Endotoxin therapeutics

Lynn, W.A. Anti-endotoxin therapeutic options for the treatment of sepsis. J Antimicrob Chemother 41 Suppl A, (1998) 71-80. 

In addition to the urgent problem of capacity, manufacturers have to cope with the operating costs of production, which are increased by the need for skilled personnel and expensive media components. Another cost driver is the inherent contamination risk when using mammalian cell culture systems. All materials must be checked closely for bacterial and viral contamination, and the presence of prions and endotoxins. This affects not only the manufacturing process, but also downstream materials and even human semm albumin (HSA) used for formulations. In the end, production costs add up to 100-1000 per gram of therapeutic protein. 

The antagonistic properties of FR 900452 have also been examined in a model of endotoxin shock, the haemodynamic and haematological manifestations of this condition closely resembling the changes induced by PAF. FR 900452 (10 mg/kg, i.v.) almost completely prevents PAF (1 /ig/kg, i.v.)-induced thrombocytopenia and leukocytopenia in rabbits [295]. It also significantly inhibits endotoxin (E. coli LPS, 30 /ig/kg, i.v.)-induced thrombocytopenia but not leukocytopenia. The same dose of FR 900452 also causes the decreased arterial blood pressure to return to normal in the endotoxin-induced rat hypotension model, an effect also reported for other PAF inhibitors [121, 274], Finally, FR 900452 has been tested for its therapeutic effect on rat nephrosis induced by aminonucleoside (puromycin, 100 mg/kg, i.p.). At 100 mg/kg twice a day orally for 6 days, the agent significantly reduces urinary protein loss in nephrotic rats [298]. 

The experience of application of various efferent methods of endotoxin elimination has led to the idea of creating selective anti-LPS hemosorbents with Polymyxin B as the bioUgand. The covalently immobilized Polymyxin B does not cause side effects characteristic of free Polymyxin B and keeps its therapeutic effects. G.W. Duff in 1982 demonstrated for the first time the sorption efficiency of Polymyxin B-immobilized sorbent in vitro [9]. In 1984 K. Hanasawa with co-workers developed PMX-F - a fibrous polystyrene based hemosorbent with immobilized Polymyxin B that could be used directly for blood perfusion [10]. In 1994 PMX-F sorbent was officially approved for patient treatment in Japan. Currently, there are several polymyxin immobilized (PMX-F) sorbents produced commercially for clinical use, such as Alteca - LPS Absorber (Sweden), Toray Industries hic. (Japan), etc. However these sorbents have poor hemocompatibility and are very expensive. 

Cmde enzyme extracts are often unsuitable for therapeutic uses because of their antigenicity, contamination with endotoxins, and rapid inactivation under physiological conditions or in fluids intended for intravenous infusion over several hours. When the enzyme used is a foreign protein, it can eHcit an immune response that alters the clearance rate or induces severe allergic reactions in the host. After an intravenous injection of an enzyme, its activity in plasma decreases with time due to distribution to other fluids and tissues, and as a consequence of proteolysis or excretion. Distribution is related to molecular size, charge, and HpophiHcity surface charges attributable to the availability of free amino, amido, or carboxyl groups may affect the rate of inactivation of some enzymes. 

Assays for media components and purification reagents are generally specific to the particular item used and will not be discussed further here. Assays for endotoxin, live virus, mycoplasma, and live microbes are relatively standard (USP, CFR, or other regulatory procedures) and likewise will not be discussed in detail. The determination of trace levels of host cell proteins in therapeutic proteins can be a formidable task, because most commonly... 

Cortiveau, C.C., Danner, R.L. Endotoxin as a therapeutic target in septic shock. Infect Agents Dis 2 (1993) 35-43. 

We had sought to address two questions (a) what is the elimination half-life of DS-96 in the murine model, and does the tV2 correspond to pharmacodynamic data shown in Fig. 12.19 (b) what is the therapeutic plasma concentration of DS-96 that corresponds to full protection against endotoxemic challenge in mice. DS-96 at a dose of 200 (xg/mouse (8 mg/kg) was administered to CF1 mice via i.p. and i.v. routes. Plasma concentrations of DS-96 were determined by LC-MS/MS using a deuterated DS-96 internal standard (Nguyen et al., 2008 Shrestha et al., 2008). The elimination tV2 in mice is about 400 min (Fig. 12.22), which is consistent with the observed pharmacodynamic (in vivo efficacy) data shown in Fig. 12.19. The observed concentration-versus-time profile of DS-96 in the mouse i.p. model suggests that a plasma concentration of 0.5-1.5 txg/mL corresponds to complete protection by a dose of 200 ng/animal of LPS in the D-galactosamine-primed model of endotoxin-induced lethality. 

Cross, A., Opal, S.M. Therapeutic intervention in sepsis with antibody to endotoxin is there a future . J Endotoxin Res 1 (1994) 57-59. 

Hurley, J.C. Antibiotic-induced release of endotoxin. A therapeutic paradox. Drug Saf 12 (1995) 183-195. 

Despite both the directness and simplicity of the hypothesis, namely that antibody directed toward a common, toxic moiety of endotoxin has therapeutic potential, the concept of anti-endotoxin antibody has been difficult to prove and mired in controversy. 

Although the clinical relevance of these observations remains to be better demonstrated, antiendotoxin strategies could have some additional therapeutic value. These preliminary observations lend credence to Ivan Bennett s insightful comment over 30 years ago that endotoxin may play a larger role in human health and disease. It has been said in jest that endotoxins will probably turn out to be the cause of most human diseases now classified as idiopathic and that they may also be the cause of human health... these possibilities have yet to be excluded (Bennett, 1964). 

Many animal and preclinical studies have showed benefit of a variety of antiendotoxin therapies, but this effect has not been translated into human trials. Since both pre-clinical rationale and studies support the targeting of endotoxin to ameliorate the pro-inflammatory and pro-coagulation response of severe sepsis, we should continue to pursue therapeutic intervention in this area. 

These impurities pose risks for the safety of proteins used as therapeutics and must be removed to a final concentration below their target limit. In addition, the product stream contacts materials such as filters and resins. Extractables, such as leachates from protein A resins, can pose an immunogenic risk to the patient and must be eliminated.75 Finally, adventitious agents such as viruses and bacterial pathogens or related contaminants such as endotoxins can lead to serious problems with the safety of the protein preparation and therefore must be minimized. Table 32.6 lists concentrations for the above impurities that are generally considered acceptable in a final protein product.76... 

When genetic engineering is used to produce the therapeutic Hb, three challenges present potential limitations to utility. First, the production scale itself presents a significant challenge. A second challenge is related to the expression system and its current limitations and third, bacterial endotoxins or other pyrogens must be removed. 

Finally, if E. coli is used as the production system, the therapeutic Hb must be purified rigorously. Both elimination of all non-Hb proteins and elimination of endotoxins are stringent requirements, because the latter lipoproteins are recognized pyrogens. Extensive chromatographic purification is used to accomplish this. 

---