The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or common general knowledge.
Heparin is a polydisperse, naturally occurring polysaccharide that inhibits coagulation, the process whereby thrombosis occurs. Heparin consists of unbranched polysaccharide chains of varying lengths and molecular weights. Chains of molecular weight from 5000 to over 40,000 daltons make up pharmaceutical grade heparin.
Heparin, which is typically derived from natural sources such as porcine intestine or bovine lung tissue, can be administered therapeutically for prevention and treatment of thrombosis. However, the effects of unfractionated heparin can be difficult to predict. Therefore, during treatment of thrombosis with unfractionated heparin, coagulation parameters must be monitored very closely to prevent over- or under-anticoagulation.
Numerous brands of heparins and low molecular weight heparins (LMWH), such as dalteparin and enoxaparin, are available for the treatments that rely on their anti-coagulant activity. A large number of in vitro and animal experimental investigations, and even clinical trials, indicate that heparin and its derivatives have beneficial properties others than those related to its anticoagulant effect. However, existing heparins and LMWH are not suitable for treating other medical conditions because of the bleeding risk associated with the anticoagulant effect.
The LMWH dalteparin has been shown to decrease protracted labour in women receiving prophylaxis for deep venous thrombosis. The mechanism is believed to involve dalteparin-induced increased levels of interleukins resulting in a favourable inflammatory reaction that promotes ripening of the cervix. Further, dalteparin has been shown to increase contractility of the uterus (Acta Obstetricia et Gynecologica, 2010; 89:147-150). However, heparin and LMWH are not suitable for preventing or treating such maladies for a number of reasons.
Firstly, heparin and LMWH have significant, well known anti-coagulant effects that restrict their use in late pregnancy and during delivery, both for prophylactic and acute use, due to the bleeding risk. For example, the use of dalteparin is strictly contraindicated when epidural anaesthesia is given, a measure frequently taken during child birth.
Secondly, heparin, and to some extent LMWH, has been associated with heparin-induced thrombocytopenia, a severe immune-mediated drug reaction that can occur in any patient exposed to heparin. It is a potentially devastating pro-thrombotic disease caused by heparin-dependent antibodies that develop either after a patient has been on heparin for five days or more, or if the patient has had previous heparin exposure.
Another undesirable possible effect of long term treatment with heparin is that it may induce demineralization of bones and cause osteoporosis.
There have been many attempts to eradicate or reduce the anticoagulant activity of heparins or low molecular weight heparins in order to provide low anticoagulant heparins
(LAHs) which aim to benefit from other potential clinical effects from the heparin chains than the anticoagulant effect, without carrying the risk of undesirable effects associated with heparin, predominantly bleeding. However, there is limited clinical experience of this type of heparins and so far no such products have been allowed for clinical use.
Heparin exerts its anticoagulant activity primarily through high-affinity binding to, and activation of, the serine proteinase inhibitor, antithrombin (AT). AT, an important physiological inhibitor of blood coagulation, neutralizes activated coagulation factors by forming a stable complex with these factors. Binding of a specific pentasaccharide within the polysaccharide chains of heparin causes a conformational change in AT that dramatically enhances the rate of inhibition of coagulation factors, thereby attenuating blood coagulation and the formation of blood clots.
European patent application EP 1 059 304 discloses enzymatically degraded or oxidized heparin resulting in a product with low anticoagulant effect, having an average molecular weight of 9 to 13 kDa, which is suggested for the treatment of neurodegenerative diseases.
U.S. Pat. No. 4,990,502 demonstrates one way of treating native heparin to cleave the pentasaccharide residues responsible for the anticoagulant effect and a following depolymerisation that results in a low anticoagulant, low molecular weight heparin with a an average molecular weight 5.8 to 7.0 kDa. However, in U.S. Pat. No. 4,990,502 time consuming methods, such as dialysis for about 15 hours, are used to terminate the oxidation process. Such processes can affect the molecular weight distribution of the final product and give rise to unfavourable structural variants, which can be seen by 1H NMR.
Controlling the molecular weight and the length of the polysaccharide chains is crucial to obtain the desired biological effect of the compound. The bioavailability of long chain heparins after subcutaneous dosing is low and the possibility of heparin induced thrombocytopenia (HIT) induction is also positively correlated to the chain lengths. To reduce these clinically undesired properties the heparin derivative should not be of full length. Heparin chains of certain molecular weight can be obtained by fractionation of standard heparin. However, the production of heparin derivatives of intermediate or low molecular weight by fractionation methods such as gel-filtration, alcohol precipitation and ion exchange chromatography is associated with a significant waste of raw material, as high molecular mass heparins are discarded.
As disclosed herein, heparin derivatives having low anticoagulant activity can be prepared using a process comprising the steps of oxidation of unfractionated heparin, depolymerisation and reduction of resulting terminal groups, which derivatives are of use in decreasing the duration of protracted labour.
In particular, we have unexpectedly found that levels of unwanted structural modifications occurring during the preparation of heparin derivatives using such processes can be minimised by controlling the time elapsed between the oxidation and reduction steps. Further, we have found that the average molecular weight of the resulting heparin derivative can be controlled by monitoring the progress of the depolymerisation step (or by reference to a previously-performed process) and adjusting the duration of the depolymerisation step accordingly.
International (PCT) application number PCT/SE2012/051433 (published as WO 2013/095279) discloses a process for the preparation of derivatives of unfractionated heparin comprising oxidation, depolymerisation and reduction of terminal groups, which derivatives have low anti-coagulant activity and are of use in decreasing the duration of protracted labour. However, this international application does not refer to monitoring the progress of the depolymerisation step to control the average molecular weight of the resulting heparin derivative.