The pharmacokinetics of 8-methoxypsoralen following i.v. administration in humans.

1. 8-methoxypsoralen (8-MOP) is a naturally occurring photoreactive substance which, in the presence of u.v. light, forms covalent adducts with pyrimidine bases in nucleic acids. For many years, 8-MOP has been used in PUVA therapy for treatment of psoriasis. Recently, the drug has been found to inactivate effectively bacteria spiked into platelet concentrates. The purpose of this study was to determine the pharmacokinetics and safety of 8-MOP administered intravenously in the bactericidal dosage range. 2. Eighteen volunteers were divided into three treatment groups to receive, respectively, 5, 10, and 15 mg 8-MOP infused over 60 min. Frequent arterial samples were gathered, and the blood and plasma were assayed for 8-MOP concentration. The pharmacokinetic parameters were determined by moment and compartmental population analysis, the latter performed with the program NONMEM. Haemodynamics, ventilatory pattern, and subjective effects were recorded throughout the study. 3. The intravenously administered 8-MOP was well tolerated in all individuals, and no acute toxicity was observed. 4. The pharmacokinetics of 8-MOP were best described by a three-compartment mammillary model in which the volumes and clearances were proportional to weight. The mean pharmacokinetic parameters for the plasma concentrations were: V1 = 0.045 1 kg-1, V2 = 0.57 1 kg-1, V3 = 0.15 1 kg-1, CL1 (systemic) = 0.010 1 kg-1 min-1, CL2 = 0.0067 1 kg-1 min-1, CL3 = 0.012 1 kg-1 min-1. The mean pharmacokinetic parameters for the blood concentrations were: V1 = 0.061 1 kg-1, V2 = 1.15 1 kg-1, V3 = 0.21 1 kg-1, CL1 (systemic) = 0.015 1 kg-1 min-1, CL2 = 0.011 1 kg-1 min-1 and CL3 = 0.015 1 kg-1 min-1. 5. The plasma pharmacokinetic model described the observations with a median absolute error of 17%, and the blood pharmacokinetic model described the observations with a median absolute error of 18%. Analysis of the relative concentration of 8-MOP between plasma and red blood cells suggested concentration-dependent partitioning. 6. The addition of 7.5 mg 8-MOP to 300 ml platelet concentrate would produce bactericidal concentrations of 25 micrograms ml-1. Simulations based upon our data show that intravenous administration of 7.5 mg over 60 min would result in systemic concentrations of 8-MOP similar to those observed with conventional PUVA therapy. We conclude that the extensive safety history established in PUVA therapy will be applicable to this new application of 8-MOP.

Post-PCR sterilization: development and application to an HIV-1 diagnostic assay.

We have developed an effective post-PCR sterilization process and have applied the procedure to a diagnostic assay for HIV-1. The method, which is based on isopsoralen photochemistry, satisfies both the inactivation and hybridization requirements of a practical sterilization process. The key feature of the technique is the use of isopsoralen compounds which form covalent photochemical adducts with DNA. These covalent adducts prevent subsequent extension of previously amplified sequences (amplicons) by Taq polymerase. Isopsoralens have minimal inhibitory effect on the PCR, are activated by long wavelength ultraviolet light, provide sufficient numbers of covalent adducts to impart effective sterilization, modify the amplified sequence such that it remains single-stranded, and have little effect on subsequent hybridization. The sterilization procedure can be applied to a closed system and is suitable for use with commonly used detection formats. The photochemical sterilization protocol we have devised is an effective and pragmatic method for eliminating the amplicon carryover problem associated with the PCR. While the work described here is limited to HIV-1, proper use of the technique will relieve the concern associated with carryover for a wide variety of amplicons, especially in the clinical setting.

Post-PCR sterilization: a method to control carryover contamination for the polymerase chain reaction.

We describe a photochemical procedure for the sterilization of polynucleotides that are created by the Polymerase Chain Reaction (PCR). The procedure is based upon the blockage of Taq DNA polymerase when it encounters a photochemically modified base in a polynucleotide strand. We have discovered reagents that can be added to a PCR reaction mixture prior to amplification and tolerate the thermal cycles of PCR, are photoactivated after amplification, and damage a PCR strand in a manner that, should the damaged strand be carried over into a new reaction vessel, prevent it from functioning as a template for the PCR. These reagents, which are isopsoralen derivatives that form cyclobutane adducts with pyrimidine bases, are shown to stop Taq polymerase under conditions appropriate for the PCR process. We show that effective sterilization of PCR products requires the use of these reagents at concentrations that are tailored to the length and sequence of the PCR product and the level of amplification of the PCR protocol.

Photochemistry of the furan-side 8-methoxypsoralen-thymidine monoadduct inside the DNA helix. Conversion to diadduct and to pyrone-side monoadduct.

We have studied the photochemical reactions of 8-methoxypsoralen (8-MOP) with calf thymus DNA. Analysis of the photoproducts formed was carried out by enzymatic digestion of the 8-MOP-modified DNA, followed by HPLC separation of photoadducts by high-pressure liquid chromatography (HPLC). The 4',5' (furan-side) monoadduct of 8-MOP bound to thymidine is converted to cross-linked thymidine-8-MOP-thymidine diadduct by 341.5 nm light with a quantum yield of 0.028 +/- 0.004. This is 4 times greater than the quantum yield for initial adduct formation (0.0065 +/- 0.0004). When low levels of 8-MOP are covalently bound to DNA by using 397.9 nm light, less than 10% of the adducts formed are diadducts yet nearly 70% are in 5'-TpA cross-linkable sites. The furan-side monoadducts in these sites can subsequently be converted to diadduct or to a lesser extent 3,4 (pyrone-side) monoadduct.