New PA 20230374055 published:CD206 DRUG DELIVERY VEHICLES with Bisphoshonate Payloads | NAVB Message Board Posts


Navidea Biopharmaceuticals, Inc.

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Msg  40535 of 40762  at  11/24/2023 3:20:28 PM  by

moneyonomics

The following message was updated on 11/29/2023 5:42:39 PM.

New PA 20230374055 published:CD206 DRUG DELIVERY VEHICLES with Bisphoshonate Payloads

  
       and why liposomes (a possible competition) or related constructs with much larger structures, etc, struggle to be successful 
 
 
 
 
 
 2022 PR
 

In this study, results demonstrate that Navidea’s macrophage-targeting Manocept platform technology, consisting of mannosylated amine dextrans (“MAD”) and carrying the therapeutic payloads paclitaxel or a novel bisphosponate, could drive the phenotype of macrophages in vitro towards a proinflammatory type (more CD80 and CD86 expression, less CD206 and CD163 expression). This is important because in tumors there exist tumor-associated macrophages (“TAMs”) that are typically of the wound healing, anti-inflammatory type, and these play a key role in paradoxically shielding the tumors from the body’s immune response. Driving the TAM phenotype more towards a proinflammatory state should enable both an immune response against the tumors as well as increase the efficacy of other therapies that can work alongside the body’s immune system against the tumors.

In addition to the in vitro work using both the paclitaxel and bisphosphonate carrying constructs, in vivo studies using the MAD-paclitaxel construct in a mouse tumor model demonstrated that this construct increased the efficacy of an approved checkpoint inhibitor therapy, anti-CTLA4, reducing tumor growth by 76% compared to a saline control. Delivery of paclitaxel and bisphosphonates by this method also reduces off-target exposure and should limit toxicity.

Future studies will examine the effect of the MAD-bisphosphonate therapy with and without anti-CTLA4 therapy in the mouse tumor model. Preclinical toxicity studies will also be conducted en route to an Investigational New Drug (“IND”) application.

 
 
 

Abstract

Provided are novel compounds containing a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a nitrogenous bisphosphonate compound coupled to the backbone via a thiol-maleimide conjugation, in addition to pharmaceutical compositions, methods of synthesizing, and methods of use. The thiol-maleimide conjugation of a bisphosphonate to a polymeric carbohydrate backbone provides for methods of using the compounds and compositions thereof for releasing the therapeutic payload when internalized into a mannose-binding C-type lectin receptor-expressing cell, such as tumor associated macrophages (TAMs) for the treatment of various diseases, including, cancer, autoimmune diseases, and inflammatory disorders.

 

BACKGROUND OF THE INVENTION

[0003] Cancer is the second leading cause of death in the United States of America, accounting for nearly one of every four deaths. Cancer is characterized by the unregulated growth and cell division of cancer cells. However, cancers benefit enormously from chronic maladaptive immune responses to tumors, and macrophages are a key mediator of that maladaptive response. Macrophages are a common cell type in all tissues of the body and are important components of innate immunity. Macrophages also contribute significantly to the maintenance of tissue homeostasis and wound repair. In general, macrophages respond to various stimuli in their local microenvironment by altering their expression patterns for many, potentially hundreds, of genes. Such phenotypically altered macrophages are said to be activated macrophages. Depending upon which stimuli a macrophage is responding to, a wide range of activated phenotypic states can be attained. Among those genes that are differentially expressed upon macrophage activation are cell surface markers (such as, for example, the macrophage mannose receptor, CD206) and various cytokines, enzymatic pathways leading to the generation of reactive oxygen species (ROS), and other signaling molecules that can regulate the behavior of other components of the immune system, such as T lymphocytes (T-cells).

[0004] Extensive literature on macrophage activation have reported experimental results showing that there are a vast number of activated phenotypes and expression of only one or a few genes cannot accurately identify any one particular phenotype. However, these activated phenotypes can be characterized for their overall immune status and can be placed on a continuum, with highly pro-inflammatory phenotypes at one end of the continuum and immunosuppressive and wound healing phenotypes at the other end. Traditionally, as referenced by historic macrophage phenotype literature, activated macrophages were divided into two phenotypes: (1) classically activated, called M1, which is highly proinflammatory, and (2) alternatively activated, called M2, which is immunosuppressive and promotes wound healing. It is now understood that a strictly dichotomous classification of activated macrophage phenotypes is overly simplistic and does not represent the true plasticity of macrophage responses to stimuli from their microenvironments; however, the concept that activated macrophages can influence a local immune response by being either proinflammatory (M1-like) or immunosuppressive (M2-like) continues to have utility when describing the role of macrophages in various pathological states.

[0005] Tumor associated macrophages (TAMs) are abundant in tumors and highly significant contributors to the maladaptive immune response associated with cancer and other conditions. TAMs are the most numerous immune cells that infiltrate tumors and can comprise from about 5% to >30% of all cells in a tumor. While both M1-like and M2-like TAMs are known, the large majority of TAMs residing in or near established tumors are immunosuppressive, i.e., M2-like activated macrophages.

[0006] Bisphosphonate drugs have been given to cancer patients with metastases to the bones for many years. The structural components of bone are constantly being turned over in a process of continuous renewal in healthy individuals, with two cell types primarily involved in this bone turnover: (1) the osteoclasts, which degrade bone, and (2) the osteoblasts, which continuously lay down new bone. As osteoclasts are a type of macrophage, treatment with bisphosphonates may reduce the number of TAMs in bone metastases and, importantly, alter the phenotype of the remaining TAMs to be more M1-like. There is also some evidence that treatment with bisphosphonate drugs can slow the growth of bone metastases, which is postulated to be due to depriving the tumors of the pro-tumoral benefits and support provided by M2-like TAMs.

[0007] However, there are several challenges with using bisphosphonates as a TAM-targeted cancer immunotherapy: (1) inefficient cell penetration due to the highly charged nature of bisphosphonates preventing diffusion of these molecules across cellular lipid membranes; (2) rapid localization to bone; and (3) off-target toxicities. Historically, to overcome these limitations, liposomal constructs carrying various bisphosphonates were used to decrease bone localization and increase their payload delivery to TAMs. Some studies involving animal models of cancers utilized liposomal constructs that carried payloads of the bisphosphonate drug, clodronate. Clodronate is a non-nitrogenous bisphosphonate drug. Clodronate induces macrophages (including TAMs and osteoclasts) to undergo apoptosis, however, has a reduced ability to alter the phenotype of macrophages compared to nitrogenous bisphosphonates, such as zoledronic acid. Clodronate containing liposomes reduced the number of TAMs, however, have had limited tumor therapeutic potential.

[0008] More recent studies have evaluated liposomal constructs carrying payloads of zoledronic acid. Several of these studies evaluated targeted delivery of zoledronic acid to TAMs. At least two of these studies involved the attempted targeted delivery of zoledronic acid to TAMs. In one study, liposomes with zoledronic acid payloads were modified to display sialic acid on their exteriors. This modification targeted the liposomes to Siglecs (Sialic acid-binding immunoglobulin-type lectins) receptors on TAMs and other Siglecs expressing cells. This construct inhibited tumor growth in a mouse model. In another study, calcium/zoledronic acid nanoparticles were encapsulated in a modified lipid membrane displaying mannose and biotin. The mannose allowed binding to CD206 on TAMs, while the biotin allowed binding to biotin receptors on cancer cells. This dual targeted construct reduced tumor growth in the A549 mouse model of lung cancer; however, since the construct could kill both cancer cells and TAMs, it remains unclear whether the observed tumor growth reduction was due to the anti-tumor cell effects or to the TAM directed effects.

[0009] An additional problem with using liposomes or related constructs—either TAM targeted or not—with the intent to avoid bone localization and deliver bisphosphonates to TAMs, is that all liposomal constructs are relatively large compared to the mannosylated amine dextran (MAD) constructs described in this disclosure. The larger constructs provide challenges with regard to tumor penetration and localization to TAMs.

[0010] Accordingly, there remains a need for compositions and methods that induce the phenotypic change of the M2-like TAMs to M1-like TAMs in order to treat cancer with greater efficacy and lower toxicity.

[0011] Other objects, advantages and features of the present disclosure will become apparent from the following specification taken in conjunction with the accompanying figures.

BRIEF SUMMARY OF THE INVENTION

[0012] Provided herein are compounds and compositions containing a carbohydrate polymeric backbone and a bisphosphonate compound coupled thereto, and methods of making, and methods of using such compounds for the treatment of diseases.

 
 

Claims

1. A compound comprising: a polymeric carbohydrate backbone; one or more mannose-binding C-type lectin receptor targeting moieties; and a nitrogenous bisphosphonate compound coupled to the polymeric carbohydrate backbone via a thiol-maleimide conjugation.

2. The compound of claim 1, wherein the compound comprises a subunit as shown in Formula (I): ##STR00021## wherein each X is independently H, L.sub.1-A-Z, or L.sub.2-R, wherein each X is bound to an OH group; each of L.sub.1 and L.sub.2 are independently amine terminated leashes; each A independently comprises a substituted or unsubstituted maleimide moiety; each Z independently comprises a bisphosphonate compound modified with a hydrazone moiety or is absent; each R independently comprises the mannose-binding C-type lectin receptor targeting moiety or H; and n is an integer greater than zero, wherein each unit of n may be the same or different; and wherein at least one A is the substituted maleimide moiety and wherein the thiol-maleimide conjugation is between A and Z.

3. The compound of claim 2, wherein at least one X is L.sub.1-A-Z, wherein at least one X is L.sub.2-R, and wherein R comprises the mannose-binding C-type lectin receptor targeting moiety.

4. The compound of claim 1, wherein the polymeric carbohydrate backbone has a molecular weight between about 1 kD to about 50 kD.

5. The compound of claim 1, wherein the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl coupling aglycon moiety, mannose, high-mannose glycans or mannose oligosaccharides, fucose, N-acetylglucosamine, peptides, galactose, or a combination thereof.

6. The compound of claim 2, wherein at least one L.sub.1 comprises —(CH.sub.2).sub.pS(CH.sub.2).sub.q—NH—, wherein p and q are integers from 0 to 5.

7. The compound of claim 2, wherein at least one L.sub.2 comprises —(CH.sub.2).sub.pS(CH.sub.2).sub.q—NH—, wherein p and q are integers from 0 to 5.

8. The compound of claim 2, wherein the bisphosphonate compound is substituted with a carbonyl functional group prior to being modified to a hydrazone moiety.

9. The compound of claim 2, wherein the hydrazone moiety comprises an acyl hydrazone.

10. The compound of claim 9, wherein the hydrazone moiety has the following structure: ##STR00022##

11. The compound of claim 10, wherein R.sub.2 comprises —R.sub.4—SH, wherein R.sub.4 is a substituted or unsubstituted, linear or branched C.sub.1-C.sub.12 alkyl, alkenyl, alkynyl, or aromatic group.

12. The compound of claim 11, wherein R.sub.2 comprises —(CH.sub.2).sub.2SH.

13. A pharmaceutical composition comprising: the compound according to claim 1; and a pharmaceutically acceptable carrier.

14. The composition of claim 13, wherein the compound comprises a subunit as shown in Formula (I): ##STR00023## wherein each X is independently H, L.sub.1-A-Z, or L.sub.2-R, wherein each X is bound to an OH group; each of L.sub.1 and L.sub.2 are independently amine terminated leashes; each A independently comprises a substituted or unsubstituted maleimide moiety; each Z independently comprises a bisphosphonate compound modified with a hydrazone moiety or is absent; each R independently comprises the mannose-binding C-type lectin receptor targeting moiety or H; and n is an integer greater than zero, wherein each unit of n may be the same or different; and wherein at least one A is the substituted maleimide moiety and wherein the thiol-maleimide conjugation is between A and Z.

15. A bisphosphonate compound of Formula (II): ##STR00024## wherein R.sub.1 are each independently H, a positively-charged counter ion, a substituted or unsubstituted, linear or branched C.sub.1-C.sub.6 alkyl group, or an acyloxyalkyl group; X.sub.1 is either H, hydroxyl, a C.sub.1-C.sub.6 alkyl, or an O—C.sub.1-C.sub.6 alkyl group; Y is absent, a C.sub.1-C.sub.6 alkyl group, or a heteroatom; and W is a linear or branched C.sub.1-C.sub.12 alkyl, alkenyl, alkynyl, aromatic or heteroaromatic group containing at least one nitrogen, wherein W is substituted with a carbonyl group.

16. The compound of claim 15, wherein W is the aromatic or heteroaromatic group, and wherein the aromatic or heteroaromatic group is selected from the group consisting of a phenyl, pyridine, pyrimidine, pyridazine, pyrazine, triazine, pyrazole, imidazole, triazole, tetrazole, thiazole, isothiazole, oxazole and isoxazole, or a pharmaceutically acceptable salt thereof.

17. The compound of claim 15, wherein W is further substituted with a C.sub.1-C.sub.8 alkyl group, an aromatic group, or a heteroaromatic group.

18. The compound of claim 15, wherein W is a thiazole, the carbonyl group is a ketone group, W is further substituted with a C.sub.1 alkyl group, Y is NH, and X.sub.1 is H.

19. The compound of claim 15, wherein W is an imidazole, the carbonyl group is a ketone group, Y is absent, and X.sub.1 is a C.sub.1 alkyl group.

20. The compound of claim 15, wherein the compound has the following formula (III): ##STR00025##

21. The compound of claim 15, wherein the compound has the following formula (IV): ##STR00026##

22. The compound of claim 15, wherein the carbonyl group is combined with an acylhydrazide to form a bisphosphonate compound modified with a hydrazone moiety.

23. The compound of claim 22, wherein the acylhydrazide has the following structure: NH.sub.2—NH—C(O)—R.sub.2.

24. The compound of claim 22, wherein the hydrazone moiety has the following structure: ##STR00027##

25. The compound of claim 23, wherein R.sub.2 comprises —R.sub.4—SH, wherein R.sub.4 is a substituted or unsubstituted, linear or branched C.sub.1-C.sub.12 alkyl, alkenyl, alkynyl, or aromatic group.

26. The compound of claim 25, wherein R.sub.2 comprises —(CH.sub.2).sub.2SH.

27. The compound of claim 22, wherein the compound has the following formula (V): ##STR00028## wherein R.sub.2 comprises —R.sub.4—SH, wherein R.sub.4 is a substituted or unsubstituted, linear or branched C.sub.1-C.sub.12 alkyl, alkenyl, alkynyl, or aromatic group.

28. The compound of claim 27, wherein R.sub.2 comprises —(CH.sub.2).sub.2SH.

29. The compound of claim 22, wherein the compound has the following formula (VI): ##STR00029## wherein R.sub.2 comprises —R.sub.4—SH, wherein R.sub.4 is a substituted or unsubstituted, linear or branched C.sub.1-C.sub.12 alkyl, alkenyl, alkynyl, or aromatic group.

30. The compound of claim 29, wherein R.sub.2 comprises —(CH.sub.2).sub.2SH.

31. A method of making the compound according to claim 1, comprising: (a) synthesizing a polymeric carbohydrate backbone having one or more amine terminated leashes attached thereto; (b) synthesizing a nitrogenous bisphosphonate compound comprising a carbonyl functional group; (c) reacting the carbonyl functional group with an acylhydrazide to form the nitrogenous bisphosphonate compound modified with a hydrazone moiety; (d) modifying one or more amine terminated leashes with a maleimide moiety; and (e) substituting one or more maleimide moieties from step (d) with the bisphosphonate compound modified with the hydrazone moiety from step (c) through a thiol-maleimide conjugation.

32. The method of claim 31, wherein step (d) may occur before step (b), after step (b), before step (c), or after step (c).

33. The method of claim 31, wherein in Formula (I), at least one X is L.sub.1-A-Z, wherein at least one X is L.sub.2-R, and wherein R comprises the mannose-binding C-type lectin receptor targeting moiety.

34. The method of claim 31, wherein the polymeric carbohydrate backbone has a molecular weight between about 1 kD to about 50 kD.

35. The method of claim 31, wherein the mannose-binding C-type lectin receptor targeting moiety comprises a mannosyl coupling aglycon moiety, mannose, high-mannose glycans or mannose oligosaccharides, fucose, N-acetylglucosamine, peptides, galactose, or a combination thereof.

36. The method of claim 31, wherein in Formula (I), at least one L.sub.1 comprises —(CH.sub.2).sub.pS(CH.sub.2).sub.q—NH—, wherein p and q are integers from 0 to 5.

37. The method of claim 31, wherein in Formula (I), at least one L.sub.2 comprises —(CH.sub.2).sub.pS(CH.sub.2).sub.q—NH—, wherein p and q are integers from 0 to 5.

38. The method of claim 31, wherein the hydrazone moiety has the following structure: ##STR00030##

39. The method of claim 38, wherein R.sub.2 comprises —R.sub.4—SH, wherein R.sub.4 is a substituted or unsubstituted, linear or branched C.sub.1-C.sub.12 alkyl, alkenyl, alkynyl, or aromatic group.

40. The method of claim 39, wherein R.sub.2 comprises —(CH.sub.2).sub.2SH.

41. A method of repolarizing a tumor associated macrophage (TAM) from an immunosuppressive (M2-like) phenotype to a proinflammatory (M1-like) phenotype, comprising: administering to a subject in need thereof an effective dose of a compound comprising a polymeric carbohydrate backbone, one or more mannose-binding C-type lectin receptor targeting moieties, and a nitrogenous bisphosphonate compound coupled to the polymeric carbohydrate backbone via a thiol-maleimide conjugation.

42. The method of 41, wherein the compound comprises a subunit as shown in Formula (I): ##STR00031## wherein each X is independently H, L.sub.1-A-Z, or L.sub.2-R, wherein each X is bound to an OH group; each of L.sub.1 and L.sub.2 are independently amine terminated leashes; each A independently comprises a substituted or unsubstituted maleimide moiety; each Z independently comprises a bisphosphonate compound modified with a hydrazone moiety or is absent; each R independently comprises the mannose-binding C-type lectin receptor targeting moiety or H; and n is an integer greater than zero, wherein each unit of n may be the same or different; and wherein at least one A is the substituted maleimide moiety and wherein the thiol-maleimide conjugation is between A and Z.

43. The method of claim 41, wherein the compound is administered in conjunction with at least one other therapy or treatment and, wherein the at least one other treatment or therapy is a chemotherapy, radiation therapy, or immunotherapy.

44. The method of claim 41, wherein the bisphosphonate compound is released from the polymeric carbohydrate backbone at a pH of below about 5.5.

45. A method of treating a disease, comprising: administering to a subject in need thereof an effective amount of a compound according to claim 1, wherein the disease is cancer, an autoimmune disease, or an inflammatory disorder.

46. The method of claim 45, wherein the compound is administered in conjunction with at least one other therapy or treatment and, wherein the at least one other treatment or therapy is a chemotherapy, radiation therapy, or immunotherapy.

47. The method of claim 45, wherein the disease is cancer.

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 

 

 

 

 

 

 

 

 


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