1 Institute of Biochemistry G. Fornaini and 2 Institute of Hygiene, University of Urbino, 61029 Urbino; 3 Department of Chemical Sciences, University of Camerino, 62032 Camerino, Italy
Received 19 December 2001; returned 26 March 2002; revised 7 May 2002; accepted 24 May 2002
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Abstract |
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Introduction |
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Among these, the bis (pivaloyloxymethyl) [bis(POM)] ester prodrug was selected as a potentially useful prodrug of PMEA,13 and clinical trials both with humans infected with HIV-1 and individuals infected with hepatitis B virus (HBV) were performed.14,15 Clinical trials with bis(POM)-PMEA (adefovir dipivoxil) in 442 patients infected with HIV have shown that the drug can add some extra anti-HIV effects to combination treatment, leading to an average decrease in viral load of just over 40% after 6 months. Furthermore, bis(POM)-PMEA works against several strains of HIV that are already resistant to AZT, ddC or ddI, and seems to have very little cross resistance with other retroviral drugs. Unfortunately, in these studies more than a third of the people who took the highest dose (120 mg/day) for more than 6 months developed signs of serious kidney damage. For this reason, at present, the development of therapeutic strategies employing bis(POM)-PMEA against HIV is going on in Europe but not in the United States, where the development of the drug is instead continuing against HBV. In particular, for this disease, bis(POM)-PMEA is now in phase III clinical trials.
In addition, some adverse effects due to the pivaloyl moiety of the prodrug have been observed. To avoid the toxicity caused by the pivaloyl group, herein we report a novel approach based on the prodrug P1,P2-bis[2-(adenin- 9H-yl)ethoxymethyl]phosphonate (Bis-PMEA), a compound consisting of two molecules of PMEA bound together by a P-O-P bond (Figure 1). We previously showed16 that Bis-PMEA acts as an efficient antiviral prodrug able to protect macrophages against in vitro HIV or HSV infections following selective targeting by means of loaded erythrocytes. In this paper, the oral bioavailability of free Bis-PMEA in mice and its antiretroviral activity in a murine model of immunodeficiency were evaluated. The results obtained show that Bis-PMEA, administered by oral gavage, acts as a prodrug for a slow delivery of PMEA in circulation, giving a higher antiretroviral activity than oral PMEA without causing increased toxicity.
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Materials and methods |
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The homodinucleotide Bis-PMEA was synthesized by coupling the morpholidate derivative of PMEA as N,N'-dicyclohexyl-4-morpholine-carboxamidinium salt with PMEA as mono n-tri-butylammonium salt. In a similar way the radiolabelled dimer [14C]Bis-PMEA was obtained. PMEA n-tri-butylammonium salt was synthesized by treatment of nucleoside in pyridine/water (1:1, v/v) with n-tri-butylamine and dried in vacuo over phosphorus pentoxide. N,N'-Dicyclohexyl-4-morpholine-carboxamidinium 2-[(adenin-9H-yl)-ethoxymethyl]morpholinophosphonate was synthesized by treatment of PMEA with morpholine and N,N'-dicyclohexylcarbodiimide (molar ratio 1/6.5) in hot aqueous tert-butyl alcohol (1:1, v/v) for 11 h as reported in Holy & Rosenberg,17 to obtain the compound as a white solid. 1H-NMR (300 MHz, D2O): 3.15, 3.30, 3.63 (3m, 8H, morpholino); 3.52 (d, J = 8.8 Hz, 2H, CH2P); 3.84 (t, J = 4.9 Hz, 2H, H-2'); 4.35 (t, J = 5.0 Hz, 2H, H-1'); 8.25 (2s, 2H, H-2, H-8). 31P-NMR (121 MHz, D2O):
18.0.
Coupling of the PMEA salt to PMEA morpholidate was carried out under anhydrous conditions (molar ratio 1/1) in N,N-dimethylformamide at 50°C for 40 h. The solvent was removed in vacuo and the oily residue was portioned between water and diethyl ether (1:2, v/v). The aqueous phase was evaporated to dryness and the residue was chromatographed on a silica gel column with iPrOH/NH4OH/H2O (80:10:10, v/v/v). Evaporation of the appropriate fractions gave a solid residue that was solubilized in aqueous ammonia and washed with diethyl ether. The aqueous phase was evaporated to dryness and co-evaporated with anhydrous ethanol. The residue was washed with ethyl ether and acetone to obtain the bis(ammonium)Bis-PMEA as a white solid, which was dried over phosphorus pentoxide (50% yield). TLC (H2O/CH3CN, 80:20, v/v): Rf = 0.68. UV (H2O): max 258 nm (
23 500). MS (API-ESI) m/z = 527.0, 263.0 [M/2H], 540.0 [M2H + Na], consistent with the expected molecular structure. All measurements were performed in the negative ion mode, and scanning in the 300800 mass range. 1H-NMR (300 MHz, D2O):
3.65 (t, J = 3.7 Hz, 4H, 2x CH2P); 3.81 (t, J = 4.5 Hz, 4H, H-2'); 4.18 (t, J = 4.5 Hz, 4H, H-1'); 7.95 (s, 2H, H-8); 7.98 (s, 2H, H-2). 31P-NMR (121 MHz, D2O):
8.8. The chemical shift values are expressed in
values (ppm) relative to tetramethylsilane and orthophosphoric acid as internal standards. The elemental analysis confirmed the structure of Bis-PMEA as a diammonium salt (Anal. Calc. for C16H28N12O7P2·2H2O: C 32.11, H 5.39, N 28.09. Found: C 32.43, H 5.48, N 27.89).
The radiolabelled dimer [14C]Bis-PMEA was obtained as above, by coupling the mono n-tri-butylammonium *PMEA [adenine-8-14C] salt to PMEA morpholidate. The *PMEA [adenine-8-14C] n-tri-butylammonium salt was synthesized from a mixture of 16.5 mg of *PMEA [adenine-8-14C] and 24.75 mg of PMEA in pyridine/water (1:1, v/v), with n-tri-butylamine (molar ratio 1/1). [14C]Bis-PMEA was characterized as described above. PMEA and *PMEA [adenine-8-14C] were kindly provided by Dr Norbert Bischofberger, Gilead Sciences, Foster City, CA, USA.
HPLC determination of Bis-PMEA and PMEA in plasma
The metabolism of Bis-PMEA in plasma was evaluated as follows: 0.14 mM Bis-PMEA was incubated in murine plasma at 37°C and at different times of incubation (15 min, 30 min, 1 h, 2 h and 5 h) 100 µL aliquots were extracted with perchloric acid, neutralized and then used for HPLC determinations of Bis-PMEA and PMEA, as described by Rossi et al.16
Pharmacokinetic studies in mice
To determine the concentration of Bis-PMEA and PMEA in circulation after oral Bis-PMEA administration, 45 mg of Bis-PMEA in 350 µL of distilled water was administered by oral gavage to four Institute of Cancer Research (ICR) mice. At different times (1, 2, 5 and 8 h), 500 µL of blood was collected from CO2-anaesthetized mice (one mouse/time point) into heparinized Pasteurs by puncture of the retro-orbital sinus. Blood was immediately centrifuged at 900g for 10 min at 4°C and 250 µL plasma samples were extracted with perchloric acid; neutralized extracts were then concentrated with a Speed Vac concentrator (Savant Instruments, Hicksville, NY, USA) to a final volume of 100 µL and used for the HPLC determinations of Bis-PMEA and PMEA, as described above.
Pharmacokinetic studies in mice were performed as follows: female ICR mice (weight 22 ± 1 g) received either [14C]PMEA by intravenous (iv) bolus injection (via the retro-orbital sinus) or oral gavage, or [14C]Bis-PMEA by oral gavage. Both compounds were given at equimolar doses of PMEA, i.e. 28 mg/kg for [14C]PMEA (with a specific activity of 11 257 cpm/nmol) and 28 mg/kg for [14C]Bis-PMEA (with a specific activity of 22 977 cpm/nmol). The [14C]PMEA used for iv administration was suspended in physiological saline solution [5 mM glucose, 5 mM KH2PO4/NaH2PO4 and 0.9% (w/v) NaCl, pH 7.4], whereas [14C]PMEA and [14C]Bis-PMEA used for oral gavage administration were suspended in distilled water and 2 M sodium hydroxide was added to obtain a final pH of 7.0. For each drug administered ([14C]PMEA iv; [14C]PMEA by mouth; [14C]Bis-PMEA by mouth) three mice were used for the determination of [14C]PMEA in plasma. At different times after drug administration, ranging from 2 min (time 0) to 48 h, blood was collected from each mouse into heparinized microhaematocrit capillary tubes by puncture of the retro-orbital sinus. Blood samples (100 µL) were immediately centrifuged for 5 min at 900g at 4°C and plasma samples were stored at 20°C until analysed. Radioactivity was counted in 50 µL of plasma in a Packard Liquid Scintillation Analyzer after addition of 5 mL of scintillation fluid (Emulsifier Scintillator Plus, Packard) and the concentrations of [14C]PMEA in plasma were evaluated.
Pharmacokinetic calculations were carried out according to the linear trapezoidal rule, evaluating the area under the curve from time zero to the time of the last measurable concentration (AUC0t last). The bioavailability of [14C]PMEA following oral administration of [14C]Bis-PMEA or [14C]PMEA was defined as 100 x (AUCpo,0t last/AUCiv,0t last), where AUCpo,0t last equals the AUC for [14C]PMEA following oral administration of [14C]Bis-PMEA or [14C]PMEA, and AUCiv,0t last equals the AUC for [14C]PMEA following iv injection of [14C]PMEA.
Virus and animals
The LP-BM5 viral mixture was kindly provided by Robert Yetter (Veterans Administration Hospital, Baltimore, MD, USA) and was maintained in a persistently infected SC-1 cell line as previously described.18 Five-week-old female C57BL/6 mice (Nossan, Milan, Italy) were infected by means of two intraperitoneal injections, one 24 h after the other, of 0.125 mL of the virus stock containing 1 U of reverse transcriptase. ICR mice (Nossan) were used to assess the pharmacokinetic studies. Mice were housed at 22 ± 1°C with a 12 h light/dark cycle, 60 ± 5% humidity, and 12 air changes per hour.
Drug administration
After viral infection (7 h after the second viral injection), mice were treated with PMEA by mouth, Bis-PMEA by mouth and PMEA intraperitoneally, 50 mg/kg for each drug tested, five consecutive days weekly for a period of 4 or 9 weeks, as already reported for other treatment regimens investigated in this animal model.19,20 PMEA and Bis-PMEA dosages were adjusted every 2 weeks on the basis of body weight.
Serum immunoglobulin determination
At different times post virus inoculation (4 and 9 weeks), blood was drawn from the mouses tail vein for use in determining serum IgG levels via an enzyme-linked immunosorbent assay (ELISA) technique, as described elsewhere.21
Semi-quantitative PCR analysis of LP-BM5 proviral DNA
Total cellular DNA was isolated from lymph nodes as previously described.20 A semi-quantitative PCR method was used to analyse BM5d proviral DNA in lymph nodes. The oligonucleotide primers and the procedure used were those described in Fraternale et al.22
Evaluation of mtDNA content
Total cellular DNA was isolated from lymph nodes of infected and infected-treated mice 9 weeks post virus inoculation as described by Rossi et al.23 The extracted DNAs were carefully quantified with an agaroseethidium bromide staining gel using the Gel Doc 1000 System. For each sample, 2 µg of DNA was digested with BamH1 restriction enzyme, analysed on a 0.8% (w/v) agarose gel and transferred to a nylon membrane. A specific 1237 bp mtDNA probe was used in hybridization experiments to detect mtDNA content. The intensities of the radiolabelled bands were quantified in a GS-250 molecular imager (Bio-Rad).
Flow cytometric analysis
The fluorescent monoclonal antibodies used for fluorescence-activated cell sorting (FACS) analysis were purchased from PharMingen, Becton Dickinson Co. (Milan, Italy). Blood samples were stained with fluorescent monoclonal antibody and analysed by FACS (Becton Dickinson, San Jose, CA, USA) as described in the study by Palamara et al.19
Lymphocyte proliferative index
[3H]Thymidine incorporation on lymphocytes stimulated by either phytohaemagglutinin (PHA) for T cells or lipopolysaccharide for B cells was determined as described by Brandi et al.24
Histological examinations
Samples of lymph node, liver and kidney tissue from three animals in each treatment group were fixed in 4% paraformaldehyde in PBS, embedded in paraffin, sectioned and stained with haematoxylineosin.
Cell cultures
U937 human monoblastoid cells and L1210 mouse leukaemia cells were grown in RPMI 1640 medium (PBI International, Milan, Italy) supplemented with 10% heat-inactivated fetal calf serum (PBI International), 2 mM L-glutamine (Sigma), penicillin and streptomycin, 100 U/mL and 100 mg/L (Bio-Whittaker, Verviers, Belgium), respectively, in 5% CO2/95% air at 37°C. U937 and L1210 cells were seeded in 96 wells at a density of 1 x 103 cells/well and incubated at 37°C, 5% CO2/95% air. PMEA toxicity was evaluated by determining the number of viable cells with the Trypan Blue dye exclusion test after exposing U937 and L1210 cells to different concentrations (04 µM) of PMEA for 8 days.
Statistical analysis
Statistical analysis of data was performed with the parametric test (t-test) of the Microcal TMOriginTM program (Microcal Software Inc., Northampton, MA, USA) with a P value <0.05 used to determine significance.
Other determinations
Haematological parameters were evaluated using ABX Micros OT (ABX, Parc Euromedicine, Montpellier, France); creatinine was evaluated in plasma samples with a test-combination kit (Boehringer-Mannheim).
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Results |
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The ability of murine plasma enzymes to convert Bis-PMEA into PMEA was evaluated. As shown in Figure 2, plasma possesses enzymes able to cleave the P-O-P bond of Bis-PMEA with the stoichiometric production of PMEA (t1/2 60 min).
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The chromatographic analysis of plasma samples obtained from mice receiving oral Bis-PMEA administration (45 mg/kg) revealed the presence of PMEA and the total absence of Bis-PMEA in circulation (data not shown). Unfortunately, the HPLC method used did not allow us to determine PMEA concentration accurately below 1 µM and thus time points after 8 h could not be evaluated. In addition, since only PMEA was found in circulation at all time points examined following Bis-PMEA administration, the chromatographic separation of these two compounds was not required for pharmacokinetic studies. For these reasons, radiolabelled compounds ([14C]PMEA and [14C]Bis-PMEA) were used and pharmacokinetic profiles obtained directly by evaluating [14C]PMEA radioactivity in plasma samples.
Figure 3 shows the time course of PMEA concentrations in plasma following the iv administration of [14C]PMEA and the oral administration of [14C]PMEA and [14C]Bis-PMEA to ICR mice at 28 mg/kg.
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Infection of C57BL/6 mice with the LP-BM5 retrovirus complex causes a disease with a pathology that resembles that of human AIDS;25 as a result, this animal model has been used extensively for preclinical studies of antiviral drugs.24,26 In the present study, LP-BM5-infected mice were treated with the antiviral agent PMEA, or its prodrug Bis-PMEA, by oral gavage administration. Drugs were administered at equimolar doses of PMEA at a concentration of 50 mg/kg. The experiment was performed as follows: one group of mice was infected but not treated, one group was infected and treated by mouth with PMEA, and another group was infected and treated by mouth with the prodrug Bis-PMEA. Both drugs were administered 5 days a week for 4 or 9 weeks. As controls, uninfected/untreated mice, and infected/intraperitoneally PMEA-treated mice at a concentration of 50 mg/kg, were used. Mice infected with LP-BM5 developed MAIDS within 4 weeks after infection. At 4 and 9 weeks post-infection, mice (three and seven, respectively) were sacrificed and several parameters characterizing the progression of the disease were evaluated. Table 2 reports the degree of lymphoadenopathy and splenomegaly. PMEA was found to be able to slightly reduce lymph node weight both after 4 weeks (10%) and after 9 weeks of treatment (11%), whereas a higher reduction was obtained following Bis-PMEA administration (36% and 27% after 4 or 9 weeks of treatment, respectively). However, these lymph node weight reductions were not statistically significant. PMEA and Bis-PMEA administration were not effective in reducing splenomegaly; moreover, an increase in the spleen weights of PMEA-treated mice as compared with infected-untreated ones was observed. The degree of hypergammaglobulinaemia is shown in Figure 4. The results obtained after 4 weeks of treatment showed a 40% reduction in hypergammaglobulinaemia following Bis-PMEA administration as compared with infected-untreated mice, whereas a lower inhibition (20%) was observed with PMEA; however, when IgG levels were determined after 9 weeks, only a 1015% reduction was observed with both treatments.
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PMEA was not approved by the Food and Drug Administration (FDA) as an anti-HIV-1 drug essentially because of its renal toxicity.2729 To evaluate renal toxicity in mice following PMEA administration for 9 weeks post-infection, the plasma concentration of creatinine was determined. As shown in Table 4, LP-BM5 infection caused a decrease (30%) in creatinine level; however, by administering PMEA by mouth or intraperitoneally, or Bis-PMEA by mouth, no additional reduction was observed. In a separate experiment the effects of PMEA and Bis-PMEA on plasma creatinine concentration in uninfected control mice were determined and found to be unmodified (data not shown). In addition, no differences were observed in histological examination of kidney tissue following drug treatments (data not shown).
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The content of mitochondrial DNA in lymph nodes was also evaluated and no decrease following PMEA (intraperitoneally or by mouth) or Bis-PMEA administration was revealed (data not shown).
Finally, PMEA toxicity was evaluated in two different cell lines: U937 human monoblastoid cells and L1210 murine leukaemia cells. The results obtained showed that PMEA had the same toxicity profile in both human and murine cells, inhibiting cell proliferation in a dose-dependent manner (IC50 = 3.88 ± 0.78 µM, L1210; IC50 = 4.3 ± 0.14 µM, U937).
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Discussion |
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Hence, a different prodrug approach aimed at increasing PMEA oral bioavailability was pursued in our laboratory. In this paper we have shown that the homodinucleotide Bis-PMEA, which consists of two PMEA molecules bound together by a phosphate bridge, can act as a prodrug of PMEA, increasing its oral bioavailability and its antiretroviral activity. We have also shown that Bis-PMEA does not reach the systemic circulation intact when administered orally to mice; in fact, only PMEA was found in plasma samples taken at different times post-Bis-PMEA administration. This is probably a consequence of hydrolysis of the P-O-P bond of the homodinucleotide during transit across the intestinal epithelium or after absorption in the portal blood. Our results in fact show that murine plasma enzymes are able to convert Bis-PMEA into PMEA and similar values were obtained in human plasma. When measuring the concentrations of free PMEA in plasma upon oral administration of equimolar doses of [14C]Bis-PMEA and [14C]PMEA, we found a PMEA oral bioavailability of 50.8% and 13.5%, respectively. The latter value is in accordance with what was found by other authors.9 Therefore, our pharmacokinetic studies demonstrate that Bis-PMEA administered by oral gavage can act as a prodrug providing a slow delivery of PMEA in circulation. Our pharmacokinetic data correlate with the antiviral results: oral administration of Bis-PMEA was in fact more effective than oral PMEA administration for the treatment of MAIDS, an AIDS-like disease caused by the LP-BM5 retrovirus complex in susceptible C57BL/6 mice. We chose the MAIDS model because it shows many similarities with human AIDS, including abnormal T- and B-lymphocyte functions, polyclona B-cell proliferation, lymphoadenophathy, splenomegaly, hypergammaglobulinaemia and enhanced susceptibility to infections.30,31 Bis-PMEA was more effective than PMEA in reducing lymphoadenopathy, hypergammaglobulinaemia and lymph node BM5 proviral DNA content (especially after 4 weeks of treatment). Moreover, no increased drug toxicity was observed upon Bis-PMEA administration, as shown by creatinine levels, histological examination and haematological parameters compared with those obtained following oral PMEA administration. It is worth noting that PMEA was not approved by the FDA as an anti-HIV-1 drug because of its renal toxicity. In our experimental model we were unable to detect alterations in either plasma creatinine concentration or kidney histological examination. This absence of signs of kidney toxicity may be due to a short treatment regimen in our murine model. A reduction in the response of B-splenocytes to mitogenic stimuli was observed with both PMEA and Bis-PMEA treatments when compared with the response in infected-untreated mice. In addition, the CD19+ level was almost the same between infected and Bis-PMEA treated mice, whereas with PMEA administration (by mouth and overall intraperitoneally) a marked reduction in the percentage of these lymphocytes as compared with the percentage in infected-untreated mice was reported. Thus, altogether, Bis-PMEA administered by mouth is more effective and less toxic than PMEA by mouth.
In conclusion, the results obtained upon oral treatment with PMEA and its prodrug Bis-PMEA were compared with those obtained by intraperitoneal PMEA administration. It had already been shown that intraperitoneal PMEA is not able to prevent MAIDS but does slow down the progression of the disease.32 In our experiments, the administration of PMEA (50 mg/kg) by intraperitoneal injection was less effective than oral Bis-PMEA in reducing lymphoadenopathy, hypergammaglobulinaemia and lymph node proviral DNA content, at least in the first weeks post-infection. Moreover, following intraperitoneal PMEA administration some signs of drug toxicity appeared (Figure 6 and Table 4). In addition, in vitro studies in two different cellular lines (murine L1210 and human U937) revealed the same toxicity profiles between murine and human cells, suggesting that mice represent a good animal model with which to evaluate the efficacy of different PMEA delivery systems.
Overall, our results show that Bis-PMEA administered by oral gavage can act as a prodrug for a better delivery of PMEA in circulation than that obtained with oral gavage PMEA, assuring higher antiviral efficacy without causing significant toxicity. Moreover, our data suggest that charged molecules other than PMEA [for example the most efficient antiretroviral drug 9-(2-phosphonylmethoxypropyl)adenine (PMPA)] can most likely be coupled by a P-O-P bond to increase their bioavailability, as shown in this paper for Bis-PMEA. It provides support to the idea that a new class of phosphonate prodrugs with good oral bioavailability, improved pharmacokinetics and reduced toxicity can be generated from the Bis-PMEA model.
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Acknowledgements |
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Footnotes |
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References |
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