Studies on the function of intracellular ribonucleases. IV. Some observations on the properties of ribosomes and polysomes from rat liver and hepatomas.

Polysome and ribosome preparations from normal rat liver and from a series of transplantable rat hepatomas of different growth rates were compared. All the hepatomas had a significantly higher percentage of RNA in a polysome preparation than did the normal liver, and the polysome preparations from the tumors, with the exception of the Dunning hepatoma which has a high lipid content, gave a greater yield of RNA and protein per gram of wet tissue than the liver did. Heavier polysomes were considerably less prevalent in the tumors than in the liver, and the tumors contained a larger proportion of monomer and dimer ribosomes than the liver did. Evidence is presented that the increased monomer and dimer ribosome population of the hepatomas studied is not an artifact of preparation, but represents the true intracellular distribution. Ribosomes from normal liver and Morris 5123-D hepatoma were readily dissociated by 20 min' treatment with 1.0 mM EDTA, but ribosomes from the Dunning, Novikoff ascites, and McCoy MDAB hepatomas were little affected by such treatment. With higher concentrations of EDTA, the ribosomes from the Novikoff ascites and McCoy MDAB hepatomas broke down and did not form specific subunits as did ribosomes from liver and the Morris 5123-D hepatoma but rather gave rise to a variety of small degradation products. This behavior is ascribed to a higher RNase content of the Novikoff and McCoy MDAB hepatomas. Dunning hepatoma ribosomes were resistant to 4 mM EDTA.

Studies on the function of intracellular ribonucleases. V. Ribonuclease activity in ribosomes and polysomes prepared from rat liver and hepatomas.

The RNase activity and properties of ribosome and polysome preparations from normal rat liver and some hepatomas have been examined. Polysome and ribosome preparations from the Novikoff, McCoy MDAB, and Dunning hepatomas had considerably higher specific RNase activity than corresponding preparations from normal rat liver, Novikoff ascites, or Morris 5123 hepatomas. The optimum pH of the RNase was approximately 8.5 for all samples tested, and the samples showed no evidence of latent RNase activity when treated with 3 M sodium chloride, EDTA, urea, or p-chloromercuribenzenesulfonic acid. The RNase activity appeared to be associated principally with breakdown products and/or subunits smaller than 80S. In the presence of Mg(++) ions, subunits could reaggregate to form monomer ribosomes indistinguishable from the natural products, but some of the reassociated ribosomes could contain RNase activity which had been bound to the smaller particles. Similar results were obtained with spermine. In the hepatomas, evidence was obtained for the preexistence of considerable amounts of the smaller, RNase-containing subunits in the cell. When a small amount of crystalline bovine pancreatic RNase was added to partly dissociated ribosomes, the RNase was found only in association with the smaller subunits, and little or no enzyme was taken up by ribosomes or polysomes. The results have led to the conclusion that RNase is not a normal constituent of the ribosome or polysome, but that RNase may become associated with these particulates if dissociation and reassociation take place. Some implications of these findings for the stability of messenger RNA and for the mechanism of its breakdown are discussed.

Abdominal wall reconstruction with large polypropylene mesh: is bigger better?

PURPOSE: Hernia repair for large and complex hernias presents challenges related to the availability of larger mesh sizes. When sizes beyond those manufactured are required, multiple meshes (MM) may be sutured to create a larger graft. With the availability of large polypropylene mesh up to 50 × 50 cm (LM), abdominal wall reconstruction (AWR) may be accomplished with a single mesh. This study evaluates clinical and economic outcomes following AWR with component separation utilizing MM and LM. METHODS: A retrospective study was performed with review of health records and cost accounting data. Patients that underwent AWR with LM were case matched 1:1 with patients undergoing MM repair based upon comorbidities, defect size and wound class. RESULTS: Twenty-four patients underwent AWR with LM. Twenty patients (10F, 10 M) who underwent AWR with LM were matched with 20 MM AWR (11F, 9 M). Age, BMI, ASA 3 + , never smoker, diabetes, and hernia characteristics were similar between LM and MM. Operative cost ($4295 vs $3669, p = 0.127), operative time (259 min vs 243 min, p = 0.817), length of stay (5.5 vs 6.2, p = 0.484), wound complication (30% vs 20%, p = 0.716), infected seroma (5% vs 5%, p = 1), and readmission (5% vs 15%, p = 0.605) were similar between LM and MM, respectively. CONCLUSIONS: This is the first report of patients undergoing AWR with a large 50 × 50 cm prolene mesh. In this small cohort, clinical outcomes were similar between those undergoing repair with multiple sutured mesh sheets and a single large mesh.

Associations between anxiolytic medications and ventral hernia repair.

PURPOSE: This study examines the relationship between anxiolytic medications (AXM) on outcomes following ventral hernia repair. METHODS: A single-center review of prospectively obtained perioperative and 30-day outcome data, including AXM use at admission, as part of the National Surgery Quality Improvement Program. RESULTS: Sixty-three of the 393 patients who presented for ventral hernia repair were taking an AXM (15.6%). AXM users were more likely to have a higher ASA class, dyspnea, and treated hypertension (p < 0.05). AXM use was associated with increased operative duration, hernia size, increased estimated blood loss, and need for component separation. After adjusting for medical comorbidities, AXM users were not found to have greater 30-day morbidity or mortality. Patients taking AXM were found to have greater length of stay and increased hospital readmissions. CONCLUSIONS: Patients taking anxiolytic medications undergoing ventral hernia repairs have higher ASA scores, more complex hernia characteristics, and require more concurrent procedures. They were found to have longer operative times, increased blood loss, greater duration of hospital stay, and increased readmissions that were associated with the increased perioperative risk factors. Further studies are required to determine causal links.

Factors affecting deoxycytidylate deaminase activity in some transplantable rat hepatomas.

Dunning hepatoma has a low activity of deoxycytidylate deaminase, comparable to that of normal adult rat liver. This activity seems inconsistent with the rapid proliferation rate of the tumor. Factors which might affect the activity of deoxycytidylate deaminase in the Dunning hepatoma have been examined in it and compared to the Novikoff hepatoma which has high activity of this enzyme. The low activity in Dunning hepatoma does not appear to be the result of any inhibition or, possibly, proteolytic enzyme as judged by mixing experiments, nor does it appear to be due to in vivo differences in nucleotide concentrations especially deoxycytidine 5'-monophosphate, deoxycytidine 5'-triphosphate, or deoxyguanosine 5'-monophosphate which might either help stabilize the enzyme, allosterically increase its activity, or inhibit it. The Dunning hepatoma does not convert cytosine deoxyriboside to uridine deoxyriboside at a significant rate, and the formation of uridine deoxyriboside from deoxyuridine monophosphate is 1% or less during a 30-min incubation of high-speed supernatant fraction from the tumor in either the presence or absence of fluoride. It is concluded that the Dunning hepatoma probably has intrinsically low deoxycytidylate deaminase activity.

The reduction of uridine 5'-diphosphate and uridine 5'-triphosphate in some transplantable rat hepatomas.

The reduction of uridine 5'-diphosphate (UDP) and uridine 5'-triphosphate (UTP) has been studied in normal adult rat liver, the Dunning hepatoma, and Morris 5123D and 7793 hepatomas. A new paper chromatographic method that separates and quantitates all the major products of the reduction and hydrolysis or other reactions of the substrate has been devised. All of the above tissues were able to reduce UDP and UTP at relatively slow rates ranging from 0.25 nmole of deoxycompound formed (deoxyuridine 5'-triphosphate) per mg protein per hr for liver to 3.5 nmoles deoxyuridine 5'-triphosphate for the Morris 7793 hepatoma when UTP was the substrate. In general, UTP was a better substrate than UDP. The method may also be used to measure cytidine 5'-diphosphate (CDP) reduction, and under the same conditions, the reduction of CDP proceeded at about 6 times the rate of UTP reduction in the Dunning hepatoma. Like CDP reduction, the reduction of UTP was strongly modulated by ATP. Reduction of UTP was insignificant with no ATP or 1.5 micronmoles ATP added to the reaction mixture and was maximal with 0.25 micronmole. The reduction of UTP was inhibited by deoxyuridine 5'-monophosphate, deoxythymidine 5'-triphosphate, deoxycytidine 5'-triphosphate, and deoxyribose 1'-phosphate. The effects of deoxyadenosine 5'-triphosphate varied, depending on its concentration in the reaction medium and whether UDP or UTP was a substrate. However, hydroxyurea did not inhibit reduction of UDP or UTP at concentrations that strongly inhibited CPD reduction. All of the tissues were able to hydrolyze [alpha-32P]deoxyuridine 5'-triphosphate readily to the diphosphate and monophosphate. It is suggested that the enzyme that reduces UTP or UDP may be different in these tissues from the enzyme that reduces CDP.

Thymidylate synthetase activity in the Novikoff hepatoma.

The mechanism by which injected methotrexate increases thymidylate synthetase activity in the Novikoff hepatoma has been studied. Folic acid injection causes a similar increase in enzyme activity in hepatoma after 16 hr but the action of folic acid and methotrexate is not additive. The increase in activity of thymidine 5'-phosphate synthetase in the hepatoma caused by methotrexate is not affected by actinomycin D, but is inhibited 50% by puromycin and 100% by cycloheximide. High-speed supernatent fraction prepared from hepatoma of animals treated with methotrexate has, initially, one-half the specific thymidine 5'-phosphate synthetase activity of untreated controls. Upon addition of increasing amounts of tetrahydrofolate, the specific enzyme activity in the supernatant fraction from the methotrexate-treated animals rises to double that of the controls. Puromycin added to homogenates of Novikoff hepatoma consistently increases enzyme activity by approximately 20%. One hypothesis consistent with these results and results reported by others is presented.

Isolation of a complementary DNA encoding the catalytic subunit of protein kinase A and studies on the expression of this sequence in rat hepatomas and regenerating liver.

A complementary DNA (cDNA) clone (B4) encoding the catalytic subunit of a cAMP-dependent protein kinase (PKAc) was isolated from a lambda gt10 rat brain cDNA library, using a synthetic oligonucleotide probe whose sequence was based on the known amino acid sequence of a bovine cardiac PKAc. Sequence analysis of this clone revealed a region of 1002 nucleotides which encodes a protein that is 92% homologous to amino acids 17-350 of the bovine cardiac PKAc protein. This clone lacks coding sequences for amino acids 1-16 of the latter protein. Nevertheless, it provided a useful probe to analyze expression of the related gene in a variety of systems. Northern blot analyses using a 32P-labeled probe prepared from a 0.6-kilobase PstI fragment of clone B4 revealed an abundant 4.6-kilobase band in rat brain RNA and lesser amounts of this 4.6-kilobase RNA in rat heart and liver. A 4.6-kilobase RNA was also detected in RNA samples obtained from mouse fibroblasts. This probe also detected homologous RNA in a variety of nonrodent species. In subsequent experiments, this cDNA was used as a probe to elucidate the role of PKAc in post-surgical hepatic regeneration and diethylnitrosamine-induced hepatomas in the rat. These experiments revealed that, following partial hepatectomy, PKAc mRNA is decreased 3-fold by 12 h, returning to normal by 72 h; hepatomas showed no consistent pattern of change in PKAc mRNA levels as compared to controls. Our results indicate that this cDNA encodes an isoform of PKAc which is distinct from PKAc-alpha isolated by Uhler et al. (Proc. Natl. Acad. Sci. USA, 83: 1300-1304, 1986) but highly homologous to PKAc-beta isolated by Showers and Maurer (J. Biol. Chem., 261: 16288-16291, 1986), that depression of cAMP-dependent protein phosphorylation may be an important mechanism in the regeneration of mature rat liver but is not a consistent alteration in chemically induced hepatoma, and that this cDNA is useful as a probe for the study of the role of PKAc gene expression in growth control, particularly in rodent species.

Phase I bioavailability and pharmacokinetic study of hexamethylene bisacetamide (NSC 95580) administered via nasogastric tube.

A Phase I clinical trial and pharmacological study of nasogastrically administered hexamethylene bisacetamide, a polar-planar compound with in vitro differentiating activity, was conducted in 14 adult patients with refractory cancer. Hexamethylene bisacetamide was administered as a 5% (w/v) solution via a nasogastric or gastrostomy tube every 4 h for 5 days, followed in 21 days by a 5-day continuous i.v. infusion at the same daily dose. Parenteral drug administration was then continued at the same interval in the absence of disease progression or unacceptable toxicity. Three patients each were treated at doses of 12 and 24 g/m2/day, while eight patients received a dose of 30 g/m2/day. Toxicity was comparable for both routes of drug administration at the above doses. Nasogastrically administered hexamethylene bisacetamide was well tolerated at the lower doses, whereas neurotoxicity and nausea and vomiting were the major, but manageable, toxicities at 30 g/m2/day. Metabolic acidosis, renal dysfunction, mucositis, and thrombocytopenia were the other commonly observed drug toxicities at this dose. No objective tumor responses were observed. Hexamethylene bisacetamide was rapidly absorbed from the gastrointestinal tract with a mean measured bioavailability of 99 +/- 15%. Pharmacokinetic parameters for hexamethylene bisacetamide and plasma concentrations of the two major metabolites, N-acetyl-1,6-diaminohexane and 6-acetamidohexanoic acid, were similar for either route of administration in individual patients. Hexamethylene bisacetamide exhibited apparent monoexponential plasma elimination after either nasogastric or parenteral administration with 27 to 60% of the administered dose being excreted in the urine as parent compound. Based on its demonstrated complete bioavailability and tolerability, nasogastric administration of hexamethylene bisacetamide can be directly and safely substituted for the comparable i.v. dose.

A phase I and pharmacokinetic study of dihydro-5-azacytidine (NSC 264880).

5,6-Dihydro-5-azacytidine (DHAC; NSC 264880) is an analogue of 5-azacytidine that does not possess the hydrolytically unstable 5,6-imino bond of the parent compound. Thus, unlike 5-azacytidine, DHAC is stable in aqueous solution and may be administered by prolonged i.v. infusion, potentially avoiding acute toxicities associated with bolus administration of 5-azacytidine. In this study, patients with advanced cancer were treated with DHAC administered as a 24-h constant i.v. infusion every 28 days. Treatment began at a dose of 1 g/sq m and was escalated to the maximum-tolerated dose of 7 g/sq m, where the limiting toxicity was pleuritic chest pain. Other toxicities included nausea and vomiting, which were not limiting. There was no evidence for myelosuppression, nephrotoxicity, or hepatotoxicity. DHAC was measured in plasma, urine, and ascites by a sensitive and specific reverse-phase high-performance liquid chromatography assay capable of detecting 50 ng of drug per ml. Steady-state plasma levels were achieved with 8 h and ranged from 10.0 to 20.5 micrograms of DHAC per ml at the maximum-tolerated dose. Total-body clearance of 311 +/- 76 ml/min/sq m and postinfusion half-lives between 1 and 2 h were observed. Between 8 and 20% of the administered dose was excreted unchanged in urine. While ascites DHAC levels in a patient with ovarian cancer were comparable to plasma levels, postinfusion elimination was slower from this compartment than from plasma. No correlation was observed between DHAC plasma levels and duration or intensity of dose-limiting pleuritic chest pain. One patient with progressive Hodgkin's lymphoma demonstrated stabilization of disease for seven treatment cycles, and two patients with aggressive lymphoma demonstrated dramatic, although transient, disease responses. A dose of 7 g/sq m is recommended for Phase II trials of DHAC using this schedule.

A pediatric phase I and pharmacokinetic study of spirohydantoin mustard.

A pediatric Phase I and pharmacokinetic study of the lipophilic alkylating agent spirohydantoin mustard (SHM) was conducted in 23 patients. The dose-limiting toxicity of SHM was neurological with disorientation, delirium, or hallucinations occurring in 9 of 23 patients. These symptoms were partially reversible and preventable with physostigmine. In 17 patients who were evaluable for response to treatment (14 of whom had central nervous system malignancies), no objective tumor responses were observed. Pharmacokinetic evaluation of SHM revealed a t1/2 alpha of 1.7 +/- 0.7 min, t1/2 beta of 16 +/- 8.3 min, and total body clearance of 2134 +/- 735 ml/min/m2. Measureable peak plasma levels were less than 40% of that which produces cytotoxicity in vitro against monolayer cultures of rat 9L brain tumor. Over 90% of SHM was protein bound, greatly limiting the free drug available for central nervous system penetration. SHM cerebrospinal fluid to plasma ratios were less than 0.047. The above suggests that in spite of its lipophilicity, SHM may not reach clinically significant levels in the central nervous system at clinically tolerable doses.

Complex ventral hernia repair with a human acellular dermal matrix.

PURPOSE: The ideal approach to complex ventral hernia repair is frequently debated. Differences in processing techniques among biologic materials may impact hernia repair outcomes. This study evaluates the outcomes of hernia repair with a terminally sterilized human acellular dermal matrix (TS-HADM) (AlloMax(®) Surgical Graft, by C. R. Bard/Davol, Inc., Warwick, RI, USA) treated with low-dose gamma irradiation. METHODS: A single-arm multi-center retrospective observational study of patients undergoing hernia repair with TS-HADM was performed. Data analyses were exploratory only; no formal hypothesis testing was pre-specified. RESULTS: Seventy-eight patients (43F, 35M) underwent incisional hernia repair with a TS-HADM. Mean follow-up was 20.5 months. Preoperative characteristics include age of 56.6 ± 11.1 years, BMI 36.7 ± 9.9 kg/m(2), and mean hernia defect size 187 cm(2). Sixty-five patients underwent component separation technique (CST) with a reinforcing graft. Overall, 21.8% developed recurrences. Recurrences occurred in 15% of patients repaired with CST. Major wound complications occurred in 31% of patients overall. Based upon CDC surgical wound classification, major wound complications were seen in 26, 40, 56, and 50% of Class 1, 2, 3, and 4 wounds, respectively. No grafts required removal. CONCLUSIONS: Hernia recurrences are not uncommon following complex abdominal wall reconstruction. Improved outcomes are seen when a TS-HADM is utilized as reinforcement to primary fascial closure.

Evidence for the presence of DNase-actin complex in L1210 leukemia cells.

L1210 leukemia cell cytosol was analysed for the presence of DNase I activity. No free activity was determined in crude cytosol. DNase I enzyme was found to occur in a latent form bound to cytoplasmic actin. DNase-actin complex was partially isolated by Sephadex filtration and DNase I-like activity was demonstrated after SDS gel electrophoresis of the complex and enzyme renaturation. The results were compared with those for synthetic complex of pancreatic bovine DNase I and chicken muscle actin.

Furanose-pyranose isomerization of reduced pyrimidine and cyclic urea ribosides.

Tetrahydrouridine (THU, 2) and other fully reduced cyclic urea ribofuranosyl nucleosides undergo a rapid, acid-catalyzed isomerization to their more stable ribopyranosyl form. This isomerization is characterized by a change in spectral properties and by a greater than 10-fold decrease in potency for those nucleosides that act as potent inhibitors of cytidine deaminase in their ribofuranose form. 1-(beta-D-Ribopyranosyl)hexahydropyrimidin-2-one (7) was synthesized and used in conjunction with its furanose isomer 6 as a model compound for more extensive 1H and 13C NMR, mass spectral, and kinetic studies of this isomerization. The 0.4 delta upfield shift and 4-Hz increase in the J1',2' coupling constant for the pyranose anomeric proton in the 1H NMR spectrum is indicative of a pyranose beta-CI conformation in which the aglycon and C-2' and C-4' hydroxyls are equatorial. The mass spectra of trimethylsilylated pyranose nucleosides also show a characteristic large shift in the m/z 204-217 abundance and the appearance of two new rearrangement ions at M-133 and M-206. For furanose 6 the rate of isomerization is pH and temperature dependent with pyranose 7 predominating by a factor of 6-9 equilibrium. At pH 1 and 37 degrees C, furanose 6 has an initial half-life of less than 12 min. Accordingly, this isomerization may explain the observed lack of enhanced ara-C levels in studies evaluating the oral administration of an ara-C and THU combination to species with an acidic stomach content.

Chemistry and anti-HIV properties of 2'-fluoro-2',3'-dideoxyarabinofuranosylpyrimidines.

The synthesis, chemistry, biochemistry, and anti-HIV activity of a series of 1-(2,3-dideoxy-2-fluoro-beta-D-threopentofuranosyl)pyrimidines have been studied in an attempt to find useful anti-AIDS drugs. Synthesis is carried out via a 2,3-dideoxyribose intermediate which facilitates the preparation of analogues by removing the sugar 3'-hydroxyl group prior to, rather than after, condensation with a uracil or cytosine aglycon. The 2'-F-dd-uridine analogues 7a-d (with H, F, Cl, and CH3 substitution in the 5-position) as well as the 4-deoxy compound (12b) are nonprotective to ATH8 or CEM cells infected with HIV-1. In the corresponding cytidine series, the 5-chloro analogue (11) is inactive. However, 2'-fluoro-2',3'-dideoxyarabinosylcytosine, 10a, and its 5-fluoro analogue, 10b, are both active. While neither compounds is a potent as ddC or 5-F-ddC (2b), 10b gives complete protection against the cytopathic effects of HIV in both host cell lines. 2'-Fluoro substitution confers increased chemical and enzymatic stability on dideoxynucleosides. Even though dideoxy pyrimidine nucleosides are inherently more stable than the corresponding purine analogues toward acid-catalyzed cleavage of the glycosidic bond, 2'-fluoro substitution (10a) still increases stabilization relative to ddC (2b). No detectable deamination by partially purified cytidine deaminase is observed with the 2'-fluoro compounds 10a, 10b, or 11 under conditions which rapidly deaminate cytidine. A small amount of 2'-F-dd-ara-U (7a) is formed from 10a in monkey plasma after greater than 24 h of exposure. The octanol-water partition coefficients for the dideoxynucleosides in this study indicate their hydrophilic character, with log P values varying from -0.28 to -1.18.

Lipophilic, acid-stable, adenosine deaminase-activated anti-HIV prodrugs for central nervous system delivery. 3. 6-Amino prodrugs of 2'-beta-fluoro-2',3'-dideoxyinosine.

A series of 6-substituted amino analogs of 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl) purines (F-ddN) has been synthesized and characterized with the objective of finding compounds which might be superior to existing drugs for the treatment of HIV in the central nervous system. These compounds are intended to be more lipophilic than the currently approved anti-HIV drugs for better blood-brain barrier penetration. Subsequent adenosine deaminase (ADA)-catalyzed hydrolysis of these prodrugs in the brain is expected to produce the anti-HIV agent, 9-(2,3-dideoxy-2-fluoro-beta-D-threo-pentofuranosyl)hypoxanthine (F-ddI). The new compounds, synthesized from the corresponding 6-chloro analog, include F-ddN which contain methylamino, ethylamino, dimethylamino, hydroxylamino, methoxyamino, benzyloxyamino, hydrazino, and nitro substituents in the 6-position. The 6-nitro analog was isolated as an unexpected product during the preparation of the 6-chloro derivative. Among the analogs with anti-HIV activity, the ethylamino and dimethylamino compounds are ca. 100 times more lipophilic than ddI or F-ddI. As expected, 2'-fluoro substitution protects the compounds from acid-catalyzed glycosylic cleavage. Only the hydroxylamino and nitro analogs underwent any nonenzymatic hydrolysis at pH 1.0 or 7.4. This reaction, however, results in hydrolysis of the group in the 6-position rather than glycosylic bond cleavage. ADA catalyzes the hydrolysis of the 6-substituents at rates which vary from slightly slower (NO2, 1.7x) to much slower (NHEt, 5000x) than F-ddA. The 6-dimethylamino analog is the only compound which possesses anti-HIV activity (ED50 18 microM) without ADA hydrolysis. With the exception of the two inactive alkoxyamino compounds, the other prodrugs exhibited cellular protection in the HIV-1/PHA-PBM system with IC50 potencies of 7-40 microM.

Acid-stable 2'-fluoro purine dideoxynucleosides as active agents against HIV.

2',3'-Dideoxy purine nucleosides have anti-HIV activity in vitro and the inosine analogue is being clinically evaluated. The instability of these compounds toward acidic conditions complicates oral administration. The effect of the addition of a fluorine atom to the 2'-position was investigated by preparing the fluorine-containing 2'-erythro and 2'-threo isomers of ddA and the threo isomer of ddI. All fluorine-containing compounds were indefinitely stable to acidic conditions which completely decomposed ddI (1) and ddA (2) in minutes. While the fluorine-containing erythro isomer, 5, was inactive, the threo isomers, 2'-F-dd-ara-A (3) and 2'-F-dd-ara-I (4), were just as potent and active in protecting CD4+ ATH8 cells from the cytopathogenic effects of HIV-1 as the parent drugs. Exposure to pH 1 at 37 degrees C prior to testing destroyed the activity of ddA and ddI but left the anti-HIV properties of 3 and 4 unchanged. The fluorinated analogues also protected cells exposed to HIV-2 and inhibited gag gene product expression but not as effectively as the parent compounds. The fluorine-containing analogues appear to be somewhat more toxic in vitro to the antigen- and mitogen-driven proliferation of immunocompetent cells than their corresponding parent compounds.

Determination of lodenosine and its major metabolite in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry.

A sensitive and selective method for the determination of 2'-beta-fluoro-2',3'-dideoxyadenosine (lodenosine, F-ddA), an experimental anti-AIDS drug, and its major metabolite, 2'-beta-fluoro-2',3'-dideoxyinosine (F-ddI), in human plasma was developed and validated. The procedure employs two internal standards and a simple ultrafiltration step followed by chromatography on a Betasil C(18) minibore column. An in-line valve is used to remove salts before reaching the ion source. Detection is by electrospray ionization tandem mass spectrometry with selected reaction monitoring. The method has a limit of quantitation of 4 ng ml(-1) (16 nM) for F-ddA and 8 ng ml(-1) (32 nM) for F-ddI with a linear range up to 2000 ng ml(-1) (7.9 microM) for each. Predicted concentrations from a three-day validation study were within 5% of the nominal values for F-ddA and 16% for F-ddI. Intra- and inter-assay precision, as measured by relative standard deviation, was 13% or better for both compounds. To achieve good reproducibility, many variables related to the electrospray ionization were optimized for both precision and sensitivity. The method was successfully employed to analyze samples and evaluate plasma pharmacokinetics from a Phase I clinical trial.

Spiromustine analogues. Relationships between structure, plasma stability, and antitumor activity.

Spiromustine is a hydantoin-containing nitrogen mustard currently in Phase I clinical trial. Since the in vitro plasma half-life of this compound (6.4 min, 37 degrees C, pH 7.4) appeared to be influenced by the hydantoin ring, analogues containing 2-5 methylene spacer groups between this ring and the nitrogen mustard moiety were prepared and evaluated for hydrolytic stability and antitumor activity. Stability correlated with structure and pKa values. The proximity of the hydantoin ring to the mustard function was a stabilizing factor. Activity against murine P-388 leukemia was demonstrated and a gradual decrease in this activity was observed as the hydrolytic instability increased. A relationship between analogue structure and a mass spectral rearrangement ion was identified.