A clinical grade poly I:C-analogue (Ampligen) promotes optimal DC maturation and Th1-type T cell responses of healthy donors and cancer patients in vitro.

Maturation of dendritic cells (DC) can be triggered in vitro by inflammatory cytokines or Toll-like receptor (TLR) ligands such as CpG or polyI:C. Corresponding, well-characterized agents which can be applied in clinical settings are sparse. We have evaluated a clinical grade, non-toxic analogue of polyI:C, poly(I:C12U) (Ampligen), as a potential adjuvant for cancer immunotherapy, for its ability to drive maturation of human myeloid DC. Our results provide evidence that poly(I:C12U) is effective in inducing optimal phenotypic (elevated levels of MHC-Class I/Class II, CD83, CCR7, CD86 and CD40 molecules) and functional maturation of human DC in vitro, capable of promoting the production of the inflammatory (Th1-type) cytokine IL-12, with significantly lower levels of IL-10 production, compared to that induced by the parent compound polyI:C. Importantly, poly(I:C12U) has a comparable effect on the maturation and function of DC derived either from healthy donors or cancer patients indicating that it is able to overcome any immune suppressive factors associated with the tumour bearing state. These characteristics make poly(I:C12U) a suitable agent for use as an adjuvant in cancer directed immunotherapeutic regimes.

Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I]:poly [C(12)U] (Ampligen R).

Human papilloma virus (HPV) found in 99.7% of cervical cancers represents an attractive immunotherapeutic target for novel adjuvant dendritic cell (DC) immunotherapy. DC primed with HPV antigens have been shown to be capable of inducing CTL responses powerful enough to eradicate established murine tumours expressing HPV16 antigen. The use of tumour lysate has been found to be an effective means of priming DC with tumour associated antigens in animal models and in clinical trials leading to significant anti-tumour responses. Autologous DC primed with sonicated HPV expressing tumour lysate have been shown to be capable of inducing HPV specific classes I and II T-cell immunity in a pilot clinical study.Synthetic double stranded polyribonucleotides are effective in vitro activation/maturation agents capable of inducing a stable mature DC phenotype producing high levels of IL12. However, the prototype polymer poly [I]:poly [G] has proved to be clinically toxic. Preliminary in vitro data have demonstrated that a novel clinically non-toxic analogue polymer poly [I]:poly [C(12)U] (Ampligen R) can effectively induce in vitro maturation of human monocyte derived DC with sustained bioactive IL12 production. Human monocyte derived DC primed with tumour lysate and matured with synthetic dsRNA may therefore offer an effective way of optimising Th1 specific anti-cancer T-cell responses in cancer patients. This strategy is currently being tested in a clinical trial in patients with cervical cancer.

Phosphoric acid triester glutathione alkyltransferase. A mechanism for the detoxification of dimethyl phosphate triesters.

1. 2-Chloro-1-(2,4,5-trichlorophenyl)vinyl dimethyl phosphate (tetrachlorvinphos) is demethylated by mammalian liver supernatant (100000g) protein in the presence of GSH. 2. GSH acts as an acceptor of the transferred methyl group to form S-methyl glutathione. 3. The enzyme that catalyses this reaction is present in the soluble fraction of liver from mouse, rat, rabbit and pig at similar activity. The enzyme was purified 45-fold from pig liver, dimethyl 1-naphthyl phosphate being used as assay substrate. 4. Methyl groups are readily removed from most of the substrates studied; ethyl groups are removed at one-fiftieth to one-hundredth of the rate for methyl groups. It is likely that the enzyme plays an important role in the detoxification of the phosphate triester pesticides containing CH(3)-O-P groups.

The oxidative dealkylation of insecticidal phosphoric acid triesters by mammalian liver enzymes.

1. The dealkylation of the insecticidal phosphoric acid triester, 2-chloro-1-(2,4-dichlorophenyl)vinyl diethyl phosphate, proceeds in mammalian liver slices via an oxidative mechanism and not by hydrolysis. 2. The enzyme that catalyses the reaction is located in the microsomal fraction of liver homogenate and is dependent for activity on molecular oxygen and NADPH. 3. There are large species differences between rat, mouse, rabbit and dog in the activity of the enzymes, the relative rates of dealkylation being 1, 8, 24 and 88 respectively in liver slices. 4. Dimethyl and di-isopropyl phosphate triesters are also dealkylated by rabbit liver microsomal preparations. 5. The mechanism of dealkylation involves hydroxylation at the alpha-carbon atom of an alkyl group, which is removed as the corresponding aldehyde, and is thus analogous to that of similar reactions catalysed by the microsomal mixed-function oxidases. 6. The relevance of these findings in the toxicology of phosphoric acid triesters is discussed.

Species specificity of phosphate triester anticholinesterases.

Two widely distributed enzyme systems degrade phosphate triester anticholinesterase agents to inactive phosphate diesters by nonhydrolytic mechanisms. The substrate specificity of these enzymes is discussed and the level of activity in various mammalian livers is described. The distribution of these enzymes is an important factor in accounting for the species specificity of at least some anticholinesterase agents.

An improved synthesis of isopentenyl pyrophosphate.

1. Isopentenol was converted into isopentenyl phosphate with phosphoryl chloride in ether containing pyridine. 2. The isopentenyl phosphate reacted in 2-methylpropan-2-ol-water with morpholine and dicyclohexylcarbodi-imide to give isopentenyl phosphoromorpholidate. 3. The isopentenyl phosphoromorpholidate, with inorganic phosphate in pyridine containing tributylamine, gave isopentenyl pyrophosphate. The yield of pyrophosphate from monophosphate was 80-85% and the yield of pyrophosphate from isopentenol 40-60%. [1-(14)C]-Isopentenyl pyrophosphate was prepared by this method. 4. The yield of isopentenyl pyrophosphate from isopentenyl phosphate was substantially improved, in comparison with the yields obtained by published methods via the phosphoramidate, by the use of the phosphoromorpholidate.