = 0.20 s) 1H MRI were performed before treatment and 2.5 and 24 h afterwards. RESULTS: The ratio beta-nucleotide triphosphate/inorganic phosphate fell by 33% within 1 h of treatment and remained constant for a further 2 h. A small but significant fall in pH (by 0.11 units) was observed at 1 h. Although an increase in the 1H MR spectroscopy signal at about 1.32 ppm (predominantly from lactate) was observed in some tumours following combretastatin treatment, this effect was not seen consistently. No changes in the intensity of T2-weighted 1H MR images or in tumour necrosis (measured histologically) were detected within 3 h of treatment. CONCLUSIONS: The reduction in tumour energetics and pH was consistent with a reduction in tumour blood flow but this occurred before any significant incidence of haemorrhagic necrosis was detected. The combretastatin dose used to achieve these effects was less than one tenth of the maximum tolerated dose in mice.     

Iodine-123 labelled nor-beta-CIT binds to the serotonin transporter in vivo as assessed by biodistribution studies in rats

Flavobacterium aurantiacum NRRL B-184 possesses the ability to degrade aflatoxin B1 in solution and in several food items. Aflatoxin B1 is a potent carcinogen that causes significant economic losses to the agricultural and food industry. The role of trace metal ions (Cu2+, Mn2+, Zn2+, and Co2+) were studied in an effort to understand the enzymatic system involved in aflatoxin B1 degradation by F aurantiacum. The effect of divalent chelators (EDTA and 1,10-phenanthroline [OPT]) in the presence of the trace metal ions was studied as well. Aflatoxin B1 (10 microg/ml) was added to 72-h cultures of F aurantiacum that had been washed and resuspended in phosphate buffer (pH 7.0). HPLC was used to determine aflatoxin B1 concentration in these cultures. Incubating cells at 30 degrees C with 1 and 10 mM Cu2+, Mn2+, and Zn2+ significantly decreased aflatoxin B degradation after 4 and 24 h (P < 0.05). Decreased degradation was also observed with 1 and 10 mM Cu2+ and Zn2+ after 48 h and with 0.1 mM Cu2+ after 24 and 48 h. Co2+ did not have a significant effect on aflatoxin B1 degradation. EDTA and OPT did not counter the inhibition in the presence of Cu2+. The addition of 1 mM EDTA countered the inhibition by 1 mM Mn2+ after 4 and 24 h, but 1 mM OPT did not counter the inhibition by 10 mM Mn2+ after 4 and 24 h. OPT countered the inhibition by 1 mM Zn2+ after 4 and 48 h. These trace elements inhibit aflatoxin B1 degradation by F aurantiacum. In addition, their presence necessitates higher concentrations (>1 mM) of EDTA and OPT for the removal of their inhibitory effect.     

Filter Sand-Phosphate Buffer Effect on 2,4-Dinitrotoluene Ozonation

The objective of this study is to evaluate the effects of different sands and phosphate buffer on ozone decomposition by measuring the observed ozone decomposition rate constant of three different natural sands at pH 7 with and without the presence of phosphate buffer and with and without sand (i.e., aqueous ozone only). In addition, this study evaluated phosphate buffer effect on the heterogeneous catalysis of ozone for the destruction of 2,4-dinitrotoluene (DNT) by measuring the observed DNT degradation rate constant (kDNT). Ozone decomposition rates in the presence of NaOH buffer and sand were up to 7.9 times greater and 3.4 times in the presence of phosphate. In the absence of phosphate, sand reduced the observed rate of DNT degradation. In the presence of phosphate, sands enhance the observed DNT degradation rate by a factor approximately proportional to their surface manganese concentration. A proposed mechanism was developed explaining the observed effects of the phosphate in a sand ozone system.     

N3-methyl-mafosfamide as a chemically stable, alternative prodrug of mafosfamide

The presence of an alkyl substituent at N3 in the oxazaphosphorine ring stabilizes N-substituted 4-(alkylthio)cyclophosphamides from spontaneous decomposition. Based on this finding, N3-methyl-mafosfamide was synthesized and examined as a chemically stable, biooxidative prodrug of mafosfamide. This prodrug was stable in aqueous buffer (pH 7.4, 37 degrees C) and underwent N-demethylation in a time dependent manner when incubated with rat hepatic microsomes. N3-Methyl-mafosfamide was 10-fold more cytotoxic in vitro than cyclophosphamide against mouse embryo Balb/c 3T3 cells (LC50 = 3.6 microM). Preliminary in vivo antitumor evaluation against L1210 leukemia in mice showed that this prodrug was active [Increase of life span (ILS) > 29%].     

DNA degradation during standard alkaline of thermal denaturation

Essential degradation 8 DNA (up to 10 per cent) with liberation of acid-soluble fragments takes place on the standard alkaline (0,01 M sodium phosphate, pH 12, 60 degrees, 15 min) or thermal (0.06 M sodium phosphate buffer, pH 6.8, 102 degrees C, 15 min) denaturation. This degradation is more or less selective: fraction of low molecular weight fragments, isolated by hydroxyapatite cromatography and eluted by 0.06 M sodium phosphate buffer, pH 6.8 is rich in adenine and thymine and contains about 2 times less 5-methylcytosine than the total wheat germ DNA. The degree of degradation of DNA on thermal denaturation is higher than on alkaline degradation. Therefore while studying reassociation of various DNA, one and the same standard method of DNA denaturation should be used. Besides, both the level of DNA degradation and the nature of the resulting products (fragments) should be taken into account.     

A comparison of the bioconversion rates and the Caco-2 cell permeation characteristics of coumarin-based cyclic prodrugs and methylester-based linear prodrugs of RGD peptidomimetics

PURPOSE: To compare the bioconversion rates in various biological media and the Caco-2 cell permeation characteristics of coumarin based cyclic prodrugs (3a, 3b) and methylester-based linear prodrugs (1b, 2b) of two RGD peptidomimetics (1a, 2a). METHODS: Bioconversion rates of the prodrugs to the RGD peptidomimetics were determined in Hank balances salt solution (HBSS), pH 7,4, at 37 degrees C and in various biological media (human blood plasma, rat liver homogenate, Caco-2 cell homogenate) known to have esterase activity. Transport rates of the prodrugs and the RGD peptidomimetics were determined using Caco-2 cell monolayers, an in vitro cell culture model of the intestinal mucosa. RESULTS. In HBSS, pH 7,4, the coumarin-based cyclic prodrugs 3a and 3b degraded slowly and quantitatively to the RGD peptidomimetics 1a and 2a, respectively (3a, t1/2 = 630+2-14 min; 3b, t1/2 = 301 +/-12 min). The methylester-based linear prodrugs 1b and 2b were more stable to chemical hydrolysis (1b and 2b, t1/2 > 2000 min). Both the coumarin-based cyclic prodrugs and the methylester-based linear prodrugs degraded more rapidly in biological media containing esterase activity (e.g., 90% human blood plasma: 1b, t1/2 < 5 min; 2b, t1/2 < 5 min; 3a, t1/2 < 91+/-1 min; 3b, 1/2 < 57+/-2 min). When the apical (AP)-to-basolateral (BL) permeation characteristics were determined using Caco-2 cell monolayers, it was found that the methylester prodrugs 1b and 2b underwent esterase bioconversion (>80%) to the RGD peptidomimetics 1a and 2a, respectively, In contrast, the cyclic prodrugs 3a and 3b permeated the cell monolayers intact. Considering the appearance of both the prodrug and the RGD peptidomimetic on the BL side, the methylester prodrugs 1b and 2b were approximately 12-fold more able to permeate than were the RGD peptidomimetics 1a and 2a. When similar analysis of the transport data for the coumarin prodrugs 3a and 3b was performed, they were shown to be approximately 6-fold and 5-fold more able to permeate than were the RGD peptidomimetics 1a and 12a, respectively. CONCLUSION: The coumarin-based cyclic prodrugs 3a and 3b were chemically less stable, but metabolically more stable, then the methylester based linear prodrugs. The esterase stability of the cyclic prodrugs 3a and 3b means that they are transported intact across the Caco-2 cell monolayer in contrast to the methylester prodrugs 1b and 2b, which undergo facile bioconversion during their transport to the RGD peptidomimetics. However, both prodrug systems successfully delivered more (5-12-fold) of the RGD peptidomimetic and/or the precursor (prodrug) than did the RGD peptidomimetics themselves.