Computational, biochemical and ex vivo evaluation of xanthine derivatives against phosphodiesterases to enhance the sperm motility.

Enhancing sperm motility in vitro has immensely benefited assisted conception methods. Phosphodiesterases (PDE) break the second messenger cAMP, and therefore, inhibition of their catalytic activity enhances the sperm motility through maintaining cAMP homeostasis in sperm. In view of identifying the molecules that could inhibit PDE functioning in spermatozoa, we aimed to evaluate the phosphodiesterase inhibitors (PDEI) - xanthine derivatives - acefylline, dyphylline and proxyphylline to repurpose them for assisted reproductive technology. These are available in the market as pharmaceutical agents to treat mainly respiratory system diseases. Based on the structure guided in silico studies, we predicted that these molecules bind to the cAMP binding catalytic pocket of PDE enzymes, and further molecular dynamics simulation analysis indicated that these molecules form the stable complexes. Isothermal titration calorimetry studies revealed that acefylline has better affinity towards PDE4A, PDE4D and PDE10A, when compared to dyphylline and proxyphylline. In addition, ex vivo studies corroborated in vitro binding studies that acefylline has much superior sperm motility enhancement property on human ejaculated spermatozoa and mouse testicular spermatozoa compared to dyphylline and proxyphylline.Communicated by Ramaswamy H. Sarma.

Chemoenzymatic synthesis of enantiomerically enriched diprophylline and xanthinol nicotinate.

A concise chemoenzymatic route toward enantiomerically enriched active pharmaceutical ingredients (API) - diprophylline and xanthinol nicotinate - is reported for the first time. The decisive step is an enantioselective lipase-mediated methanolysis of racemic chlorohydrin-synthon acetate, namely 1-chloro-3-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)propan-2-yl acetate, performed under kinetically-controlled conditions on a preparative 500 mg-scale. The best results in terms of reaction enantioselectivity (E = 14) were obtained for the enantiomers resolution performed with lipase type B from Candida antarctica immobilized on acrylic resin (CAL-B, Novozym 435) suspended in homophasic acetonitrile-methanol mixture. The elaborated biocatalytic system furnished the key chlorohydrin intermediate (in 71% ee and 38% yield), which was then smoothly converted into enantioenriched active agents: (R)-(-)-diprophylline (57% ee) and (S)-(+)-xanthinol nicotinate (65% ee). To support the assignment of absolute configurations of EKR-products as well as to confirm the stereochemical outcome of the remaining reaction steps, docking studies toward the prediction of enantiomers binding selectivity in CAL-B active site as well as the respective chemical correlations with enantiomerically enriched analytical standards obtained from commercially available (R)-(-)-epichlorohydrin, were applied. In addition, single-crystal X-ray diffraction (XRD) analyses were performed for the synthesized optically active APIs furnishing by this manner a first crystal structures of nicotinic acid salt of xanthinol.

Comparative study of the binding of pepsin to four alkaloids by spectrofluorimetry.

The interactions between pepsin and four alkaloids, including caffeine (Caf), aminophylline (Ami), acefylline (Ace), diprophylline (Dip), were investigated by fluorescence, UV-visible absorption, resonance light scattering, synchronous fluorescence spectroscopy and 3D spectroscopy under mimic physiological conditions. The results revealed that Caf (Ami/Ace/Dip) caused the fluorescence quenching of pepsin by the formation of Caf (Ami/Ace/Dip)-pepsin complex. The binding constants and thermodynamic parameters at three different temperatures, the binding locality and the binding power were obtained. The hydrophobic and electrostatic interactions were the predominant intermolecular forces to stabilize the complex. Results showed that aminophylline was the stronger quencher and bound to pepsin with higher affinity than other three alkaloids.

Synthesis and activity against HBV of novel Tetra-seconucleoside analogues of dyphlline having the acyclic chains of ACV and HBG.

Selective alkylation of dyphylline (1) with (2-acetoxyethoxy)methyl bromide (2a) or 4-acetoxybutyl bromide (2b) afforded 3'-O-[(acetoxyethoxy)methyl]dyphylline (3a) and 3'-O-(4-acetoxybutyl)-dyphylline (3b), respectively. A trans esterification process rather than alkylation of the dihydroxy-propyl side chain in 1 had taken place during the reaction with 2-p-toluoyloxy)ethyl chloride (5) to afford the respective 3'-toluoyloxy derivative 7 and not the anticipated 3'-O-[(p-toluoyloxy)ethyl]-dyphylline (6). Deacylation of 3a,b and 7 afforded 4a,b and 1, respectively. Viral screening of selected compounds against HBV has been investigated.

Convulsant effects of some xanthine derivatives in genetically epilepsy-prone rats.

The behavioural and electrocorticographic (ECoG) convulsant effects of several xanthine derivatives injected intraperitoneally (i.p.) were studied in genetically-epilepsy prone rats. The aim of the study was to evaluate the relationship among convulsant potency, molecular structure and lipophilicity of some xanthines. Animals were injected i.p. with various doses (250-1000 micromol/kg) and a different convulsant potency was observed among the various xanthines tested. IBMX (3-isobutyl-1-methylxanthine), theophylline (1,3-dimethylxanthine) and caffeine (1,3,7-trimethylxanthine) induced an epileptogenic pattern that consisted in an initial phase characterized by wet-dog shakes followed by head tremor, nodding, clonic convulsion and they appeared to be the most potent xanthines among those studied. During seizures, the electrocortical activity was usually characterized by single or multiple sharp- or spike-wave episodes followed by polyspike discharges. After the highest doses of IBMX, theophylline and caffeine, the animals react with falling down, transient tonic clonic seizures, escape response and generalized seizures followed by post-ictal period. Equimolar doses of 8-chlorotheophylline and theobromine (3,7-dimethylxanthine) produced less evident epileptic responses in comparison to previous compounds, whereas no epileptic signs were observed following the administration of enprofylline (3-propylxanthine), etofylline [7-(2-hydroxyethyl)theophylline], diprophylline [7-(2,3-dihydroxy-propyl)theophylline] and doxofylline [7-(1,3-dioxolan-2-ylmethyl) theophylline]. Lipophylicity of the compounds was determined, but no convincing correlations were found between the rank order of lipophilicities and the convulsant potencies of the compounds studied. On the other hand, structure-activity relationship was also investigated. We suggest that the substitution pattern on the xanthine nucleus may explain, in part, the different convulsant potency of the compounds studied. Furthermore, a selective antagonism of adenosine subtype receptors should be considered.

Probenecid inhibition of the renal excretion of dyphylline in chicken, rat and man.

The effect of probenecid on the renal excretion of dyphylline was studied in chicken, rat and man. Dyphylline was found to be actively excreted when measured by the Sperber preparation in hens, the isolated perfused kidney of the rat and clearance studies in man. In each study probenecid significantly decreased dyphylline excretion demonstrating that dyphylline occupied the renal organic anion transport system. This same drug interaction, at the level of the renal excretory system, in these three species occurred at comparable concentrations of dyphylline and probenecid.

Bioavailability of dyphylline and dyphylline-guaifenesin tablets in humans.

A six-way-crossover bioavailability study was conducted with twelve healthy male volunteers to evaluate the relative bioavailability of three tablet formulations containing dyphylline and three tablet formulations containing dyphylline-guaifenesin. Each subject was administered two tablets of each product with greater than or equal to 3 d separating each dose. Blood samples were obtained just prior to each dose and at 0.25, 0.5, 0.75, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, and 10.0 h following each dose. An HPLC method was used to assay dyphylline in the serum. The mean tmax ranged from 0.6 to 1.0 h for the six products. The mean values for Cmax differed by 29%, and the AUC values differed by less than 8%. It was noted that the dyphylline-guaifenesin products exhibited a lower bioavailability than the products which only contained dyphylline. It was concluded that the three combination products were bioequivalent, as were the three dyphylline products.

Effect of probenecid on dyphylline elimination.

Dyphylline is a methylxanthine bronchodilator with such a short a biologic t 1/2 that development of practical dosing regimens has been difficult. Because its rapid renal elimination suggests active secretion, the effect of 1 gm probenecid on single-dose elimination kinetics of dyphylline was determined. Twelve subjects (six male, six female) participated in a crossover design. Subjects were their own controls and received dyphylline, 20 mg/kg orally, alone and after probenecid. The dyphylline t 1/2 increased from 2.57 +/- 0.45 to 4.88 +/- 1.2 hr, whereas the elimination rate constant decreased from 0.276 +/- 0.056 to 0.150 +/- 0.037 hr-1 after probenecid. There was no significant change in the dyphylline apparent volume of distribution. Dyphylline total body clearance fell from 173 +/- 20 to 95 +/- 12 ml/kg . hr. The combined use of these drugs may lead to a practical dyphylline dosage schedule in aminophylline-hypersensitive patients or those incapacitated by theophylline gastrointestinal side effects.

Pharmacokinetics and bioavailability of three dyphylline preparations.

The pharmacokinetics and bioavailability of 3 oral dyphylline preparations, solution (S), regular (R) and sustained release (SR), were studied in 8 healthy subjects (mean age 25 years). A single dose of each preparation, 20 mg X kg-1, was given at one week intervals and multiple serum samples obtained over 24 h. Drug levels were measured by high performance liquid chromatography. No adverse effects were found. The dyphylline half-life for the solution was 2.16 +/- 0.18 h and for the tablet 2.59 +/- 0.56 h. The mean clearance rate for S was 13.6 +/- 1.7 h-1 and volume of distribution 43.0 +/- 3.91. Peak concentration (Cmax, micrograms X ml-1), time of peak (Tmax, h), area under the curve (AUC, micrograms X ml-1 X h) and relative bioavailability (RB, %), were determined for three preparations: Cmax S, 33.7 +/- 3.7; R, 27.7 +/- 4.2; SR, 10.4 +/- 1.5 Tmax: S, 0.33 +/- 0.0; R, 0.66 +/- 0.0; SR, 2.13 +/- 1.1 AUC: S, 108.4 +/- 12.1; R, 113.9 +/- 25.2; SR, 104.0 +/- 30.8 RB: Reference Product R, 105.00 +/- 16.00; SR, 100.00 +/- 25.00 The data confirm the short half-life of dyphylline, demonstrate a lack of toxicity for the 20 mg X kg-1 dose and establish bioequivalence for the products studied.

Pharmacokinetics of dyphylline elimination by uremic patients.

The elimination kinetics of dyphylline were investigated in four uremic patients receiving chronic hemodialysis treatment. Dyphylline (1000 mg) was administered to each patient orally 2 hr before hemodialysis. Serial arterial and venous blood samples as well as outflowing dialysate samples were collected and analyzed by high-pressure liquid chromatography for dyphylline. The extraction efficiency of the hollow-fiber dialyzers averaged 44.6%. The mean dialysis clearance was 108.7 ml/min which compares favorably to the 57 ml/min metabolic clearance of the drug. A mean of 278.6 mg or 28.34% of the administered dose was recovered in the dialysate during the 4-hr dialysis interval. Half-lives for dyphylline during dialysis ranged from 3.43 to 7.62 hr. The off-dialysis half-lives for two of the four patients studied on a separate occasion were 10.6 and 13.2 hr. The overall clearance calculation projects an average half-life reduction of approximately 65% during hemodialysis. We conclude that dyphylline is dialyzable. Hemodialysis patients receiving dyphylline treatment may require a dosage regimen alteration. In addition, hemodialysis may be useful for the treatment of dyphylline overdose because of its effectiveness in accelerating the removal of the drug.

Utilization of intravenous dihydroxypropyl theophylline (dyphylline) in an aminophylline-sensitive patient, and its pharmacokinetic comparison with theophylline.

The pharmacokinetics and urinary excretion of intravenously administered 7-(2,3-dihydroxypropyl) theophylline (dyphylline), were studied in a 37-yr-old asthmatic woman with ethylene diamine sensitivity who manifested intolerance to intravenous aminophylline on three separate occasions. In this subject, intravenously administered dyphylline was tolerated very well and was effective in the subsequent management of acute bronchospastic episodes. Dyphylline was significantly concentrated in the urine. This, coupled with its rapid clearance, suggests potential clinical application in patients with hepatic dysfunction. Though aminophylline sensitivity is rare, ethylene diamine sensitivity should be considered in untoward reactions to this drug.

Comparative pharmacokinetics of theophylline and dyphylline following intravenous injection in rabbits.

The pharmacokinetics of theophylline and dyphylline in four rabbits were investigated following intravenous injection. Each rabbit received theophylline (10 mg/kg) first then dyphylline (25 mg/kg) two weeks afterwards as an intravenous bolus injection. The serum concentration-time data for both theophylline and dyphylline were best fitted to the two compartment open model. The half-lives of theophylline and dyphylline were 5.5 +/- 1.3 and 0.74 +/- 0.10 hr, respectively. The apparent volume of distribution (Vdbeta) and total body clearance (TBC) of dyphylline (Vdbeta = 1008 +/- 156 ml/kg; TBC = 942 +/- 118 ML/HR/KG)a = 1008 +/- 156 ml/kg; TBC = 942 +/- 118 ml/hr/kg) were much higher than those of theophylline (Vdbeta = 545 +/- 160 ml/kg; TBC = 69 +/- 10 ml/hr/kg). Thus both theophylline and dyphylline showed pronounced difference in their pharmacokinetic profile.