Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing – Multienzyme complexes or mixtures of enzymes
Reexamination Certificate
1996-04-10
2001-09-18
Bui, Phuong T. (Department: 1638)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
Multienzyme complexes or mixtures of enzymes
C424S094300, C424S094500, C435S455000, C435S458000, C435S194000, C514S04400A, C536S023200
Reexamination Certificate
active
06290952
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to pharmaceutical compositions suitable for treating or curing clinical complications induced by infections with Gram-negative bacteria, including sepsis. In particular the invention is directed at systemically applicable compositions. The compositions contain components suitable for detoxifying bacterial-wall derived lipopolysaccharides (also known as endotoxins) rendering these products less deleterious to mammals such as humans, in particular to patients with sepsis, optionally in combination with reduced host-defence resistance, i.e. after organ transplantations, during leucopenia (ref. 1) associated with cancer or chemotherapeutic treatment of cancer or during AIDS and AIDS-related diseases (ref. 2).
The invention also relates to pharmaceutical compositions suitable for stimulating bone formation, e.g. for mending broken bone or for prophylaxis or therapy of metabolic bone diseases such as osteoporosis and osteomalacia and also pharmaceutical compositions for decreasing or inhibiting undesired bone formation.
BACKGROUND INFORMATION
Endotoxin is a negatively charged lipopolysaccharide present in the capsule of Gram-negative bacteria (ref. 3). Endotoxins are complexes of phospholipid (lipid A) and polysaccharide. The endotoxins produced by different bacteria differ in their antigenicity but they all have the same biological effects which are mainly due to lipid A. For the purposes of this description the term endotoxin also comprises enterotoxins. In addition to the negatively charged sugar moieties, an endotoxin contains two phosphate groups which are essential for its toxicity (ref. 3, 4).
Although it is an ubiquitous molecule in the external environment as well as in the gastro-intestinal tract of many species, an endotoxin can be extremely deleterious to these species once it leaves the gastro-intestinal tract e.g causing sepsis and inflammation such as in an abcess even in amounts as low as 10 picogrammes. Yet so far, no important endotoxin detoxifying mechanism has been found in vivo (ref. 5).
Endotoxin is known to induce serious even lethal complications (ref. 5 and 6). In fact, despite the use of antibiotics, this bacterial product is the major cause of death in intensive-care units in Western society.
There are numerous different endotoxins produced by various microorganisms and consequently the actions of endotoxin in vivo are numerous as are the ways it can enter the organism. The symptoms associated with Gram negative infections therefore also vary widely among patients (ref. 7). These symptoms may be further complicated by septic shock of which hypotension, peripheral vasodilation and diffuse intravascular coagulation are the main characteristics (ref. 8). Subsequently organs such as heart (acute heart failure), lungs (adult respiratory distress syndrome). kidney (acute tubular necrosis) and brain may be affected (ref. 8). Endotoxin mediated pathology also comprises the syndrome of multiple organ failure and any other syndrome generally accepted in the art to be directly or indirectly caused by endotoxin.
To date, antibodies directed against endotoxin are the only endotoxin detoxifying proteins known to reduce toxicity irreversibly, but the clinical value of these antibodies remains to be established. Other substances which are able to bind endotoxin, such as lipopolysaccharide binding protein and high density lipoproteins (HDL) (ref. 9), exhibit the major drawback of forming reversible complexes in vivo. Upon dissociation of these complexes, the native (toxic) molecule is produced again. Furthermore although the detoxifying activity of plasma has been noted for some time (ref. 10) efforts to isolate or characterise the substance(s) responsible for this activity have not been successful. Other experimental approaches to treat sepsis include the application of preparations which antagonize the activities of cytokines (e.g. TNF-&agr;), which are important mediators of endotoxin-induced shock, aggravating the effects of endotoxin in vivo. A major disadvantage of this approach is that these preparations do not detoxify the causative agent but rather block one of the reactions of the body to this toxin. In addition, antagonizing naturally occurring cytokines may cause multiple side effects.
Alkaline phosphatase (EC 3.1.3.1) is a common enzyme present in many species, including man and has been studied extensively. The DNA sequence encoding alkaline phosphatase has even been obtained, but so far no commercial exploitation thereof has occurred. Although the enzyme is routinely applied as antibody label or as a marker for liver and neutrophil function, it's biological relevance is still unknown. Recombinant alkaline phosphatase enzymes with improved specific activity used as indicator reagents are disclosed, e.g. in EP-A-0 441 252. This patent application however mentions nothing regarding anti-endotoxin activity or bone formation of alkaline phosphatase. The cited European patent application describes a number of derivatives in which one amino acid differs from the wild type. The substituents include replacement of Thr 100 by Val or Ile, replacement of Lys 328 by Arg, replacement of Val 99 by Ala, replacement of Thr 107 by Val, replacement of Asp 101 by Ser, replacement of Val 377 by Ala and replacement of Ser 115 by Gly as well as replacement of Ala 103 by Asp. Other derivatives described in the cited patent application are derivatives with M maleimidobenzoyl-N-hydroxysuccinimide ester for carrying out a sandwich EIA and a thiolated mutant of alkaline phosphatase which can be derived through use of succinimidyl-4-N-maleimidomethyl-1-thicapramide cyclohexanecarboxylate. Of all these derivatives no mention is made of the charge carried by the alkaline phosphatase derivative. It is pointed out that all the mentioned derivatives with the exception of the replacement of Ala 103 by Asp have been calculated by us as resulting in a more positive netto charge or an equal netto charge in comparison to the corresponding native alkaline phosphatase.
Alkaline phosphatase is a membrane-bound ecto-enzyme which is known to dephosphorylate extracellular molecules. The enzyme is present in many organs, including intestine, kidney, osteoblasts and neutrophils (ref. 11, 12 and 13). in vitro, it exhibits a pH optimum of approximately 10.5. (ref. 12). This extremely high pH optimum has hampered recognition of its biological relevance (ref. 12-14), because it was felt that this pH level does not occur in biological tissues of the intact organism.
In a number of publications a derivative of alkaline phosphatase and collagen, in particular fibrillar collagen is described. Nothing is mentioned about the netto negative charge of such a derivative, however, we have calculated that the netto charge is positive in comparison to a non-derivatized alkaline phosphatase.
In U.S. Pat. No. 4,409,332 (1983) collagen sutures derivatized with alkaline phosphatase are described as reducing the inflammatory characteristics of collagen. The collagen induced inflammation is not an inflammatory reaction due to endotoxins, it is an inflammation that is generally caused by damage of tissue that has occurred, by the fact that collagen is a heterologous protein which is foreign to the body and by the fact that collagen always induces coagulation in vivo which can subsequently activate inflammatory cells in a number of manners. An inflammation due to infections of the wound is not likely as the authors themselves frequently state that they worked in a sterile environment, using sterile solutions. A person skilled in the art cannot derive from this cited patent publication how alkaline phosphatase coupled to collagen can inhibit the inflammation usually caused by collagen. A number of manners can however be postulated such as, for example by protection of antigens for cells of the specific immunoreaction, thereby prohibiting recognition or by binding positively charged mediators of the non-specific immune reaction as alkaline phosphatase contains negatively charged
Bakker Winston Willem
Hardonk Machiel Josephus
Meijer Dirk Klaas Fokke
Poelstra Klaas
Bui Phuong T.
Fitch Even Tabin & Flannery
Rijksuniversiteit Groningen
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