WO1996039148A1

WO1996039148A1 - Methods and compositions for inhibiting cytomegalovirus replication

Info

Publication number: WO1996039148A1
Application number: PCT/US1996/008029
Authority: WO
Inventors: Charles E. Mckenna
Original assignee: University Of Southern California
Priority date: 1995-06-06
Filing date: 1996-05-30
Publication date: 1996-12-12
Also published as: CA2223874A1; AU5953696A; EP0831842A4; EP0831842A1

Abstract

Methods and compositions for use in inhibiting CMV replication in mammalian patients, wherein an effective amount of TPFA is administered to a patient in need of such treatment. TPFA is demonstrated to be at least as effective as PFA in inhibiting CMV viral replication, while having substantially improved bioavailability relative thereto.

Description

METHODS AND COMPOSITIONS FOR INHIBITING CYTOMEGALO VIRUS REPLICATION Background of the Invention

The present invention relates generally to the fields of biology and medicine. More particularly, the present invention relates to compositions and methods for use in treating a mammalian patient infected with particular viruses, such as cytomegalovirus (CMV).

A cytopathic retrovirus, human immunodeficiency virus (HIV-1), has been linked to pathogenesis of the fatal immunosuppressive disease known as Acquired Immune Deficiency Syndrome (AIDS). Replication of the virus is dependent on an RNA-directed DNA polymerase (reverse transcriptase; RT). Compounds that selectively inhibit HIV RT relative to human DNA polymerases provide a basis for anti-viral chemotherapy of pre- AIDS and AIDS patients. AIDS is believed to have been first observed as a medical phenomenon in the summer of 1981 in Los Angeles. The primary cause of ALDS is well established to be infection with HIV. It is estimated that as many as 1,000,000 persons are infected ("HIV-positive¹¹) in the USA alone. After infection, the average progression to symptoms of the disease syndrome is 7 years or longer; however, death usually follows within 18-24 months of diagnosis [Stine, G. J. Acquired Immune Deficiency Syndrome: Biological, Medical, Social and Legal Issues ; Prentice-Hall: Englewood, New Jersey, 1993; Vol. 462].

HIV is principally transmitted by sexual contact, IV drug use with contaminated needles, and transfusion with contaminated blood. The virus attacks human T4 cells, which are essential to normal function of the immune system. As the T4 cell count diminishes, loss of the immune response leads to uncontrolled bacterial, fungal, parasitic and/or viral infections, known as Opportunistic Infections (OI). Chemotherapy of AIDS involves drugs directly targeting HIV, such as AZT (Zidovudine^*), and drugs targeting various opportunistic infections. Ideally, drugs to treat any disease caused by an infectious pathogen should be effective against the target organism at reasonably low concentration (i.e., have high potency). They should also have low toxicity and minimal side effects, exhibit good stability under standard storage conditions, be readily bioavailable when taken orally (avoiding discomfort and cost from injection), and be inexpensive to manufacture and administer. These considerations are particularly important with respect to AIDS chemotherapy. Because infection is permanent, incurable and lethal if untreated, medication must be taken for the life of the patient; this enhances the importance of toxicity and cost. For the same reasons, the path of drug administration is especially important. Because AIDS patients must be treated for HIV infection and simultaneously for one or more opportunistic infections, drug compatibility is also a key issue. This is a matter of particular importance where the OI is viral in origin. For example, AZT (to treat HIV) and ganciclovir (Cytovene*, an anti-viral used to treat CMV) are only compatible because they reinforce each other's toxic effects.

CMV causes blindness in a substantial portion of AIDS patients (CMV retinitis). One estimate [Stine 1993, supra] suggests that 46% of AIDS patients who have reached the latter stages of the disease suffer from CMV retinitis; other estimates range from 20% to 76% [Keijer, W. J. et al. Ocular complications of the acquired immunodeficiency syndrome. Focus on the treatment of cytomegalovirus retinitis with ganciclovir and foscarnet. Pharm World Sci 1993, 15, 56-67] and 11% to 40% [Hansen, L. L. {Retinal diseases in AIDS}. Ophthalmologe 1993, 90, 239-49]. A CMV vaccine is still in the early stages of development and antiviral agents will continue to play a major role in CMV infection management for the foreseeable future [Sasadeusz, J. J.; Sacks, S. L. Systemic antivirals in herpesvirus infections. Dermatologic Clinics 1993, 11, 171-85]. CMV possesses a virus-specific DNA polymerase.

One drug recently approved by the FDA for treatment of AIDS-related CMV-retinitis is foscarnet (Astra Pharmaceuticals), also known as Foscavir^* and PFA (phosphonoformic acid) [Stine 1993, supra]. Like another drug approved for the same condition, ganciclovir, PFA does not cure the condition, but it can significantly delay progression to blindness and increase patient survival time [Gumbel, H. et al. {Therapeutic alternative or 2d choice drug. Trisodium phosphonoformate in cytomegalovirus retinitis}. Fortschritte der Ophthalmologie 1991, 88, 731-4; Brockmeyer, N. H. et al. Foscarnet treatment in various cytomegalovirus infections. International Journal of Clinical Pharmacology, Therapy, and Toxicology 1993, 31, 204-7; Polis, M. A et al. Increased survival of a cohort of patients with acquired immunodeficiency syndrome and cytomegalovirus retinitis who received sodium phosphonoformate (foscarnet). American Journal of Medicine 1993, 94, 175-80]. Both drugs have an initial efficacy of induction therapy of 80-90%, and maintenance therapy is always needed to prevent a relapse [Keijer et al. 1993, supra]. About 40% of AIDS patients with CMV retinitis cannot tolerate the side effects of ganciclovir, which include inhibited production of bone marrow white blood cells.

Although PFA also shows toxic side effects, notably reversible kidney damage and abnormal blood electrolyte levels [Ryrfeldt, A. et al. Hypocalcemia induced by foscarnet (Foscavir) infusion in dogs. Fundamental and Applied Toxicology 1992, 18, 126-30; Gearhart, M. 0.& Sorg, T. B. Foscarnet-induced severe hypomagnesemia and other electrolyte disorders. Annals of Pharmacotherapy 1993, 27, 285-9], its toxicity profile is very different from that of ganciclovir. This difference is important particularly for patients also receiving AZT, with which PFA is more compatible than ganciclovir, and foscarnet-AZT combination chemotherapy has been demonstrated to prolong the lives of ALDS-CMV patients relative to the lives of ganciclovir-treated historical controls [Polis et al. 1993, supra]. It has also been reported that concurrent use of ganciclovir and foscarnet in cases of failure of either alone in AIDS CMV infections was as effective as standard therapy with single agents and may be of value in cases of clinically-resistant CMV retinitis in AIDS [Flores-Aguilar, M. et al. Pathophysiology and treatment of clinically resistant cytomegalovirus retinitis. Ophthalmology 1993, 100, 1022-31].

Foscarnet was further shown to induce remission of CMV gastrointestinal disease in 67% of a group of AIDS patients when ganciclovir induction had failed. Foscarnet has been shown to penetrate the blood-brain barrier and is stated to be the drug of choice for CMV encephalitis [Hengge, U. R. et al. Foscarnet penetrates the blood-brain barrier: rationale for therapy of cytomegalovirus encephalitis. Antimicrobial Agents and Chemotherapy 1993, 37, 1010-4]. Efforts to circumvent problems of systemic (infusion) administration of foscarnet have included transscleral iontophoresis [Sarraf, D. et al. Transscleral iontophoresis of foscarnet. American Journal of Ophthalmology 1993, 115, 748-54]; nonetheless, there remains a need for agents which may be administered in a more convenient manner, and preferably orally.

U.S. Patents 5,072,032 and 5,183,812 to McKenna, the entire disclosures of which are hereby incorporated by reference, disclose the use of TPFA as an anti-HIV drug. A novel, simple synthesis of trisodium TPFA from trimethyl phosphonoformate was also reported. The compound has been completely characterized by elemental analysis, ³¹P, ^DC and Η NMR, UV, LR and X-ray crystallographic analysis [see, e.g., McKenna, C. E. et al. Design and Synthesis of Organophosphorus Compounds with Antiviral and Other Bioactivities. Phosphorus Sulfur 1990, 49, 183-186; McKenna, C. E. et al. Sodium Thiophosphonoformate, a Selective HTV RT Inhibitor: Facile Synthesis and Effects in HlV-Infected Cell Culture. Annals NY Acad. Sci. 1990, 616, 569-572]. TPFA was found to inhibit HIV-1 reverse transcriptase with a very similar potency to that of PFA (IC₅₀ near 1 μM) [McKenna et al. 1990, supra]. TPFA was significantly less inhibitory than PFA for DNA polymerases specific for Herpes simplex virus in these experiments and somewhat less inhibitory than PFA for a group of human DNA polymerases. In a comparison of TPFA vs. PFA for dose-related inhibition of HIV-1 infected human H9 cells in culture (p24 expression assay), it was found that the two drugs had virtually identical inhibition curves. An NMR study further showed that under model assay conditions, almost all the TPFA was converted in situ into PFA. It is noteworthy that TPFA was the only simple pyrophosphate analog so far reported to equal the potency of PFA

In an HPLC study on the pharmacokmetics of TPFA vs. PFA in the cat [Straw, J. A et al. Pharmacokinetics of potential anti-AIDS agents thiofoscarnet and foscarnet in the cat. Journal of Acquired Immune Deficiency Syndromes 1992, 5, 936-42], it was reported that TPFA had a shorter renal clearance than PFA (42 min. vs. 172 min.), and that TPFA was partly converted to PFA in vivo; an inactive metabolite, thiophosphonate (TPA) was also detected. The 6-h cumulative urinary excretion was 42% of an intravenous dose, including 23.5% as TPFA 14% as PFA and 5% as TPA. A key experiment compared oral bioavailability of the two drugs. TPFA was about 3x more bioavailable than PFA when administered by SSL enteric coated capsules. When administered orally by gavage, TPFA also had a higher mean oral bioavailability, 33% (with greater variability). Based on 6-h urinary excretion of total drug, the mean oral bioavailability with TPFA was 44%, similar to that calculated from the plasma (intravenous) data.

In another study [Straw, J. A. et al. Thiofoscarnet (TPFA) and Foscarnet (PFA) Pharmacokinetics in Beagle Dogs. Proc. Am. Assoc. Cancer Res. 1992, 33, 530], it was found that 95% of i.v. administered TPFA was excreted in the urine, in the form of 78% TPFA 8% PFA and 9% TPA. Oral bioavailability of TPFA was only 13%, but was increased to 44.5% by cimetidine pretreatment (the latter, an anti-ulcer drug, decreases stomach acidity and presumably thereby increases TPFA stability in the stomach). In comparison, the oral bioavailability of PF 12%, was only slightly increased by antacid pretreatment.

It is an object of the present invention to provide compositions and methods for treating CMV infection which do not suffer from all of the drawbacks of the heretofore-known methods and compositions.

Summary of the Invention

Pursuant to the present invention, there are provided methods and compositions for use in inhibiting CMV replication in mammalian patients, wherein an effective amount of TPFA is administered to a patient in need of such treatment. TPFA is demonstrated herein to be at least as effective as PFA in inhibiting CMV viral replication, while having substantially improved bioavailability relative thereto. Brief Description of the Drawing

The invention may be better understood with reference to the accompanying drawing, in which:

Fig. 1 illustrates inhibition of hCMV replication in human cells by added TPFA and PFA Detailed Description of the Invention

Pursuant to the present invention, compositions and methods are provided for inhibiting CMV replication in mammalian patients using an effective amount of TPFA Because it has been reported (for example, in the aforementioned U.S. Patents 5,072,032 and 5,183,812) that TPFA was substantially less effective than PFA with respect to viruses other than HIV (e.g., herpes simplex Types I and II, Epstein-Barr, Herpes Virus 6), it is quite surprising that substantial efficacy against CMV has now been found. Given the completely unpredictable nature of the antiviral inhibitory activity of TPFA it clearly could not have been predicted that TPFA would be efficacious in the manner disclosed and claimed herein.

As would be immediately apparent to those skilled in the art, TPFA and/or its addition salts (as described, for example, in the aforementioned U.S. Patents 5,072,032 and 5,183,812) may be administered to mammals (including humans) in an amount effective to inhibit viral replication. The optimum rate of administration for a given formulation (e.g., the free compound or a physiologically acceptable salt form thereof) and mode of delivery may routinely be determined empirically. In general, an effective amount of the active agent is in the range of about 1 μM to about 10 mM per kilogram of patient body weight. The compound may be administered orally, parenterally, topically or by other standard routes of administration; it is presently preferred to administer the compound orally. Typically, compositions for use in accordance with the present invention comprise a pharmaceutically acceptable carrier, adjuvant or excipient; details concerning some suitable types of formulations may be derived from, e.g., U.S. Patent 4,665,062 to Eriksson et al., the entire disclosure of which is hereby incorporated by reference. Additionally, TPFA may suitably be administered in accordance with the present invention in conjunction with other bioactive compounds, such as AZT, ddC, ddl, antibiotics, etc. Preliminary experiments have compared the effects of TPFA and PFA on creatinine titers in the animal model. PFA caused significant suppression of creatinine levels, which correlate with normal renal function, whereas TPFA at similar dose had little or no effect, compared to a control. This result could signify decreased kidney toxicity by TPFA vs. PFA TPFA has already been shown to have similar anti-HIV-1 potency to PFA but different pharmacological characteristics in animal models which indicate that TPFA might have advantages as a replacement for PFA Pursuant to the present invention, it is demonstrated that TPFA also has activity against CMV, further enhancing its potential as an AIDS-related anti- viral agent.

The invention may be better understood with reference to the accompanying examples, which are intended for purposes of illustration only and should not be construed as in any sense limiting the scope of the invention as defined in the claims appended hereto. Example 1

Synthesis of the target compound, TPFA depended upon the preparation of the intermediate Me₃TPFA which was effected as described in the aforementioned U.S. Patents 5,072,032 and 5,183,812. Thionation of trimethyl phosphonoformate (Me₃PFA) by Lawesson's reagent in acetonitrile under N₂ afforded analytically pure Me₃TPFA in 75-80% yield on a small (10 mmole) or large scale (1.5 mole). The structure of Me₃TPFA was verified by Η, ^UC, and ^3,P NMR (Table 1). Table 1. NMR Properties of Me₃TPFA and Na₃TPFA Samples

CαφOUld Solvent ^Λ NMR ¹³C NMR ³¹P NMR d (pp ) d (ppi) d (ppa)

Me₃TPFA C0C1₃ 3.85(d, 6H) 52.7(s) 3.88(s, 3H) 54.3<d, ²/--.=7 Hz)

167<d, -^226 Hz) 65.0 (s)

TPFA D- no signal 183.3(d, V--.=181 Hz) 37.8 (s)

Hydrolysis of Me₃TPFA with 10 M NaOH gave, after purification, the drug in 24-30% yield and analytical purity. The structure was verified by Η, ^UC, and ³¹P NMR (Table 1) and elemental analysis (CNa₃0₄PS 1.5H₂0, found: C, 5.24; H, 1.50; S, 13.68; Na, 29.24; calcd: C, 5.11; H, 1.29; S, 13.64; Na, 29.34). UV vis spectroscopy (H₂0, pH=8.8) gave e_2S4mn=0.9xl(f, e_233nπι=3.6xl0³, e_∞5mB=6 xlϋⁱ. Titration of TPFA was carried out at both 25°C and 0°C with 0.0837 M standardized HC1 using a Corning 125 pH meter. The pKa₃ was determined to be ca. 7.5.

TPFA (LZIII.1.19) (0.1175 g) was dissolved in pure water (HPLC Grade) and diluted to 5.00 mL (volumetric flask). Two 0.100 M solutions were prepared identically. Aliquots of each solution (2 x 3.00 mL) were employed in the CMV inhibition assay described in Example 2. The remaining solutions were stored in a freezer (-20°C), and an equivalent solution of PFA sodium salt (from Alfa products) was also prepared and used as a positive control.

To determine the chemical behavior of TPFA in the assay for inhibition of CMV in infected human cell cultures, it was necessary to separate the drug from the very numerous components of the assay mixture, particularly those contributed by the DMEM medium which contains 14 amino acids, 9 inorganic salts, 8 vitamins, and 3 other compounds. Previous work had used different sample matrices (blood, urine) [Straw, J. A et al., Journal of Acquired Immune Deficiency Syndromes 1992, 5, 936-42; Straw, J. A. et al., Proc. Am. Assoc. Cancer Res. 1992, 33, 530], requiring development of a method compatible with the DMEM medium. The goal was to develop a method permitting quantitative determination of IO^'3 - 10^"* M TPFA in drug assay samples. TPFA absorbs UV weakly, and PFA is undetectable by UV. Neither compound is directly detectable by emission (fluorescence) spectroscopy. These considerations suggested electrochemical detection (ECD) as the most promising direct-detection method for TPFA. ECD can be highly selective and also very sensitive for specific analytes. The analyte passes through an electrochemical cell, where it undergoes oxidation or reduction at the working electrode, and the resultant current is detected. In these experiments, oxidation mode was used, corresponding to a positively charged working electrode. The earlier work was based on the use of an obsolete detector and provided inadequate sensitivity for present purposes. Competitive tests demonstrated that the Waters 464 detector would best suit the requirements of these experiments; in addition to equivalently high sensitivity and ease of use, the Waters 464 afforded a larger selection of sensitivity ranges, corresponding to the need to conduct analyses over a wide span of drug concentrations.

As expected, TPFA - with its more easily oxidized C-P=S bonds - gave a stronger response than PFA To ensure detection of PFA, a relatively high potential of +1.2 V was adopted. In reversed phase HPLC, electrolysis of water limits the working electrode potential at pH 7 to around + 1.2 V. At this potential, the background current was ca. 1 μA, and the most sensitive detection current range was 0.5 - 10 μA

ECD is more sensitive to changes in eluting conditions, including the eluent, than detection based on light absorption or emission. To ensure the integrity of the results, such variables as pH, ionic strength and solvent were studied for their effect on mixture resolution and detection.

Reversed-phase ion-pair chromatography (IPC) has quickly gained widespread acceptance as a versatile and efficient method for the separation of ionized and easily ionizable analytes. It is complementary to ion- exchange chromatography which is used to separate similar samples. An important advantages of IPC is its ability to simultaneously separate samples containing neutral and ionized molecules. Method development in IPC is generally more flexible since the type and capacity of the stationary phase for ion interactions can be varied by changes in the composition of the mobile phase. Retention and selectivity in reversed phase IPC are influenced by a large number of experimental variables, including the type and hydrophobicity of the counterion, the concentration of ion-pairing reagent, the type and concentration of the buffer, the pH, ionic strength, concentration of organic modifier, temperature and the sorptive properties of the stationary phase.

In the HPLC system used herein, a 25 cm x 4.6 μm 5 μm Spherisorb ODS-2 column (Rainin) was employed. The normal operating pH range for this column is 2.5-8.0. It was found that injection of DMEM culture medium samples without filtration resulted in rapid reduction in separation efficiency together with increasing back pressure. Consequently, medium samples were filtered before injection. For this purpose, samples were centrifuged using Microcon microconcentrators model 3, with membrane MWCO (molecular weight cut off in Daltons) at 3000, spun in an Eppendorf 5415C microcentrifuge at 14,000 rpm for 25 min. In spite of this precaution, some reduction in column efficiency was seen after two weeks of intensive medium sample injections (around 30 samples per day). Therefore, every two weeks the column was regenerated by pumping 30 volumes (125 mL) of HPLC- grade water, acetonitrile, chloroform, acetonitrile, water and finally the mobile phase each. A standard approach to prevent column performance deterioration is the use of guard columns. However, this led to significant deterioration in TPFA peak shape (guard column with Ultrapack ODS 10 μm).

All compounds and solvents were of high purity (solvents, HPLC grade). For oxidative detection, removal of dissolved air from the mobile phase is necessary to prevent air bubble formation at the column outlet, which disturbs the electrolysis process. Solvents were degassed by vacuum filtration through a Versapor 450 membrane filter (0.45 μm) and argon sparging. All new stainless steel components (among them a pulse damping system, which was incorporated between pump and injector for some experiments) of the HPLC system (excluding the detector flow cell) were passivated with 6 M nitric acid by pumping at 1 mL/min, 20 mL water, 20 mL nitric acid (6M), 20 mL glacial acetic acid, 20 mL water, 20 mL mobile phase.

With daily use, the detector lost sensitivity after about one month, chiefly due to contamination of the working glassy-carbon electrode. Glassy-carbon working electrodes were re-activated by immersion in a solution of 1 g chromic acid in 10 mL reagent grade sulfuric acid for 15 min

(background current ~ 0.50 μA instead of ~ 1.0/1.2 μA). It is also necessary to add reference electrode filling solution to reference electrode once monthly.

To maintain consistency with data on TPFA in blood and urine, 7-hydroxybenzoic acid (POBA) was utilized as the internal HPLC standard. Detector output was charted and integrated using a Hewlett-Packard 3390A integrator. Chromatograms and NMR spectra were scanned using a Hewlett- Packard CIIx Scanner and Deskscan II software. NMR spectra were obtained on a Bruker 360 MHz instrument, using D_zO as internal or external lock solvent.

Initially, the phosphate-buffered eluent at pH 6.8 was used. This gave good resolution of PFA the POBA standard, and TPFA (all at 100 μM) in an aqueous sample. A stronger response from TPFA vs. PFA was evident. In a separation of DMEM medium at eluent pH 7.02, strikingly few peaks (considering the many medium components) were detected, and 'windows' were observed in the 4-6 min and >7 min regions of the chromatogram. The major peak at 6.39 min was identified using a kit of amino acid standards as tryptophan, a medium component. Medium background could be reduced by sample dilution, at a corresponding cost in total sensitivity. TPFA peak broadening was found to be dependent upon the medium concentration; fortunately, it could be abated simply by moderate dilution of samples with H₂0. Subsequent samples were diluted by 1 : 1 or more, which gave single TPFA peaks.

It was found that a slight reduction of pH broadened the TPFA peak, which at pH 6.55 split into two peaks. Resolution of the POBA standard from tryptophan was only fair at this pH. At pH 7.02, the standard was nicely resolved, but tryptophan now co-eluted with TPFA Optimal resolution was obtained at about pH 6.8-6.9.

Initially, a phosphate buffer concentration of 0.1 N was used, which provides good resolution of 7-hydroxybenzoic acid (POBA - standard), TPFA and PFA in aqueous solutions. However, to optimize medium-drug resolution with respect to the tryptophan interference, other concentrations of phosphate buffer and the organic modifier (acetonitrile) were investigated to resolve the standard, TPFA and tryptophan peaks satisfactorily. Optimal separation was achieved using a phosphate concentration of 0.07 N, with 7% acetonitrile. The concentration of tetrabutylammonium phosphate (TBAP), set at 2.4 mM, significantly influenced resolution of the TPFA POBA and tryptophan peaks. As pointed out above, with prolonged usage the resolving power of the column deteriorates. However, it proved possible to re-optimize resolution by modifying the concentration of the ion-pair agent over a range of 2.4 -3.2 mM. Pyrophosphoric acid also can be used as a component of the buffer (cone. = 1 mM) to sharpen the PFA peak.

Calibration curves were developed using the optimized conditions for HPLC-ECD analysis of TPFA and PFA in water. Good linearity of response was observed over drug concentration ranges of 0 to 75-100 μM. The sensitivity ratio for PFA vs. TPFA was 0.0285. A similar response slope was obtained with TPFA in the medium (dilution 1:1) vs. water. The stability of TPFA in medium was monitored by both HPLC (low, medium concentrations) and ³¹P NMR (high concentration). TPFA was dissolved in the medium and incubated at 37±0.5 °C on a VWR 400 HPS hot plate with temperature probe. The ³¹P NMR spectrum of the medium alone reveals a single peak, attributed to phosphate, which was easily distinguishable from TPFA and PFA at pH 8.3 (sample concentrations 25 mM).

At high initial concentration in medium, TPFA is slowly converted to PFA in a zero order process with a half-life of >150 h. At a concentration of 1 μM (HPLC analysis), conversion of TPFA to PFA is much more rapid, but still apparently linear, possibly with some admixed non-linear component; the half-life is ca. 6 h. At 150 μM, the process is seen to be 1st order-like, showing a half-life of ca. 3 hr (half life = 161 min). The same behavior was seen at 75 μM, with a similar but not identical half-life (186 min).

The synthesis described in the aforementioned U.S. Patents

5,072,032 and 5,183,812 provided a TPFA sodium salt sample indistinguishable from past preparations spectroscopically. Freshly prepared sample differed from a sample subjected to prolonged storage in a vial in being virtually free of odor. It was also pure by HPLC. The HPLC methodology for the analysis was successfully developed using a new Waters ECD and systematic examination of elution conditions. Despite the complexity of the assay mixture, conditions were established for the determination of TPFA and PFA in micromolar concentrations, permitting study of TPFA 'prodrug' behavior in the assay medium. Example 2

Experiments using TPFA and PFA to inhibit replication of hCMV were carried out and the data were analyzed substantially as described in the literature [Angulo, A et al. Retinoid Activation of Retinoic Acid Receptors but Not of Retinoid X Receptors Promotes Cellular Differentiation and Replication of \human Cytomegalovirus in Embryonal Cells. /. Virology 1995, 69, 3831-3837]. The results indicated that the activities of TPFA and PFA in inhibiting hCMV DNA synthesis are similar under the conditions used, at least at highly effective concentrations. Additional experiments showed that PFA was unchanged during the experiments, whereas TPFA was partly transformed into PFA and into a metabolite. The metabolite was identified as a compound called thiophosphonic acid, or TPA TPA was identified in reported experiments on TPFA metabolism in dogs and cats [Straw et al. (1992), supra], TPA was shown herein to have some activity of its own, a finding which has heretofore not been reported.

Chemically pure TPA was synthesized as a control; new TPFA PFA and TPA samples were prepared and used in the protocol previously described herein.

The % viral DNA replication for treated (+drug) and untreated (-drug) virus-infected cells is plotted as a function of initial drug concentration in Fig. 1. The positive control drug, PFA is seen to suppress viral DNA synthesis from about 0.1-1 mM, with virtually complete suppression observed at 1 mM drug. In the TPFA-treated cells, a similar pattern is observed, but apparently at slightly lower overall concentrations. Several paired points which show a variation in the replication value of ±10% or more are included; however, the data indicate that TPFA has at least comparable activity to that of PFA The observed activity of TPFA in Fig. 1 is probably due to a combined effect of TPFA PFA and TPA (i.e., the summed inhibitions of TPFA and its two metabolites); no attempt has been made to factor out the individual contributions to activity.

Chemical analysis of TPFA and PFA in assay samples and controls using electrochemical analysis methods as described in Example 1 revealed the presence of a new peak in the HPLC trace, identified by use of authentic standard as thiophosphonic acid (TPA). The TPA was confirmed by NMR experiments. PFA was also identified in the TPFA-treated, virus-infected cells. The results are summarized in Tables 2-5. Data for two concentrations of drug are presented. At 1 mM drug, about 1/2 the original TPFA was found in the cell culture (after 24 hours), and a little less in the infected cell cultures. TPFA was stable in the initially prepared medium ("DMEM"). The sum of TPA remaining TPFA and PFA was in good agreement with the initial TPFA concentration, ruling out a major additional metabolite. PFA was stable under the same conditions.

From the foregoing description, one skilled in the art can readily ascertain the essential characteristics of the invention and, without departing from the spirit and scope thereof, can adapt the invention to various conditions. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient, and any specific terms employed herein are intended in a descriptive sense and not for purposes of limitation. TABLE 2

Normalized Results of Drug Metabolism Experiments:

TPFA in HCMV-Infected Human Cells

Compound Initial concentration Found TPFA, PFA and

(H) TPA Concentration (M) in Infected Cells

6 h 24 h 72 h xlO -3 x10^"3 x10^"3 x10^"3

TPFA 1 0.53 0.41 0.15 TPA 0 0.10 0.18 0.29 PFA 0 0.37 0.41 0.56

TPA 0.08 0.20 0.35 PFA 0.36 0.45 0.55 x10 -4 x10^"4 x10^"* x10^"4

TPFA 1 0.47 0.41 0.07 TPA 0 0.11 0.18 0.39 PFA 0 0.42 0.41 0.54

TABLE 3

Normalized Results of Drug Metabolism Experiments:

TPFA and PFA in Human Cells

Compound Initial concentration Found TPFA, PFA and

(H) TPA Concentration (M) in Cel ls

6 h 24 h 72 h xlO x10^"3 x10^"3 x10^"3

TPFA 1 0.56 0.43 0.27

TPA 0 0.09 0.17 0.27

PFA 0 0.35 0.41 0.46

TPA 0 0.13 0.18 0.36 PFA 0 0.43 0.43 0.44 x10^" x10 -3 x10^"3 xlO^"3

PFA 1 1 1

TABLE 4

Results of Drug Metabolism Experiments:

TPFA in HCMV- Infected Himβn Cells

Compound Initial concentration

(M) Found TPFA, PFA and

TPA Concentration (M) in Infected Cel ls

6 h 24 h 72 h x10^" x10^"3 xlO^-3 x10^"3

TPFA 1 0.44l0.02 0.4010.02 0.1810.01 TPA 0 0.0810.01 0.1810.01 0.3510.02 PFA 0 0.3110.01 0.4010.03 0.6810.01

x0.5x10^"3 xO.SxIO^*3 x0.5x10^"3 x0.5x10^"3

TPFA 1 0.4810.02 0.3410.04 0.1210.02 TPA 0.0710.01 0.19ι0.02 0.4010.02 PFA 0.3010.01 0.4310.04 0.6410.05 x10 -4 xlO^-4 xlO^"4 x10^"4

TPFA 1 0.4710.02 0.4010.02 0.0810.01 TPA 0 O. H O.01 0.1710.01 0.4710.03 PFA 0 0.4310.02 0. 010.04 0.6510.01

TABLE 5

Results of Drug Metabolism Experiments:

TPFA and PFA in Human Cells

Compound Initial concentration

(M) Found TPFA, PFA and

TPA Concentration (M) in Cel ls

6 24 h 72 h x10 -3 x10^"3 x10^'3 x10^"3

TPFA 1 0.5110.01 0.4310.01 0.31 0.01 TPA 0 0.0810.01 0.1710.01 0.3010.03 PFA 0 0.3210.02 0.41ι0.03 0.5210.04 H 00 x10 -4 x10^-4 xlO^*4 xlO^"4

TPFA 1 0.4610.02 0.4210.02 0.2110.02 TPA 0 0.14ι0.01 0.19ι0.01 0.3810.01 PFA 0 0.4610.02 0.4610.03 0.47ι0.05 x10 -3 xlO^-3 x10^"3 xlO^"3

PFA 0.8110.07 0.9310.04 1.0810.05

Claims

WHAT IS CLAIMED IS:

1. A method of inhibiting CMV replication in a mammalian patient in need of such treatment, said method comprising administering to the patient an amount of TPFA effective to inhibit viral replication.

2. A composition for use in inhibiting CMV replication in a mammalian patient, comprising an effective amount of TPFA and a suitable carrier or excipient.