US008980907B2
(12) United States Patent (10) Patent No.: US 8,980,907 B2 Baker, Jr. et al. (45) Date of Patent: Mar. 17, 2015
(54) DENDRIMER CONJUGATES 4,892,935 A 1/1990 Yoshida et al. 4.914,021 A 4, 1990 Toth (71) Applicant: E" of the try is of 4,918,1644,921,789 A 4,5/1990 1990 SalemHellstrom et al. et al. ichigan, Ann Arbor, MI (US) 4,921,790 A 5/1990 O’Brien 4,939,240 A 7, 1990 Chu et al. (72) Inventors: James R. Baker, Jr., Ann Arbor, MI 4.946,778 A 8, 1990 Ladner et al. (US); Xue-min Cheng, Ann Arbor, MI 4,963.484. A 10/1990 Kufe (US); Abraham F. L. Van Der Spek 4,965,128 A 10, 1990 Greidanus Ann Arbor,s MI (US);O - O Baohua Mark s 5,041,5165,053.489 A 10/19918, 1991 KifeFrechet et al. Huang, Ann Arbor, MI (US); Thommey 5,110,911. A 5/1992 Samuel et al. P. Thomas, Dexter, MI (US) 5,270,163 A 12/1993 Gold et al. 5,338,532 A 8, 1994 Tomalia et al. (73) Assignee: The Regents of the University of 3: A g 3: R etd 1 Michigan,O O Ann Arbor, MI (US) 5,393,795- w A 2/1995 HedstrandSa etca. al. 5,393,797 A 2f1995 Hedstrand et al. (*) Notice: Subject to any disclaimer, the term of this 5,475,096 A 12/1995 E. s a patent is extended or adjusted under 35 5,512,443 A 4/1996 Schlom et al. U.S.C. 154(b) by 0 days. 5,527,524. A 6/1996 Tomalia et al. (Continued) (21) Appl. No.: 14/107,817 FOREIGN PATENT DOCUMENTS (22) Filed: Dec. 16, 2013 CA 2187921 11, 1995 (65) Prior Publication Data CA 2386998 4/2001 US 2014/0128327 A1 May 8, 2014 (Continued) OTHER PUBLICATIONS Related U.S. Application Data (63) Continuation of application No. 12/570977, filed on Maini, et al., “Infliximab (chimeric anti-tumour necrosis factor Sep 30, 2009 s- s monoclonal antibody) versus placebo in rheumatoid arthritis patients p. 3U, receiving concomitant methotrexate: a randomised phase III trial.” (60) Provisional application No. 61/101.461, filed on Sep. Lancet (1999) vol. 354, pp. 1932-1939. 30, 2008. Chandrasekar, Durairaj, et al., “The development of folate-PAMAM dendrimer conjugates for targeted delivery of anti-arthritic drugs and (51) Int. Cl. their pharmacokinetics and biodistribution in arthritic rats.” A 6LX3/553 (2006.01) Biomaterials (2007) vol. 28, pp. 504-512. A6 IK3I/485 (2006.01) Mullen, Douglas G., “A Quantitative Assessment of Nanoparticle— A 6LX3/5377 (2006.01) Ligand Distributions: Implications for Targeted Drug and Imaging CO7D 489/02 (2006.015 Delivery in Dendrimer Conjugates.” ACS Nano (2010), 4(2), pp. CO7D 413/06 (2006.01) 657-670. inued A6 IK 47/48 (2006.01) (Continued) (52) U.S. Cl. CPC ...... A61K (2013.0);47/483 (2013.01); A61K31/485 A61K47/48207 (2013.01) Primary Examiner - Anoop Singh USPC ...... 514/282, 514/237.2, 424/78.3:546/46 Assistant Examiner - Anna Falkowitz (58) Field of Classification Search (74) Attorney, Agent, or Firm — Casimir Jones S.C. USPC ...... 514/282,237.2: 424/78.3:546/46 See application file for complete search history. (57) ABSTRACT (56) References Cited The present invention relates to novel therapeutic and diag U.S. PATENT DOCUMENTS nostic dendrimers. In particular, the present invention is directed to dendrimer-linker conjugates, methods of synthe 4,161,948 A 7, 1979 Bichon sizing the same, compositions comprising the conjugates, as 4,507,466 A 3, 1985 Tomalia et al. well as systems and methods utilizing the conjugates (e.g., in 4,558,120 A 12/1985 Tomalia et al. diagnostic and/or therapeutic settings (e.g., for the delivery of 4,568,737 A 2, 1986 Tomalia et al. 4,587,329 A 5, 1986 Tomalia et al. therapeutics, imaging, and/or targeting agents (e.g., in disease 4,631,337 A 12/1986 Tomalia et al. (e.g., cancer) diagnosis and/or therapy, pain therapy, etc.)). 4,694,064 A 9, 1987 Tomalia et al. Accordingly, dendrimer-linker conjugates of the present 4,708,930 A 1 1/1987 Kortright et al. invention may further comprise one or more components for 4,713,975 A 12/1987 Tomalia et al. targeting, imaging, sensing, and/or providing a therapeutic or 4,737,550 A 4, 1988 Tomalia et al. 4,743,543 A 5/1988 Kortright diagnostic material and/or monitoring response to therapy. 4,827,945 A 5, 1989 Groman 4,857,599 A 8, 1989 Tomalia et al. 4,871,779 A 10, 1989 Killat et al. 5 Claims, 50 Drawing Sheets US 8,980.907 B2 Page 2
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0.3 h. Taxo-linker-control Concentration of free mana axo-inker-2hr taxo~29 M24hr) rTaxo-linker-24hr. 0.25 - SO - 40% ------0.2 - - 8 ...K...... --r a O, 15 - 89% -1 9. O1 |----
0.05 -
0-m 5
- S -
Where Y axis is Amount Released (arbitrary units) Where X axis is Time (minutes)
U.S. Patent Mar. 17, 2015 Sheet 16 of 50 US 8,980,907 B2
FG. 4
2O 2O
> O N-ron-a-rorurO OH .9. > OStra-ror- O ~roro O O O O A oTaxo 1. BH THF Linker-drug conjugate 1 O 1. FA r bo------> Dendriner conjugate 2. p-NO,PhOCOC ...: 2. Gs-COH 3. TaxoMAP > -1No1a-O-1 onto O O U.S. Patent Mar. 17, 2015 Sheet 17 Of 50 US 8,980,907 B2
F.G. 15
H O 2O eO O--N-1-1--O-1 1-Br HO Dr O O B HN -- K2CO, Acetone OH Ök O separate regio isomer and diacylated product
O eo BH, THF oN-o-o-o-oO H are r-2. p-NOPhOCOCa a a a r- r a a P ro re blk 3. Taxol, DMAP O f O. 1Taxol TFA > in-1o-n-O-1N O 9 ------se Dendrimer conjugate O 2. Gs-COH Linker-drug conjugate 2 (k U.S. Patent Mar. 17, 2015 Sheet 18 Of 50 US 8,980,907 B2
F.G. 6
Me Clk Me Me > O r S1o 1\- On-ror OH ON ON H H2N O A O -O-> -e ------e EtN PC OH (k ök O O Me OH o, R. O NBS o, > r N1c1a1 n-roy a Y as a a > ~ron-o-ror O O 2. AgOAc O O ok ck O O O. Taxol
- s > N-1on-O-1orn O 2. p-NO,PhOCOC O O 3. Taxo, DMAP Linker-drug conjugate 3 (k O
a- Dendrimer conjugate 2. G-COH U.S. Patent Mar. 17, 2015 Sheet 19 Of 50 US 8,980,907 B2
F.G. 7
O M p-( N Paclitaxel A. HOC p-()- M -/)-dO HO HO OHN Glucuronidase induced cleavage U.S. Patent Mar. 17, 2015 Sheet 20 Of 50 US 8,980,907 B2
FG, 8
O O O O -Drugr OH1N ?on-Urug O H H Hypoxia n N Drug R-4 - SP'- R- 2 -- R- 2 H2N
O OH OH U.S. Patent Mar. 17, 2015 Sheet 21 of 50 US 8,980,907 B2
FG, 9
OH HX-Drug Hwpoxiypoxia -- --
NO2 C CO NHR NHR X = O, NH Rs H, OH U.S. Patent Mar. 17, 2015 Sheet 22 of 50 US 8,980,907 B2
F.G. 2
s*so es. Drug Tririgger O 8) OH Y. HY, ( a X-Drugis Y. HY Activation 14-elimination -a------as
Y CO
O X O s Drug s x Drug O y OH HX-Drug Activation 1,6-elimination -e- -Ho- -- -a-
Y ( CO, Y YH N rigger X, Y = O. NR U.S. Patent Mar. 17, 2015 Sheet 23 Of 50 US 8,980,907 B2
FG, 2. Add R for steric effect s X Drug Ya () Trigger
Electronic and steric effect of the substituents Factors that affect the effectiveness of the linker U.S. Patent Mar. 17, 2015 Sheet 24 of 50 US 8,980,907 B2
F.G. 22 Drug Drug Trigger 1 N. O Activation Nu O Spontaneous - e Hee O
X = O, NR U.S. Patent Mar. 17, 2015 Sheet 25 Of 50 US 8,980,907 B2
FG, 23
X Drug N O X O
O
OH Hip- r NH2 He NH U.S. Patent Mar. 17, 2015 Sheet 26 of 50 US 8,980,907 B2
FG, 24
is eitsiafi
- / ? A Paclitaxel ANYX N-14 Ny cyclization Glucuronidase induced cleavage U.S. Patent Mar. 17, 2015 Sheet 27 Of 50 US 8,980,907 B2
FIG. 25
Trigger Ya activation (overall)
activation spontaneous
pOntaneous : s U.S. Patent Mar. 17, 2015 Sheet 28 Of 50 US 8,980,907 B2
F.G. 26A
PAMAM PAMAM Y O Y O O o1J.---to NN1\-1 1 rigger o1 NN1\1Y. O O O 2 Xeo Xeo X X Drug Drug
X = O. NH Additional Self-degradable linkers Y (CH)nX PAMAM Y PAMAM O Y OH O HX, O - OH - Drug X=o HO X M Drug
FG, 2.6B
PAMAM Y O O PAMAM a a l Trigger Y - O O 1- HX M A ?" - a * Drug O O M A OH O HO X VDrug
Xs O, NH Additional self-degradable linkers Y = (CH)nX U.S. Patent Mar. 17, 2015 Sheet 29 Of 50 US 8,980,907 B2
FG, 27 Simple esters
O HO2 H O 1n Drug Esterase (i. 1N l -- HX-O Gs (in x1 a G1\, S.-D9 - > Gs ()n OH - Drug X as O.
Esters with an 1,6-elimination spacer will drive the hydrolysis to completion: O O O ---Drug EsteraseHO bH te,H r l -Drug l -o- 9. SY G1 N(1o ----- G1)(frg \a minimal b
O b l OH HO- G1 Nt in OH -- HO -- CO -- HX-Drug X is O, NR U.S. Patent Mar. 17, 2015 Sheet 30 Of 50 US 8,980,907 B2
F.G. 28
10-Hydroxycamptothecin (SN-38) / NN
On O O On O O s n N s n N N N1 M A O N? M A O O S. N N O O ( )n
Irinotecan -i?O SPAMAM wf or whout triggering unit inserted U.S. Patent Mar. 17, 2015 Sheet 31 Of 50 US 8,980,907 B2
FG, 29
CHO CHO MeO MeO Na-MF MeO O-M. 1. DMFi PoC1 Y-Me y-Me 2. NaOACl2O N 2. Br-coat N COEt
NO2. CHO N2 CHO cho AcOH/HNO Meo Snic MeO Fremy's Salt MeO W Me W Me W Me N N N Scott co,Et 8 sco,Et
O O O OH NaBH Meo N io MeO ------> N \ Me N O \- COEt O co-h
U.S. Patent Mar. 17, 2015 Sheet 33 Of 50 US 8,980,907 B2
FG. 3
O O N.-- l rig: R y-r to in Hypoxic N R w cancer cells O R idolesiose linker
N on--| O. ? : ~r ::g in Hypoxic - y O –- O \U/ cancer cells Nitriidazole like U.S. Patent Mar. 17, 2015 Sheet 34 of 50 US 8,980,907 B2
FG. 32
Periphera Approach: Centra: Approach: Ketan ineflorazepamitoxapram orphinei Naioxone targeting
&etatise U.S. Patent Mar. 17, 2015 Sheet 35 of 50 US 8,980,907 B2
CNS
in Wiwo Oxidation
Constant release tlocking module by esterase M. Morphine N. Naloxone M M M. M. U.S. Patent Mar. 17, 2015 Sheet 36 of 50 US 8,980,907 B2
F.G. 34 Step 4: Sangle Pre-treatinert
Step 3: Sarping
Stag 3: inciatio Step 2: Ciezavage cue to S3Se
alos Paralysis
roposed Strategy to analyze drag conjugated to the deficiner scaffold with easte: tinkage sing Pig. U.S. Patent Mar. 17, 2015 Sheet 37 Of 50 US 8,980,907 B2
FIG. 35
HO O H OH HO O H O Morphine Hydromorphone U.S. Patent Mar. 17, 2015 Sheet 38 of 50 US 8,980,907 B2
FG, 36
Me N.Me N.Me
O O - O O OH O O OH Prodrug B
O Prodrug A Aromatic ester - A O Aliphatic8. ester
A-/ Prodrug C O Aliphaticw ester O / with PEG chain
BocN U.S. Patent Mar. 17, 2015 Sheet 39 Of 50 US 8,980,907 B2
FG, 37 a21 N OH
O O J N^-No- O H O MeO O N Me N O O \-2 O
Indolequinone-based Naloxone Pro-drug U.S. Patent Mar. 17, 2015 Sheet 40 of 50 US 8,980,907 B2
FG 38
U.S. Patent Mar. 17, 2015 Sheet 41 of 50 US 8,980,907 B2
FG, 39
-8- Pro-drug A, 8 mg -e-Pro-drug A Dendrimer Complex, 35 mg *** Pro-drug A Dendrimer Conjugate, 4.8g
() 5 O 5 2O 25 Incubation time with Fresh Frozen Plasma (hours) U.S. Patent Mar. 17, 2015 Sheet 42 of 50 US 8,980,907 B2
FG. A.
30
5 - S - Naloxone Indolequinone Pro-drug, 78 mg O
5
O 5 O 15 20 25 Incubation Time with Fresh Frozen Plasma at a pO, of 18 mmHg (hours) U.S. Patent Mar. 17, 2015 Sheet 43 of 50 US 8,980,907 B2
FG, 4.
Drug molecules (8) U.S. Patent Mar. 17, 2015 Sheet 44 of 50 US 8,980,907 B2
FG, 42.
Surface Modified Prodrugs Dendrimer Conjugates Dendrimers U.S. Patent Mar. 17, 2015 Sheet 45 of 50 US 8,980,907 B2
FIG. 43
is: exix::::::::t:atic: (); ki:gi::::::::
ecrease it tig S-1 coicestratiot due to distrinitia and eitiatio:
iiia cit::gicaxiing to stair sissies girag ca:kaitiatic: it: sixxi.
Title
U.S. Patent Mar. 17, 2015 Sheet 48 of 50 US 8,980,907 B2
FG. 46
U.S. Patent Mar. 17, 2015 Sheet 49 Of 50 US 8,980,907 B2
F. 47
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US 8,980,907 B2 1. 2 DENDRMER CONUGATES There exists a need for compositions, methods and systems for delivering agents (e.g., diagnostic and/ortherapeutic (e.g., CROSS-REFERENCE TO RELATED cancer and/or pain therapeutics) to Subjects that provide APPLICATIONS effective therapy (e.g., disease treatment, symptom relief, etc.) with reduced or eliminated side effects, even when The present application is a continuation of U.S. patent administered in high doses. application Ser. No. 12/570977, filed Sep. 30, 2009, which claims priority to U.S. Provisional Patent Application No. SUMMARY 61/101.461, filed Sep. 30, 2008, hereby incorporated by ref. erence in their entireties. 10 Battlefield trauma covers a range of injuries caused by different mechanisms resulting at times in severe distur STATEMENT REGARDING FEDERALLY bances of vital functions, disability, fear and pain (see, e.g., SPONSORED RESEARCH ORDEVELOPMENT Conventional warfare: ballistic, blast, and burn injuries. 1990, Department of the Army, Office of the Surgeon Gen This invention was made with government Support under 15 eral, Borden Institute. 396; herein incorporated by reference CA1 19409 awarded by the National Institutes of Health and in its entirety). Depending on the military situation, initial W911NF-07-1-0437 awarded by the Army/ARO. The Gov reliance for first aid must either be administered by oneself, ernment has certain rights in the invention. other troops or field medics. It is not until the injured reach a FIELD OF THE INVENTION Forward Surgical Team (FST) or a Battalion Aid Station (BAS) that trained personnel can administer traditional pain The present invention relates to novel therapeutic and diag and anxiolytic medications due to the need for frequent nostic dendrimers. In particular, the present invention is assessment of the medications side effects and need for directed to dendrimer-linker conjugates, methods of synthe Supportive measures to prevent drug-induced deterioration of sizing the same, compositions comprising the conjugates, as 25 Vital functions (see, e.g., Anesthesia and perioperative care of well as systems and methods utilizing the conjugates (e.g., in the combat casualty. 1995, Department of the Army, Office of diagnostic and/or therapeutic settings (e.g., for the delivery of the Surgeon General, Borden Institute. 931; herein incorpo therapeutics, imaging, and/or targeting agents (e.g., in disease rated by reference in its entirety). Unfortunately, the time for (e.g., cancer) diagnosis and/or therapy, pain therapy, etc.)). an injured soldier to arrive at a FST or BAS unit can be Accordingly, dendrimer-linker conjugates of the present 30 delayed for hours to days. Adequate pain therapy may be invention may further comprise one or more components for markedly delayed resulting in problems such as long term, targeting, imaging, sensing, and/or providing a therapeutic or psychological-psychiatric effects, chronic pain syndromes diagnostic material and/or monitoring response to therapy. and Post Traumatic Stress Disorders. This prolonged expo Sure of service members to inadequately treated pain is asso BACKGROUND OF THE INVENTION 35 ciated with considerable cost to the soldier and society (see, e.g., Bloodworth, D., Phys Med Rehabil ClinNAm, 2006. Cancer remains the number two cause of mortality in the 17(2): p. 355-79; herein incorporated by reference in its United States, resulting in over 500,000 deaths per year. entirety). Despite advances in detection and treatment, cancer mortality If one were to attempt to administer potent pain and anxi remains high. New compositions and methods for the imag 40 olytic/amnestic medications to injured soldiers on the battle ing and treatment (e.g., therapeutic) of cancer may help to field, it is important to understand the compensatory physi reduce the rate of mortality associated with cancer. ological responses required to preserve vital functions during Severe, chronic pain is observed a variety of subjects. For trauma and evaluate how these medications side effects example, there exist large numbers of individuals with sever could potentiate combat-induced pathophysiology. Ventila pain associated with arthritis, autoimmune disease, injury, 45 tion with oxygenation and perfusion of vital organs—the cancer, and a host of other conditions. heart, lungs and brain—must be preserved in the face of major A vast number of different types of pain medications exist. injuries and blood loss, while at the same time perfusion of For example, a number natural and synthetic alkaloids of other organ systems may virtually shut down. Such a reduced opium (i.e., opioids) are useful as analgesics for the treatment flow state may be maintained for hours or days depending on of severe pain. However, a number of severe side effects 50 the soldier's condition, environment and other factors. Dis associated with opioid and other pain medication usage exist. turbance of this delicate cardio respiratory balance by pain For example, administration of opioid agonists often results medications needs to be considered carefully lest it be detri in intestinal dysfunction due to action of the opioid agonist mental or even fatal to the injured. Reports of blood gasses of upon the large number of receptors in the intestinal wall. injured personnel taken at the time of admission to the FST Opioids are generally known to cause nausea and Vomiting as 55 unit illustrate the degree of cardio, respiratory and metabolic well as inhibition of normal propulsive gastrointestinal func compromise that occur in non-fatal injuries. Approximately tion in animals, resulting in side effects such as constipation. 35% of injured entrants had pH levels in the range of 7.0 to Pain medication (e.g., opioid)-induced side effects are a 7.20, and as low as 6.9 while pCO2 levels were often in the serious problem for patients being administered pain medi 50-60 mmHg range and could be as high as 80-90 mmHg in cations (e.g., opioid analgesics) for both short term and long 60 severe trauma. Oxygen saturation levels were as low as 40% term pain management. For instance, more than 250,000 ter while the lowest hematocrit encountered was 9 mg/dl. This minal cancer patients each year take opioids, such as mor shows the dire nature of combat injuries, and provides an phine, for pain relief, and about half of those patients expe insight into why the administration of analgesic and anxi rience severe constipation. At present, patients receiving olytic/amnestic medications with vasodilatory and respira opioid pain medications face the difficult choice of suffering 65 tory depressive side effects has been avoided. It also suggests burdensome adverse effects (e.g., constipation) or ineffective the need for physiologically triggered feedback regulation to analgesia. prevent worsening of these derangements. US 8,980,907 B2 3 4 The unique conditions of battlefield trauma require a com are co-administered with additional agents designed to pre plex pain relief solution. For example, there is a need for a vent adverse side effects of pain relief agents (e.g., respiratory form of sustained therapeutic that could relieve pain over failure, adverse cardiovascular consequences). In some many hours or even days. This situation is further compli embodiments, the compositions comprising dendrimer mol cated by the need for battlefield therapeutics to be easily ecules conjuguated to one or more pain relief agents (e.g., administered, preferably without the technical challenge of narcotic prodrugs) are co-administered with a pain relief intravenous access or continuous administration. Therapy agent antagonist (e.g., a narcotic antagonist that is modulated should not require monitoring, since uninjured soldiers will to permit analgesia while preventing respiratory depression). be involved in ongoing combat. Some effective pain medica In some embodiments, a feedback system is employed so as tions (e.g., narcotic analgesics) have serious limitations. For 10 to prevent respiratory failure and adverse cardiovascular con example, narcotic analgesics are short acting, the therapeutic sequences while still maintaining analgesia. In some embodi index is relatively narrow, and the doses of drug that cause ments, the “feedback’ component is a rapid-acting narcotic analgesia are not greatly separated from those that cause antagonist (e.g., Naloxone) released only upon detection of serious side effects, including respiratory depression and symptoms of respiratory depression. In some embodiments, hypotension due to vasodilatation (see, e.g., Bloodworth, D., 15 the biomarker used to monitor respiratory depression and Phys Med Rehabil ClinNAm, 2006. 17(2): p. 355-79; herein trigger the release of the antagonist is hypoxia (low pC2). incorporated by reference in its entirety). Such side effects Hypoxia is a sensitive and important marker as it is the direct can actually worsen the physiological derangements of acute cause of tissue injury from respiratory failure. It is also more trauma. Respiratory depression leads to respiratory acidosis specific than lowered serum pH, a marker of respiratory aci that can cause metabolic derangements. This can also exac dosis, since this is also observed in acute trauma situations as erbate metabolic acidosis due to traumatic injury, and Sus a result of metabolic derangements. A fast acting antagonist tained depression can lead to death from hypoxia due to released in response to hypoxia rapidly reverses respiratory respiratory failure. Hypotension can trigger shock and car depression, which thereby increases the pC2, reversing the diovascular collapse, especially in an individual who has hypoxia and stopping the release of the antagonist. In con already suffered traumatic blood loss. Thus, to provide 25 trast, the narcotic itself would continue to be released at a autonomously effective narcotic analgesia over long periods slow and predictable rate. In this way, appropriate analgesia is of time in the battlefield, a need exists for a sustained release achieved that is reversed only when absolutely necessary to narcotic with a widened therapeutic index allowing for Sus prevent respiratory failure. This would allow maintenance of tained analgesia in the absence of respiratory depression. This analgesia in the battlefield for prolonged periods of time formulation should also be easily administered (e.g., through 30 without monitoring of the wounded. In some embodiments, intramuscular auto-injector). Finally, lack of sedation and an as shown in FIG. 32A, the present invention provides com absence of systemic side effects are acutely important given positions comprising a plurality of pain relief agents coupled the fact that individuals with traumatic injuries in the battle to dendrimers with a linkage agent connected to a trigger field may need to participate in self-extraction to safety. agent that slowly degrades in a biological system (e.g., ester The present invention provides compositions and related 35 linkage) (as shown in FIG. 32A, the trigger agent is an ester methods addressing Such needs. In particular, the present bond that is released by serum esterases to mediate Sustained invention provides compositions comprising dendrimer mol release analgesia). When administered together, the plurality ecules (e.g., polyamideamine (PAMAM) dendrimers, of pain relief agents (e.g., Ketamine and Lorazepam) have polypropylamine (POPAM) dendrimers, or PAMAM-PO favorable analgesic and anxiolytic/amnestic qualities, and PAM dendrimers) conjuguated to one or more pain relief 40 relatively broad therapeutic indexes. Compositions compris agents (e.g., prodrug analgesic molecules, prodrug anxiolytic ing a plurality of pain relief agents (e.g., Ketamine and drugs, prodrug amnestic drugs). In some embodiments, the Lorazepam) provide analgesia without the cardiovascular dendrimers conjugated to one or more pain relief agents are effects of opioids and minimize their major complication, that configured for controlled and/or Sustained release of the pain being respiratory depression. As a feedback mechanism, relief agents (e.g., through use of targeting agents, linking 45 compositions of the invention comprising a pain relief agent agents, and/or trigger agents conjugated to the dendrimer antagonist (e.g., Doxapram) are complexed with a dendrimer and/or pain relief agent). In some embodiments, the pain through charge interaction (e.g., that is releasable by, for relief agent conjugated to the dendrimer is active upon admin example, acidosis). While acidosis may be observed from istration to a subject. In some embodiments, Sustained release causes other than respiratory depression, this feedback (e.g., slow release over a period of 24-48 hours) of the pain 50 mechanism is unique in that Doxapram stimulates respira relief drug is accomplished through conjugating the pain tions without reducing analgesia. As such, the present inven relief drug to the dendrimer through, for example, a linkage tion provides release of Doxapram regardless of the source of agent connected to a trigger agent that slowly degrades in a the acidosis (e.g., traumatic injuries, hemorrhagic shock, biological system (e.g., ester linkage). In some embodiments, burns or rhabdomyolysis). Thus, the present invention pro constitutively active release of the pain relief drug is accom 55 vides a safe analgesia with easily achieved physiological plished through conjugating the pain relief drug to the den feedback. drimer through, for example, a linkage agent connected to a In some embodiments, targeting agents are conjugated to trigger agent that renders the pain relief agent constitutively the dendrimers for delivery of the dendrimers to desired body active in a biological system (e.g., amide linkage, ether link regions (e.g., to the central nervous system (CNS). The tar age). In some embodiments, Sustained release (e.g., a slow 60 geting agents are not limited to targeting specific body release mechanism that achieves analgesic concentrations regions. In some embodiments, the targeting agents target the over a period of, for example, 24–48 hours) of the pain relief central nervous system (CNS). In some embodiments, target agent prevents adverse side effects of the pain relief agent ing agents target the peripheral nervous system, specific (e.g., respiratory failure, adverse cardiovascular conse nerves (e.g., perception nerves, pain nerves, pressure nerves, quences). 65 etc.), muscles, and/or tendons. In some embodiments, where In certain embodiments, the compositions comprisingden the targeting agent is specific for the CNS, the targeting agent drimer molecules conjugated to one or more pain relief agents is transferrin (see, e.g., Daniels, T. R. et al., Clinical Immu US 8,980,907 B2 5 6 nology, 2006. 121(2): p. 159-176; Daniels, T.R., et al., Clini hypoxia to prevent respiratory failure. In some embodiments, cal Immunology, 2006. 121(2): p. 144-158; each herein incor the antagonist and agonist are attached to two identical popu porated by reference in their entireties). In some lations of dendrimers in a very consistent manner, and admin embodiments, the targeting agents target neurons within the istered together (or separately) to form a single drug delivery central nervous system (CNS). In some embodiments, where system. the targeting agent is specific for neurons within the CNS, the Accordingly, the present invention provides compositions, targeting agent is a synthetic tetanus toxin fragment (e.g., a 12 systems and methods for treating and/or managing pain in a amino acid peptide (Tet 1)) (see, e.g., Liu, J. K., et al., Neu Subject through use of dendrimers conjugated to pain relief robiology of Disease, 2005. 19(3): p. 407-418; herein incor agents and/or pain relief agent antagonists. The following porated by reference in its entirety). In some embodiments, 10 discussion describes individual component parts of the den locking agents designed to retain the dendrimer within a drimer and methods of making and using the same in some particular body region are conjugated to the dendrimer (e.g., embodiments of the present invention. To illustrate the design locking agents designed to prevent back diffusion of a den and use of the systems and compositions of the present inven drimer across the bloodbrain barrier (BBB) (e.g., pyridinium tion, the discussion focuses on specific embodiments of the molecule, which when activated by enzymatic reduction, 15 use of the compositions in the treatment and reduction of pain becomes charged and locks the dendrimer in the CNS)). In suffered by a subject. These specific embodiments are Some embodiments, trigger agents are conjugated to the den intended only to illustrate certain preferred embodiments of drimers so as to permit a controlled release of a particular the present invention and are not intended to limit the scope agent (e.g., a narcotic and/or narcotic antagonist). The den thereof. For example, although some discussion of the present drimers are not limited to particular types of trigger agents. In invention involves battlefield injuries, other types apply (e.g., Some embodiments, Sustained release (e.g., slow release over general trauma settings). a period of 24–48 hours) of the pain relief drug is accom In certain embodiments, the present invention provides plished through conjugating the pain relief drug to the den compositions comprising a dendrimer linked to a moiety drimer through, for example, a linkage agent connected to a comprising a trigger agent, a linkage agent, a targeting agent, trigger agent that slowly degrades in a biological system (e.g., 25 and at least one therapeutic agent, wherein the therapeutic ester linkage). In some embodiments, constitutively active agent is a pain relief agent designed to reduce and/or elimi release of the pain relief drug is accomplished through con nate pain in a Subject and/or a pain relief agentantagonist. The jugating the pain relief drug to the dendrimer through, for composition are not limited to particular dendrimers. In some example, a linkage agent connected to a trigger agent that embodiments, the dendrimeris, for example, a polyamideam renders the pain relief agent constitutively active in a biologi 30 ine (PAMAM) dendrimer, a polypropylamine (POPAM) den cal system (e.g., amide linkage, ether linkage). In some drimer, and a PAMAM-POPAM dendrimer. The composi embodiments, the trigger agent is designed to permit release tions are not limited to particular linkage agents (e.g., a spacer of the drug conjugated to the dendrimer in the presence of comprising between 1 and 8 straight or branched carbon brain enzymes (e.g., the trigger agent indolequinone is chains). In some embodiments, the linkage agent is Substi reduced by brain enzymes Such as, for example, diaphorase). 35 tuted or unsubstituted straight or branched carbon chain. In In some embodiments, the trigger agent is designed to permit Some embodiments, straight or branched carbon chains are release of the drug conjugated to the dendrimer upon detec substituted with alkyls. In some embodiments, the dendrim tion of reduced pC2 concentrations (e.g., through use of a ers are acetylated. trigger agent that detects reduced pC2 levels (e.g., a re-dox The compositions are not limited to particular trigger linker)). The use of a re-dox linker provides direct physiologi 40 agents. In some embodiments, the trigger agents are config cal feed back in order to prevent consequences of opoid ured to delay release of the pain relief agent from the moiety induced respiratory depression (e.g., cerebral hypoxia). (e.g., an ester bond). In some embodiments, the trigger agents FIG. 32B shows two dendrimer conjugates designed for are configured to constitutively release the therapeutic agent pain management in a Subject. One of the dendrimers is from the moiety (e.g., an amide bond, an ether bond). In some conjugated to a morphine drug through a linkage agent con 45 embodiments, the trigger agent is configured to release the nected to a trigger agent (e.g., ester linkage) permitting Sus therapeutic agent from the moiety under conditions of acido tained release. The other dendrimer is conjugated to a mor sis. In some embodiments, the trigger agent is configured to phine antagonist (e.g., Naloxone) through a linkage agent release the therapeutic agent from the moiety under condi connected to a trigger agent (e.g., re-doX linker) permitting tions of hypoxia (e.g., indoquinones, nitroheterocyles, and release of the morphine antagonist upon detection of reduced 50 nitroimidazoles). In some embodiments, the trigger agent is pO2 levels. Each of the dendrimers are targeted for CNS configured to release the therapeutic agent from the moiety in delivery through conjugation of targeting agents specific for the presence of a brain enzyme (e.g., the trigger agent is the CNS (e.g., transferrin, a synthetic tetanus toxin fragment). indolequinone and the brain enzyme is diaphorase). Each of the dendrimers are designed for retention within the The compositions are not limited to particular targeting CNS through conjugation of locking agents designed to pre 55 agents. In some embodiments, the targeting agent is config vent back diffusion of the dendrimer across the BBB (e.g., ured to permit the composition to cross the bloodbrainbarrier pyridinium molecule, which when activated be enzymatic (e.g., transferrin). In some embodiments, the targeting agent reduction, becomes charged and locks the dendrimer in the is configured to permit the composition to bind with a neuron CNS). within the central nervous system (e.g., the targeting agent is These two characteristics differentiate the sustained 60 a synthetic tetanus toxin fragment (e.g., an amino acid peptide release of a narcotic, and the feedback release of a narcotic fragment (e.g., HLNILSTLWKYR (SEQID NO:1))). antagonist. For example, in some embodiments, the narcotic In some embodiments, the moiety further comprises a is linked such that it would remain constitutively active while locking agent. The compositions are not limited to particular coupled to the polymer or continuously released over time locking agents. In some embodiments, the locking agent, (e.g., through triggering agents designed to permit Sustained 65 upon activation, prevents transfer of the composition across release) to provide prolonged activity, whereas, in some the blood brain barrier. In some embodiments, the locking embodiments, the antagonist is active when released during agent is a pyridinium molecule which is activated by enzymes US 8,980,907 B2 7 8 specific to the central nervous system. In some embodiments, Enflurane, Halothane, Isoflurane, Nitrous oxide, Sevoflurane, the locking agent is a re-dox system. In some embodiments, Xenon, intravenous anesthetics, Barbiturates, amobarbital the re-dox system is the 1,4- (Amytal), pentobarbital (Nembutal), secobarbital (Seconal), dihydrotrigonellines strigonelline (coffearine) re-doX sys Phenobarbital, Methohexital. Thiopental, Methylphenobar tem, wherein conversion of lipophilic 1,4-dihydroform (L) in bital, Metharbital, Barbexaclone)), Benzodiazepines, alpra vivo to the hydrophilic quaternary form (L) by oxidation Zolam, bromazepam (Lexotan), chlordiazepoxide (Librium), prevents the composition from diffusing across the blood Clobazam, Clonazepam, CloraZepate, Diazepam, Mida brain barrier. Zolam, Lorazepam, Nitrazepam, temazepam, nimetazepam, The compositions are not limited to particular pain relief Estazolam, Flunitrazepam, oxazepam (Serax), temazepam agents. In some embodiments, the pain relief agents include, 10 (Restoril, Normison, Planum, Tenox, and Temaze), Triaz but are not limited to, analgesic drugs, anxiolytic drugs, anes olam, Etomidate, Ketamine, and Propofol. thetic drugs, antipsychotic drugs, hypnotic drugs, sedative In Some embodiments, the antipsychotic drugs include, but drugs, and muscle relaxant drugs. are not limited to, butyrophenones, haloperidol, phenothiaz In some embodiments, the analgesic drugs include, but are ines, Chlorpromazine (Thorazine), Fluphenazine (Prolixin), not limited to, non-steroidal anti-inflammatory drugs, COX-2 15 Perphenazine (Trilafon), Prochlorperazine (Compazine), inhibitors, and opiates. In some embodiments, the non-ste Thioridazine (Mellaril), Trifluoperazine (Stelazine), roidal anti-inflammatory drugs are selected from the group Mesoridazine, Promazine, Triflupromazine (Vesprin), consisting of Acetylsalicylic acid (Aspirin), Amoxiprin, Levomepromazine (Nozinan), Promethazine (Phenergan)), Benorylate/Benorilate, Choline magnesium salicylate, thioxanthenes, Chlorprothixene, Flupenthixol (Depixol and Diflunisal, Ethenzamide, Faislamine, Methyl salicylate, Fluanxol). Thiothixene (Navane), Zuclopenthixol (Clopixol Magnesium salicylate, Salicyl salicylate, Salicylamide, ary & Acuphase)), clozapine, olanzapine, Risperidone (Risper lalkanoic acids, Diclofenac, Aceclofenac, Acemethacin, dal), Quetiapine (Seroquel), Ziprasidone (Geodon), Amisul Alclofenac, Bromfenac, Etodolac, Indometacin, Nabume pride (Solian), Paliperidone (InVega), dopamine, bifeprunoX, tone, Oxametacin, Proglumetacin, Sulindac, Tolimetin, norclozapine (ACP-104), Aripiprazole (Abilify), Tetrabena 2-arylpropionic acids, Ibuprofen, Alminoprofen, Benoxapro 25 Zine, and Cannabidiol. fen, Carprofen, Dexibuprofen, Dexketoprofen, Fenbufen, In some embodiments, the hypnotic drugs include, but are Fenoprofen, Flunoxaprofen, Flurbiprofen, Ibuproxam, not limited to, Barbiturates, Opioids, benzodiazepines, alpra Indoprofen, Ketoprofen, Ketorolac, Loxoprofen, Naproxen, Zolam, bromazepam (Lexotan), chlordiazepoxide (Librium), Oxaprozin, Pirprofen, Suprofen, Tiaprofenic acid), N-arylan Clobazam, Clonazepam, CloraZepate, Diazepam, Mida thranilic acids, Mefenamic acid, Flufenamic acid, Meclofe 30 Zolam, Lorazepam, Nitrazepam, temazepam, nimetazepam, namic acid, Tolfenamic acid, pyrazolidine derivatives, Phe Estazolam, Flunitrazepam, oxazepam (Serax), temazepam nylbutazone, Ampyrone, AZapropazone, Clofezone, (Restoril, Normison, Planum, Tenox, and Temaze), Triaz KebuZone, Metamizole, Mofebutazone, Oxyphenbutazone, olam, nonbenzodiazepines, Zolpidem, Zaleplon, Zopiclone, PhenaZone, Sulfinpyrazone, oxicams, Piroxicam, Droxicam, ESZopiclone, antihistamines, Diphenhydramine, Doxy Lornoxicam, Meloxicam, Tenoxicam, Sulphonanilides, 35 lamine, Hydroxy Zine, Promethazine, gamma-hydroxybu nimeSulide, licofelone, and omega-3 fatty acids. In some tyric acid (Xyrem), Glutethimide, Chloral hydrate, Ethchlo embodiments, the COX-2 inhibitors are selected from the rvynol, Levomepromazine, Chlormethiazole, Melatonin, and group consisting of Celecoxib, Etoricoxib, Lumiracoxib, Alcohol. Parecoxib, Rofecoxib, and Valdecoxib. In some embodi In some embodiments, the sedative drugs include, but are ments, the opiate drugs are selected from the group consisting 40 not limited to, barbituates, amobarbital (Amytal), pentobar of natural opiates, alkaloids, morphine, codeine, thebaine, bital (Nembutal), secobarbital (Seconal), Phenobarbital, semi-synthetic opiates, hydromorphone, hydrocodone, oxy Methohexital. Thiopental, Methylphenobarbital, Methar codone, oxymorphone, desomorphine, diacetylmorphine bital, Barbexaclone), benzodiazepines, alprazolam, bro (Heroin), nicomorphine, dipropanoylmorphine, diamor mazepam (Lexotan), chlordiazepoxide (Librium), Clobazam, phine, benzylmorphine, Buprenorphine, Nalbuphine, Penta 45 Clonazepam, CloraZepate, Diazepam, Midazolam, Zocine, meperidine, diamorphine, ethylmorphine, fully Syn Lorazepam, Nitrazepam, temazepam, nimetazepam, Esta thetic opioids, fentanyl, pethidine, Oxycodone, Zolam, Flunitrazepam, oxazepam (Serax), temazepam (Re Oxymorphone, methadone, tramadol. Butorphanol, Levor storil, Normison, Planum, Tenox, and Temaze), Triazolam, phanol, propoxyphene, endogenous opioid peptides, endor herbal sedatives, ashwagandha, catnip, kava (Piper methysti phins, enkephalins, dynorphins, and endomorphins. 50 cum), mandrake, marijuana, Valerian, Solvent sedatives, chlo In some embodiments, the anxiolytic drugs include, but are ral hydrate (Noctec), diethyl ether (Ether), ethyl alcohol (al not limited to, benzodiazepines, alprazolam, bromazepam coholic beverage), methyl trichloride (Chloroform), (Lexotan), chlordiazepoxide (Librium), Clobazam, Clon nonbenzodiazepine sedatives, esZopiclone (Lunesta), Zale azepam, CloraZepate, Diazepam, Midazolam, Lorazepam, plon (Sonata), Zolpidem (Ambien), Zopiclone (Imovane, Nitrazepam, temazepam, nimetazepam, Estazolam, Fluni 55 Zimovane)), clomethiazole (clomethiazole), gamma-hy trazepam, oxazepam (Serax), temazepam (Restoril, Normi droxybutyrate (GHB), Thalidomide, ethchlorvynol son, Planum, Tenox, and Temaze, Triazolam, serotonin 1A (Placidyl), glutethimide (Doriden), ketamine (Ketalar, agonists, Buspirone (BuSpar), barbituates, amobarbital Ketaset), methaqualone (Sopor, Quaalude), methyprylon (Amytal), pentobarbital (Nembutal), secobarbital (Seconal), (Noludar), and ramelteon (Rozerem). Phenobarbital, Methohexital. Thiopental, Methylphenobar 60 In some embodiments, the muscle relaxant drugs include, bital, Metharbital, Barbexaclone), hydroxyzine, cannabidiol, but are not limited to, depolarizing muscle relaxants, Succi Valerian, kava (Kava Kava), chamomile, Kratom, Blue Lotus nylcholine, short acting non-depolarizing muscle relaxants, extracts, Sceletium tortuosum (kanna) and bacopa monniera. Mivacurium, Rapacuronium, intermediate acting non-depo In some embodiments, the anesthetic drugs include, but are larizing muscle relaxants, Atracurium, Cisatracurium, Rocu not limited to, local anesthetics, procaine, amethocaine, 65 ronium, Vecuronium, long acting non-depolarizing muscle cocaine, lidocaine, prilocaine, bupivacaine, levobupivacaine, relaxants, Alcuronium, Doxacurium, Gallamine, Metocu ropivacaine, dibucaine, inhaled anesthetics, Desflurane, rine, Pancuronium, Pipecuronium, and d-Tubocurarine. US 8,980,907 B2 10 The compositions are not limited to particular pain relief FIG. 4 shows a diagram of a dendrimer conjugate provided agent antagonists. In some embodiments, the pain relief agent in some embodiments of the present invention. antagonists include drugs that counter the effect of a pain FIG. 5 shows a diagram of a dendrimer conjugate provided relief agent (e.g., an anesthetic antagonist, an analgesic in some embodiments of the present invention. antagonist, a mood Stabilizer antagonist, a psycholeptic drug 5 FIG. 6 shows a diagram of a dendrimer conjugate provided antagonist, a psychoanaleptic drug antagonist, a sedative drug in some embodiments of the present invention. antagonist, a muscle relaxant drug antagonist, and a hypnotic FIG. 7 shows the release of a therapeutic compound from a drug antagonist). In some embodiments, pain relief agent dendrimer conjugate in one embodiment of the invention. antagonists include, but are not limited to, a respiratory stimu FIG. 8 shows the release of a therapeutic compound from a lant, Doxapram, BIMU-8, CX-546, an opiod receptor antago 10 nist, Naloxone, naltrexone, nalorphine, levallorphan, dendrimer conjugate in one embodiment of the invention. cyprodime, naltrindole, norbinaltorphimine, buprenorphine, FIG.9A, FIG.9B, FIG.9C and FIG.9D show a diagram of a benzodiazepine antagonist, flumazenil, a non-depolarizing a dendrimer conjugate provided in Some embodiments of the muscle relaxant antagonist, and neostigmine. present invention. In some embodiments, the moiety comprises a plurality of 15 FIG. 10 shows the release of a therapeutic compound from therapeutic agents (e.g., 2, 3, 4, 5, 10, 15, 50, 100, at any esterase sensitive linker-dendrimer conjugate in one embodi desired ratio). In some embodiments, the moeity comprises a ment of the invention (Scheme 1). plurality of pain relief agents (e.g., ketamine andlorazepam). FIG. 11 shows examples of several (A, B, and C) elimina In some embodiments, the therapeutic agent is a pain relief tion linkers designed for esterase triggered cleavage. agent, wherein the pain relief agent is morphine. In some FIG. 12 shows the characterization of therapeutic com embodiments, the therapeutic agent is a pain relief agent pound release from dendrimer conjugates of the present antagonist, wherein the pain relief agent antagonist is Dox invention. apram. In some embodiments, the therapeutic agent is a pain FIG. 13 shows dendrimer conjugate and methods of syn relief agent antagonist, wherein the pain relief agent antago thesizing the same in some embodiments of the invention. nist is Naloxone. 25 FIG. 14 shows a diagram of a dendrimer conjugate pro In certain embodiments, the present invention provides vided in some embodiments of the present invention. methods for reducing pain in a Subject (e.g., cat, dog, human, FIG. 15 shows a diagram of a dendrimer conjugate pro monkey, ape, cow, etc.) comprising administering to the Sub vided in some embodiments of the present invention. ject at least one composition comprising a dendrimer linked FIG. 16 shows a diagram of a dendrimer conjugate pro to a moiety comprising a trigger agent, a linkage agent, a 30 vided in some embodiments of the present invention. targeting agent, and at least one therapeutic agent, wherein FIG. 17 shows an example of a dendrimer conjugate the therapeutic agent is a pain relief agent designed to reduce designed for glucuronidase triggered cleavage in one embodi and/or eliminate pain in a subject and/or a pain relief agent ment of the present invention. antagonist (as described above). FIG. 18 shows an example of a dendrimer conjugate In some embodiments, the two compositions are adminis 35 designed for hypoxia induced activation in one embodiment tered to the Subject Such that one of the compositions com of the present invention. prises a pain relief agent and one of the compositions com FIG. 19 shows that, in some embodiments, a heteroaro prises a pain relief agent antagonist. In such embodiments, for matic nitro compound present in a dendrimer conjugate of the example, the pain relief agent is morphine and the pain relief present invention is reduced to either an amine or a hydroxy agent antagonist is Naloxone. In such embodiments, for 40 lamine, thereby triggering the spontaneous release of a thera example, the pain relief agent is ketamine and/or lorazepam peutic agent/drug. and the pain relief agent antagonist is Doxapram. FIG. 20 depicts the activation of a dendrimer conjugate In some embodiments, the present invention provides comprising either a 1.4 or a 1.6 elimination linker in embodi methods for treating cancer localized with a Subject’s central ments of the present invention. nervous system (e.g., brain) administering to the Subject at 45 FIG. 21 shows that a spacer (R2) can be used to decrease least one composition comprising a dendrimer linked to a steric hindrance in a dendrimer conjugate in Some embodi moeity comprising a trigger agent (e.g., a trigger agent that is ments of the present invention. sensitive to (e.g., is cleaved by) hypoxia) (e.g., a trigger agent FIG. 22 depicts a dendrimer conjugate comprising a that is sensitive to (e.g., is cleaved by) tumor associated cyclization based linker in Some embodiments of the present enzymes), a linkage agent, a targeting agent configured to 50 invention. cross the blood brain barrier, and at least one therapeutic FIG. 23 depicts cyclization based linkers in some embodi agent configured for treating cancer. In some embodiments, ments of the invention. the dendrimers are designed for retention within the CNS FIG.24 depicts a linker utilized in a dendrimer conjugate in through conjugation of locking agents designed to prevent Some embodiments of the present invention. back diffusion of the dendrimer across the BBB (e.g., pyri 55 FIG.25 shows branched self-elimination linkers utilized in dinium molecule, which when activated by enzymatic reduc a dendrimer conjugate in Some embodiments of the present tion, becomes charged and locks the dendrimer in the CNS). invention. FIG. 26A and FIG. 26B depicts dendrimer conjugates pro DESCRIPTION OF THE DRAWINGS vided in some embodiments of the present invention. 60 FIG. 27 shows a dendrimer comprising a simple ester (top FIG. 1 shows a diagram of a dendrimer conjugate provided portion of figure) and a dendrimer conjugate comprising an in Some embodiments of the present invention. elimination linker (e.g., a 1, 6, elimination linker/spacer as FIG. 2A, FIG. 2B and FIG. 2C show a diagram of a den shown in the bottom portion). drimer conjugate provided in some embodiments of the FIG.28 shows a dendrimer conjugate comprising hydroxy present invention. 65 camptothecin in some embodiments of the invention. FIG.3 shows a diagram of a dendrimer conjugate provided FIG. 29 shows a synthesis scheme for generating a den in Some embodiments of the present invention. drimer comprising a hypoxia induced linker. US 8,980,907 B2 11 12 FIG. 30 shows a synthesis scheme for generating a den is not known. The term further includes people who once had drimer comprising a hypoxia induced linker. cancer (e.g., an individual in remission). A “Subject Suspected FIG.31 shows a diagram depicting a mechanism of release of having cancer is sometimes diagnosed with cancer and is ofatherapeutic agent from a dendrimer comprising a hypoxia Sometimes found to not have cancer. sensitive linker. 5 As used herein, the term “subject diagnosed with a cancer' FIG. 32 shows two dendrimer conjugates. refers to a subject who has been tested and found to have FIG.33 shows a schematic representation of CNS locking cancerous cells. The cancer may be diagnosed using any targeted drug. Suitable method, including but not limited to, biopsy, X-ray, FIG. 34 shows a strategy to analyze drug conjugated to the blood test, and the diagnostic methods of the present inven dendrimer scaffold with ester linkage using HPLC. 10 tion. FIG. 35 shows structures of pain relief agents. As used herein, the term “initial diagnosis' refers to a test FIG. 36 shows structures of morphine pro-drugs. result of initial cancer diagnosis that reveals the presence or FIG. 37 shows the structure of a Naloxone pro-drug where absence of cancerous cells (e.g., using a biopsy and histol the length of the spacer was varied to produce 3 additional ogy). Naloxone pro-drugs. 15 As used herein, the term “identifying the risk of said tumor FIG.38 shows drug formulations. metastasizing” refers to the relative risk (e.g., the percent FIG. 39 shows time-dependent release kinetics of Mor chance or a relative score) of a tumor metastasizing. phine compounds incubated in fresh frozen plasma. As used herein, the term “identifying the risk of said tumor FIG. 40 shows time-dependent release kinetics of Nalox recurring refers to the relative risk (e.g., the percent chance one pro-drug incubated in fresh frozen plasma under hypoxia. or a relative score) of a tumor recurring in the same organ as FIG. 41 shows a schematic depicting the complexation of the original tumor. drug to G5 PAMAM dendrimer. As used herein, the term “subject at risk for cancer refers FIG. 42 shows a scheme depicting synthesis used to form to a subject with one or more risk factors for developing a pro-drug dendrimer conjugates. specific cancer. Risk factors include, but are not limited to, FIG. 43 shows a schematic showing the initial loading and 25 gender, age, genetic predisposition, environmental expose, Subsequent decrease of drug concentration in blood due to and previous incidents of cancer, preexisting non-cancer dis distribution and elimination. Models are used to determine an eases, and lifestyle. appropriate mixture of compounds needed to maintain the As used herein, the term "characterizing cancer in Subject’ desired drug concentration. refers to the identification of one or more properties of a FIG. 44 shows in vitro sustained release of morphine using 30 cancer sample in a subject, including but not limited to, the a morphine pro-drug. presence of benign, pre-cancerous or cancerous tissue and the FIG. 45 shows the release kinetics of free morphine from stage of the cancer. the prodrug in the various plasma samples. As used herein, the term “stage of cancer refers to a FIG. 46 shows that naloxone is released from an indole qualitative or quantitative assessment of the level of advance quinone based naloxone prodrug only under low oxygen con 35 ment of a cancer. Criteria used to determine the stage of a ditions. cancer include, but are not limited to, the size of the tumor, FIG. 47 shows sustained release of morphine in the guinea whether the tumor has spread to other parts of the body and pig model over a six hour period with prodrug A. where the cancer has spread (e.g., within the same organ or FIG. 48 shows in vivo studies with a guinea pig model region of the body or to another organ). demonstrating that naloxone is release from naloxone-pro 40 As used herein, the term “providing a prognosis' refers to drug only under low oxygen conditions. providing information regarding the impact of the presence of cancer on a subjects future health (e.g., expected morbidity DEFINITIONS or mortality, the likelihood of getting cancer, and the risk of metastasis). To facilitate an understanding of the present invention, a 45 As used herein, the term "characterizing tissue in a Subject’ number of terms and phrases are defined below: refers to the identification of one or more properties of a tissue As used herein, the term “subject” refers to any animal sample (e.g., including but not limited to, the presence of (e.g., a mammal), including, but not limited to, humans, non cancerous tissue, the presence of pre-cancerous tissue that is human primates, rodents, and the like, which is to be the likely to become cancerous, and the presence of cancerous recipient of a particular treatment. Typically, the terms “sub 50 tissue that is likely to metastasize. ject' and “patient are used interchangeably herein in refer As used herein, the term “providing a prognosis' refers to ence to a human Subject. providing information regarding the impact of the presence of As used herein, the term “antagonist' or “pain relief agent cancer (e.g., as determined by the diagnostic methods of the antagonist” refers to an agent able to counter the effect of a present invention) on a subject’s future health (e.g., expected pain relief agent and/or the effect of a pain relief agent (e.g., 55 morbidity or mortality, the likelihood of getting cancer, and respiratory distress, cardiovascular distress). the risk of metastasis). As used herein, the term "subject Suspected of having As used herein, the term “non-human animals' refers to all cancer refers to a subject that presents one or more symp non-human animals including, but not limited to, Vertebrates toms indicative of a cancer (e.g., a noticeable lump or mass) Such as rodents, non-human primates, ovines, bovines, rumi or is being screened for a cancer (e.g., during a routine physi 60 nants, lagomorphs, porcines, caprines, equines, canines, cal). A subject Suspected of having cancer may also have one felines, ayes, etc. or more risk factors. A subject Suspected of having cancer has As used herein, the term “sample' is used in its broadest generally not been tested for cancer. However, a “subject sense. In one sense, it is meant to include a specimen or Suspected of having cancer encompasses an individual who culture obtained from any source, as well as biological and has received a preliminary diagnosis (e.g., a CT scan showing 65 environmental samples. Biological samples may be obtained a mass) but for whom a confirmatory test (e.g., biopsy and/or from animals (including humans) and encompass fluids, Sol histology) has not been done or for whom the stage of cancer ids, tissues, and gases. Biological samples include blood US 8,980,907 B2 13 14 products, such as plasma, serum and the like. Environmental jugate that comprises a degradable linkage of the present samples include environmental material Such as Surface mat invention is capable of generating hRNase that is free (e.g., ter, soil, water, crystals and industrial samples. Such completely or partially free) of the polymer (e.g., in Vivo after examples are not however to be construed as limiting the hydrolysis of the linkage). sample types applicable to the present invention. A "physiologically cleavable' or “hydrolysable' or As used herein, the term "drug is meant to include any “degradable bond is a bond that reacts with water (i.e., is molecule, molecular complex or Substance administered to an hydrolyzed) under physiological conditions. The tendency of organism for diagnostic or therapeutic purposes, including a bond to hydrolyze in water will depend not only on the medical imaging, monitoring, contraceptive, cosmetic, nutra general type of linkage connecting two central atoms but also ceutical, pharmaceutical and prophylactic applications. The 10 on the Substituents attached to these central atoms. Appropri term “drug is further meant to include any such molecule, ate hydrolytically unstable or weak linkages include but are molecular complex or Substance that is chemically modified not limited to carboxylate ester, phosphate ester, anhydrides, and/or operatively attached to a biologic or biocompatible acetals, ketals, acyloxyalkyl ether, imines, orthoesters, pep Structure. tides and oligonucleotides. As used herein, the term “purified’ or “to purify” or “com 15 An "enzymatically degradable linkage” means a linkage positional purity refers to the removal of components (e.g., that is Subject to degradation by one or more enzymes. contaminants) from a sample or the level of components (e.g., A“hydrolytically stable' linkage or bond refers to a chemi contaminants) within a sample. For example, unreacted moi cal bond (e.g., typically a covalent bond) that is Substantially eties, degradation products, excess reactants, or byproducts stable in water (i.e., does not undergo hydrolysis under physi are removed from a sample following a synthesis reaction or ological conditions to any appreciable extent over an preparative method. extended period of time). Examples of hydrolytically stable The terms “test compound” and “candidate compound linkages include, but are not limited to, carbon-carbon bonds refer to any chemical entity, pharmaceutical, drug, and the (e.g., in aliphatic chains), ethers, amides, urethanes, and the like that is a candidate for use to treat or prevent a disease, like. illness, sickness, or disorder of bodily function (e.g., cancer). 25 As used herein, the term "click chemistry” refers to chem Test compounds comprise both known and potential thera istry tailored to generate Substances quickly and reliably by peutic compounds. A test compound can be determined to be joining Small modular units together (see, e.g., Kolb et al. therapeutic by Screening using screening methods known in (2001) Angewandte Chemie Intl. Ed. 40:2004-2011; Evans the art. (2007) Australian J. Chem. 60:384-395; Carlmark et al. As used herein, the term "NAALADase inhibitor” refers to 30 (2009) Chem. Soc. Rev. 38:352-362; each herein incorpo any one of a multitude of inhibitors for the neuropeptidase rated by reference in its entirety). NAALADase (N-acetylated-alpha linked acidic dipepti As used herein, the term "one-pot synthesis reaction” or dase). Such inhibitors of NAALADase have been well char equivalents thereof, e.g., "1-pot”, “one pot', etc., refers to a acterizied. For example, an inhibitor can be selected from the chemical synthesis method in which all reactants are present group comprising, but not limited to, those found in U.S. Pat. 35 in a single vessel. Reactants may be added simultaneously or No. 6,011,021, herein incorporated by reference in its sequentially, with no limitation as to the duration of time entirety. elapsing between introduction of sequentially added reac As used herein, the term "nanodevice' or "nanodevices' tantS. refer, generally, to compositions comprising dendrimers of As used herein, an “ester coupling agent” refers to a the present invention. As such, a nanodevice may refer to a 40 reagent that can facilitate the formation of an ester bond composition comprising a dendrimer and metal nanoparticles between two reactants. The present invention is not limited to (e.g., iron oxide nanoparticles (e.g., poly(styrene Sulfonate) any particular coupling agent or agents. Examples of cou (PSS)-coated iron oxide nanoparticles)) of the present inven pling agents include but are not limited to 2-chloro-1-meth tion that may contain one or more functional groups (e.g., a ylpyridium iodide and 4-(dimethylamino) pyridine, or dicy therapeutic agent) conjugated to the dendrimer. A nanodevice 45 clohexylcarbodiimide and 4-(dimethylamino) pyridine or may also refer to a composition comprising two or more diethyl azodicarboxylate and triphenylphosphine or other different dendrimers of the present invention. carbodiimide coupling agent and 4-(dimethylamino)pyri As used herein, the term “degradable linkage,” when used dine. in reference to a polymer (e.g., PEG-hRNase conjugate of the As used herein, the term “glycidolate” refers to the addition present invention), refers to a conjugate that comprises a 50 ofa 2,3-dihydroxylpropyl group to a reagent using glycidolas physiologically cleavable linkage (e.g., a linkage that can be a reactant. In some embodiments, the reagent to which the hydrolyzed (e.g., in vivo) or otherwise reversed (e.g., via 2,3-dihydroxylpropyl groups are added is a dendrimer. In enzymatic cleavage). Such physiologically cleavable link some embodiments, the dendrimer is a PAMAM dendrimer. ages include, but are not limited to, ester, carbonate ester, Glycidolation may be used generally to add terminal carbamate, Sulfate, phosphate, acyloxyalkyl ether, acetal, and 55 hydroxyl functional groups to a reagent. ketal linkages (See, e.g., U.S. Pat. No. 6,838,076, herein As used herein, the term “ligand’ refers to any moiety incorporated by reference in its entirety). Similarly, the con covalently attached (e.g., conjugated) to a dendrimer branch; jugate may comprise a cleavable linkage present in the link in preferred embodiments, such conjugation is indirect (e.g., age between the polymer and hRNase, or, may comprise a an intervening moiety exists between the dendrimer branch cleavable linkage present in the polymer itself (e.g., such that 60 and the ligand) rather than direct (e.g., no intervening moiety when cleaved, a small portion of the polymer remains on the exists between the dendrimer branch and the ligand). Indirect hRNase molecule) (See, e.g., U.S. Pat. App. Nos. attachment of a ligand to a dendrimer may exist where a 20050158273 and 20050181449, each of which is herein scaffold compound (e.g., triazine scaffold) intervenes. In pre incorporated by reference in its entirety). For example, a PEG ferred embodiments, ligands have functional utility for spe polymer comprising an ester linkage can be utilized for con 65 cific applications, e.g., for therapeutic, targeting, imaging, or jugation to hRNase to create a PEG-hRNase conjugate (See, drug delivery function(s). The terms “ligand”, “conjugate'. e.g., Kuzlowski et al., Biodrugs, 15, 419-429 (2001). A con and “functional group' may be used interchangeably. US 8,980,907 B2 15 16 DETAILED DESCRIPTION OF THE INVENTION embodiment, a linker may comprise a chemical structure that allows, for example, conjugation of a targeting moiety and a The present invention relates to novel therapeutic and diag therapeutic compound to the linker. Thus, in some embodi nostic dendrimers. In particular, the present invention is ments, a dendrimer conjugate of the present invention permits directed to dendrimer-linker conjugates, methods of synthe control of the Stoichiometry between targeting agent and sizing the same, compositions comprising the conjugates, as therapeutic compound (e.g., generation of one to one ratio, well as systems and methods utilizing the conjugates (e.g., in two to one ratio, one to two ratio, one to three ratio etc. diagnostic and/or therapeutic settings (e.g., for the delivery of between targeting and therapeutic moieties). therapeutics, imaging, and/or targeting agents (e.g., in disease In some embodiments, a dendrimer conjugated to a linker (e.g., cancer) diagnosis and/or therapy, pain therapy, etc.)). 10 that is conjugated to a targeting agent and/ortherapeutic agent Accordingly, dendrimer-linker conjugates of the present comprises a linker that is configured to be irreversibly invention may further comprise one or more components for degraded (e.g., that is non-reversible (e.g., that permits drug targeting, imaging, sensing, and/or providing a therapeutic or delivery at the correct time and/or at the correct place)). diagnostic material and/or monitoring response to therapy. In some embodiments, the present invention provides den Accordingly, in Some embodiments, the present invention 15 drimer molecules (e.g., polyamideamine (PAMAM) den provides a linker conjugated to an agent (e.g., therapeutic drimers, polypropylamine (POPAM) dendrimers, or agent, imaging agent, targeting agent, triggering agent) (e.g., PAMAM-POPAM dendrimers) conjuguated to one or more that can be conjugated to a dendrimer (e.g., for specific tar pain relief agents (e.g., prodrug analgesic molecules). In geting and/or therapeutic use of the dendrimer)). Thus, in Some embodiments, the dendrimers conjugated to one or Some embodiments, the present invention provides methods more pain relief agents (e.g., prodrug analgesic molecules) of synthesizing dendrimer conjugates (e.g., PAMAM den are configured for controlled and/or sustained release of the drimers) comprising linkers (e.g., conjugated to a trigger pain relief agents (e.g., through use of targeting agents, link moiety, therapeutic moiety and/or other type of moiety), com ing agents, and/or trigger agents conjugated to the dendrimer positions comprising the same, and methods of using the and/or pain relief agent). In some embodiments, the pain same in the diagnosis, imaging and treatment of disease (e.g., 25 relief agent conjugated to the dendrimer is active upon admin cancer, inflammatory disease, chronic pain, etc.). istration to a subject. In some embodiments, Sustained release The present invention provides a multiplicity of linkers and (e.g., slow release over a period of 24-48 hours) of the pain agents (e.g., therapeutic agent, imaging agent, targeting relief agent is accomplished through conjugating the pain agent, triggering agent) that find use in the present invention. relief agent to the dendrimer through, for example, a linkage Indeed, the present invention is not limited to any particular 30 agent connected to a trigger agent that slowly degrades in a linker or to any particular targeting agent or to any particular biological system (e.g., ester linkage). In some embodiments, dendrimer. In some embodiments, the present invention pro constitutively active release of the pain relief agent is accom vides a dendrimer conjugated to a linker that is conjugated to plished through conjugating the pain relief agent to the den an agent (e.g., therapeutic agent, imaging agent, targeting drimer through, for example, a linkage agent connected to a agent, triggering agent), and methods of generating and using 35 trigger agent that renders the pain relief agent constitutively the same (e.g., to treat cancer, pain and/or inflammation, etc.). active in a biological system (e.g., amide linkage, ether link In some embodiments, a dendrimer conjugated to a linker that age). In some embodiments, the dendrimers conjugated to is conjugated to an agent (e.g., therapeutic agent, imaging one or more pain relief agents simultaneously configured for agent, targeting agent, triggering agent) decreases the number Sustained release (e.g., a slow release mechanism that of conjugation steps required to form a dendrimer (e.g., a 40 achieves analgesic concentrations over a period of, for dendrimer conjugate (e.g., a dendrimer conjugated to a tar example, 24–48 hours) of the pain relief agent prevents geting agent, imaging agent, therapeutic agent and/or trigger adverse side effects of the pain relief agent (e.g., respiratory ing agent)). For example, in Some embodiments, the present failure, adverse cardiovascular consequences). The present invention provides a customizable dendrimer wherein one or invention further provides systems and methods for treating a plurality of linkers (e.g. attached to one or a plurality of 45 and/or managing pain through utilization of dendrimers con targeting agents, triggering agents and/or therapeutic agents) jugated to one or more pain relief agents. are conjugated to a dendrimer, thereby decreasing the number In some embodiments, as shown in FIG. 32A, the present of conjugation steps used to form a dendrimer (e.g., versus a invention provides compositions comprising a plurality of dendrimer that is conjugated to a targeting moiety in one step pain relief agents (e.g., Ketamine and Lorazepam) coupled to and that is separately conjugated to a linker (e.g., comprising 50 dendrimers with a linkage agent connected to a trigger agent a therapeutic agent, imaging agent, triggering agent or other that slowly degrades in a biological system (e.g., amide link moiety) in an additional conjugation step). In some embodi age, ester linkage, ether linkage) (as shown in FIG. 32A, the ments, a linker conjugated to one or more agents (e.g., thera trigger agent is an ester bond that is released by serum peutic agents, imaging agents, targeting agents, triggering esterases to mediate Sustained-release analgesia). When agents) is conjugated to one or more additional moieties 55 administered together, Ketamine and Lorazepam have favor including, but not limited to, a therapeutic agent, a triggering able analgesic and anxiolytic/amnestic qualities, and rela agent, an imaging agent, a triggering agent, etc. Thus, in some tively broad therapeutic indexes. Such compositions com embodiments, the present invention provides a dendrimer prising Ketamine and Lorazepam provide analgesia without with increased load capacity (e.g., increased load of thera the cardiovascular effects of opioids and minimize their peutic, imaging agent, etc. on the dendrimer). In some 60 major complication, that being respiratory depression. As a embodiments, two or more linkers (e.g., conjugated to one or feedback mechanism, compositions comprising a pain relief a plurality of targeting agents) are conjugated to a dendrimer agent antagonist (e.g., Doxapram) complexed with dendrimer via the same or different linkage (e.g., covalent linkage). through charge interaction that would be released by, for Several different schemes were evaluated for generating example, acidosis. While acidosis could be observed from dendrimer conjugates wherein a dendrimer is conjugated to 65 causes other than respiratory depression, this feedback one or more linkers that comprise multiple sites for binding mechanism is unique in that Doxapram stimulates respira (e.g., covalent binding) moieties. For example, in one tions without reducing analgesia. As such, there would not be US 8,980,907 B2 17 18 an issue in releasing the Doxapram regardless of the source of Daniels, T. R. et al., Clinical Immunology, 2006. 121(2): p. the acidosis. Indeed, the increased respiratory drive would be 159-176; Daniels, T. R. et al., Clinical Immunology, 2006. of benefit to compensate acidosis even when caused by meta 121(2): p. 144-158; each herein incorporated by reference in bolic Sources from traumatic injuries, hemorrhagic shock, their entireties). In some embodiments, the targeting agent for burns or rhabdomyolysis. One advantage to this approach is neuron targeting is a 12 amino acid peptide (Tet 1) (see, e.g., that it provides safe analgesia with easily achieved physi Liu, J. K., et al., Neurobiology of Disease, 2005. 19(3): p. ological feedback. 407-418; hereinincorporated by reference in its entirety). The For example, in some embodiments. G5 dendrimers with dendrimer conjugates are not limited to particular locking different percentages of Succinamic acid termini (Scheme 3) agents. In some embodiments, the locking agent for locking 10 the dendrimer conjugate within the CNS is the 1,4- wherein Doxapram molecules are encapsulated in the interior dihydrotrigonellines strigonelline (coffearine) re-dox system of dendrimers. In Such embodiments, the negative charges on where the lipophilic 1,4-dihydroform (L) is converted in vivo the dendrimer surface prevent the release of the drug due to to the hydrophilic quaternary form (L) by oxidation to pre the strong electrostatic interaction with the positively charged vent the dendrimer conjugate from diffusing back into the Doxapram hydrochloride. In Such embodiments, the drug is 15 circulation (see, e.g., Bodor, N. and P. Buchwald, Drug Dis released once the dendrimer Surface carboxyl groups are pro covery Today, 2002. 7(14): p. 766-774; herein incorporated tonated with decreasing pH. by reference in its entirety). In some embodiments, the den drimer conjugate device is eliminated from the CNS (e.g., because of acquired hydrophilicity due to loss of the quater Scheme 3. Molecular structure of Doxapram nary form). In some embodiments, the pain relief agent and/ CH3 or pain relief agent antagonist is attached to the dendrimer through, for example, triggering agents designed for delayed release (e.g., ester bonds, amide bonds, ether bonds). In some K N O embodiments wherein the dendrimer conjugate comprises a O \ / 25 pain relief agent antagonist (e.g., Naloxone), the pain relief agent antagonist is attached to the dendrimer through a link age agent connected to a trigger agent (e.g., re-dox linker) permitting release of the pain relief agent antagonist upon detection of reduced pCO2 levels. 30 In some embodiments, the present invention provides a dendrimer conjugate as shown in FIG. 1. For example, FIG. 1 In some embodiments, as shown in FIG. 32B shows two shows a targeting agent (T.A.) conjugated to a linker that is dendrimer conjugates designed for pain management in a also conjugated to a drug, wherein the linker conjugated to a Subject. One of the dendrimers is conjugated to a morphine drug and targeting agent is conjugated to a dendrimer conju drug through a linkage agent connected to a trigger agent 35 gated to an imaging agent (I.A.). In some embodiments, the (e.g., ester, amide) permitting Sustained release. The other present invention provides a dendrimer conjugate as shown in dendrimer is conjugated to a morphine antagonist (e.g., FIG. 2 (e.g., possessing targeted anticancer therapeutic moi Naloxone) through a linkage agent connected to a trigger ety). For example, FIG. 2 shows several structures of den agent (e.g., re-dox linker) permitting release of the morphine drimer conjugates, wherein R1, R2, R3 and R4 are each antagonist upon detection of reduced pCO2 levels. Each of the 40 independently selected from hydrogen, halogen, and alkyl. In dendrimers are targeted for CNS deliver through conjugation Some embodiments, the alkyl is straight or cyclic, unsubsti of targeting agents specific for the CNS (e.g., transferrin, a tuted or Substituted (e.g., by from 1 to 4 substituents (e.g., synthetic tetanus toxin fragment). Each of the dendrimers are selected from the group comprising, but not limited to, halo designed for retention within the CNS through conjugation of gen, amino, monoalkylamino, dialkylamino, hydroxy, locking agents designed to prevent back diffusion of the den 45 alkoxy, nitro, aryl, cyano, carboxyl, carboxamide, monoalky drimer across the BBB (e.g., pyridinium molecule, which lcarboxamide, dialkylcarboxamide, thiol, thioalkyl and sul when activated be enzymatic reduction, becomes charged and fonic acid)). In some embodiments, the “U” moiety is present locks the dendrimer in the CNS). or absent. In some embodiments, when the “U” moiety is In some embodiments, as shown in FIG. 33, the present absent, one of the R1,R2, R3 and/or R4 groups is linked to a invention provides dendrimer conjugates configured to pro 50 targeting agent through a linker and/or spacer. In some vide effective analgesia (e.g., narcotic analgesia (e.g., Mor embodiments, R5 is an alkyl (e.g., that is straight chained, phine)) over prolonged time periods. In some embodiments, branched, cyclic (e.g., that is substituted or unsubtituted)). In the dendrimer conjugates shown in FIG.33 are transported to Some embodiments, R6 is a hydrogen oran alkyl (e.g., of 1-4 the CNS, and retained in the CNS to provide, for example, carbons (e.g., that are straight chained or cyclic (e.g., that is constitutive narcotic analgesia. Such embodiments permit the 55 substituted or unsubtituted)). In some embodiments, Ra, Rb, use of smaller analgesic doses, while reducing the peripheral Rc, Rd and Re are each independently selected from hydro adverse effects. In some embodiments, as shown in FIG.33, gen, halogen, and alkyl. In some embodiments, the alkyl is the dendrimer conjugates comprise i) a targeting agent that straight or cyclic, unsubstituted or Substituted (e.g., by from 1 enables the conjugate to cross the BBB and target neurons, ii) to 4 substituents (e.g., selected from halogen, amino, a locking agent (e.g., a re-dox locking module) to prevent the 60 monoalkylamino, dialkylamino, hydroxy, alkoxy, nitro, aryl, dendrimer conjugate from diffusing back across the BBB. cyano, carboxyl, carboxamide, monoalkylcarboxamide, and iii) a pain relief agent (e.g., narcotic analgesic (Mor dialkylcarboxamide, thiol, thioalkyl and sulfonic acid. In phine)) or pain relief agent antagonist (e.g., Naloxone) some embodiments, the “U” moiety is present or absent. In coupled by different linking agents and triggering agents. The some embodiments, when the “U” moiety is absent, one of the dendrimer conjugates are not limited to particular targeting 65 Ra, Rb, Ric, Rd and Re groups is linked to a targeting agent agents. In some embodiments, the targeting agent for CNS through a linker and/or spacer. In some embodiments, “Y” is targeting through crossing the BBB is transferrin (see, e.g., an oxygenatom. In some embodiments, “Y” is two hydrogen US 8,980,907 B2 19 20 atoms. In some embodiments. G5 is a generation five poly the linker (B) proceeds through a spontaneous chemical (amidoamine) (PAMAM) dendrimer (e.g., conjugated to one breakdown (e.g., to a point where the therapeutic drug is or more imaging agents (e.g., FITC, etc.), although higher released from the dendrimer linker conjugate) at the target (e.g., G6, G7, G8, G9, G10 or higher, or lower, G4, G3, or G2 site, releasing (e.g., irreversibly releasing) the therapeutic dendrimers may also be used. In some embodiments, “W' is drug at the target site. In some embodiments, cleavage of the a linker comprising 1-8 carbon and/or nitrogen atoms (e.g., trigger and Subsequent linker breakdown is not necessary to straight chanined, branched, or cyclic, unsubstituted or Sub deliver the therapeutic drug to the target site. Several design stituted by “R” groups as described above. processes for generating a dendrimer conjugate comprising a In some embodiments, the present invention provides a trigger are shown in FIGS. 7 and 8. The dendrimer conjugates dendrimer conjugate as shown in FIGS. 3 and 4. In particular, 10 for the present invention (e.g., the dendrimer conjugates as a dendrimer conjugate as shown in FIG. 3 comprises a den shown in FIGS. 32, 3 and 4) are not limited to any particular drimer (e.g., a G5 PAMAM dendrimer conjugated to an imag dendrimer. Indeed, the conjugates may comprise a variety of ing agent (e.g., FITC) and/or targeting agent) conjugated to a different types of dendrimers. In some embodiments, the trigger molecule that is conjugated to a linker that is conju dendrimer is a PAMAM dendrimer (e.g., G3, G5 or G7 den gated to a therapeutic. A dendrimer conjugate as shown in 15 drimer). In some embodiments, one or more amino groups FIG. 4 comprises a dendrimer (e.g., a G5 PAMAM dendrimer present on the dendrimer are linked (e.g., through a covalent conjugated to an imaging agent (e.g., FITC) and/or targeting bond) to one or more targeting agents (e.g., folic acid) and/or agent) conjugated to a linker that is conjugated to a trigger and imaging agents (e.g., FITC) (e.g., as described in U.S. Pat. to a therapeutic moiety. The conjugates of FIGS. 3 and 4 are Nos. 6,471.968 and 7,078.461; U.S. Patent Pub. Nos. configured to be non-toxic to normal cells. For example, the 200201651.79 and 2007004 1934 and WO 06/033766, each of conjugates are configured in Such a way so as to release their which is hereby incorporated by reference in its entirety for therapeutic agent only at a specific, targeted site (e.g., through all purposes). activation of a trigger molecule that in to leads to release of In some embodiments, the present invention provides a the therapeutic agent) For example, once a conjugate arrives dendrimer conjugate as shown in FIG. 9. In particular, a at a target site in a Subject (e.g., a tumor, or a site of inflam 25 dendrimer conjugate as shown in FIG. 9 comprises a den mation), components in the target site (e.g., a tumor associ drimer (e.g., a G5 PAMAM dendrimer conjugated to an imag ated factor, or an inflammatory or pain associated factor) ing agent (e.g., FITC) and/or targeting agent) conjugated to a interacts with the trigger moiety thereby initiating cleavage of trigger molecule that is conjugated to a linker that is conju this unit from the linker. In some embodiments, once the gated to a therapeutic, or a dendrimer (e.g., a G5 PAMAM trigger is cleaved from the linker (e.g., by a target associated 30 dendrimer conjugated to an imaging agent (e.g., FITC) and/or moiety, the linker proceeds through spontaneous chemical targeting agent) conjugated to a linker that is conjugated to a breakdown thereby releasing the therapeutic agent at the tar trigger and to a therapeutic moiety). For example, FIG. 9 get site (e.g., in its active form). The present invention is not shows several structures of dendrimer conjugates, wherein limited to any particular target associated moiety (e.g., that R1, R2, R3 and R4 are each independently selected from interacts with and initiates cleavage of a trigger). In some 35 hydrogen, halogen, and alkyl. In some embodiments, the embodiments, the target associated moiety is a tumor associ alkyl is straight orcyclic, unsubstituted or Substituted (e.g., by ated factor (e.g., an enzyme (e.g., glucuronidase and/or plas from 1 to 4 Substituents (e.g., selected from the group com min), a cathepsin, a matrix metalloproteinase, a hormone prising, but not limited to, halogen, amino, monoalkylamino, receptor (e.g., integrin receptor, hyaluronic acid receptor, dialkylamino, hydroxy, alkoxy, nitro, aryl, cyano, carboxyl, luteinizing hormone-releasing hormone receptor, etc.), can 40 carboxamide, monoalkylcarboxamide, dialkylcarboxamide, cer and/or tumor specific DNA sequence), an inflammatory thiol, thioalkyl and sulfonic acid. In some embodiments, R5 is associated factor (e.g., chemokine, cytokine, etc.) or other an alkyl that is straight, branched or cyclic, that is unsubsti moiety. tuted or substituted. In some embodiments, R6 is a hydrogen Although an understanding of a mechanism of action is not or alkyl of 1-4 carbons that are straight, branched or cyclic, necessary to practice the present invention, and the present 45 that is unsubstituted or substituted. In some embodiments, the invention is not limited to any particular mechanism of action, two R6 are connected together to form a ring of 306 members. in Some embodiments, a dendrimer conjugate as described in In some embodiments, R', R", R" and R" are each indepen FIG.3 or 4 provides a therapeutic to a site by a mechanism as dently selected from hydrogen, halogen, and alkyl. In some shown in FIG. 5 or 6. For example, as shown in FIG. 5, a embodiments, the alkyl is straight or cyclic, unsubstituted or dendrimer conjugate comprising a dendrimer (e.g., a G5 50 Substituted (e.g., by from 1 to 4 Substituents (e.g., selected PAMAM dendrimer conjugated to an imaging agent (e.g., from the group comprising, but not limited to, halogen, FITC) and/or targeting agent) conjugated to a trigger mol amino, monoalkylamino, dialkylamino, hydroxy, alkoxy, ecule that is conjugated to a linker that is conjugated to a nitro, aryl, cyano, carboxyl, carboxamide, monoalkylcar therapeutic (A) interacts with a target associated moiety boxamide, dialkylcarboxamide, thiol, thioalkyl and sulfonic thereby activating the trigger and initiating cleavage of same, 55 acid. In some embodiments, X, X2 and X3 are either oxygen releasing the linker therapeutic drug conjugate. Once cleav or “NR', wherein “N” is a nitrogen atom, and “R” is an alkyl age of the trigger occurs, the linker (B) proceeds through a that is straight or branched or cyclic (e.g., Substituted or spontaneous chemical breakdown at the target site, releasing unsubstituted). In some embodiments, “Y” is an oxygenatom (e.g., irreversibly releasing) the therapeutic drug at the target or two hydrogen atoms. In some embodiments, A-B is an site. In some embodiments, as shown in FIG. 6, a dendrimer 60 ethylene group (e.g., unsubstituted or Substituted by alkyls conjugate comprising a dendrimer (e.g., a G5 PAMAM den (e.g., straight or cyclic). In some embodiments, A-B are con drimer conjugated to an imaging agent (e.g., FITC) and/or nected by a carbon chain (e.g., of 2, 3, 4, 5, or more carbons) targeting agent) conjugated to a linker that is conjugated to a and/or hetero atoms (e.g., forming a saturated or unsaturated trigger and to a therapeutic moiety (A) interacts with a target aromatic ring structure (e.g., comprising Substituents such as associated moiety thereby activating the trigger and initiating 65 R1,R2, R3 and R4). In some embodiments, G5 is a dendrimer cleavage of same, releasing a dendrimer-linker-therapeutic (e.g., a G5 PAMAM dendrimer conjugated to an imaging moiety from the trigger. Once cleavage of the trigger occurs, agent (e.g., FITC) and/or targeting agent). As described US 8,980,907 B2 21 22 herein, the present invention is not limited to any particular drimers with a linkage agent connected to a trigger agent that dendrimer. In some embodiments, “W' is a linker (e.g., com degrades upon detection of reduced pC2 concentrations (e.g., prising a carbon or nitrogen chain (e.g., 2, 3, 4, 5, 6,7,8,9, or through use of a re-doX linker). more carbons or nitrogens (e.g., straight or branched or cyclic The concept of prodrug systems in which the pharmacoph (e.g., Substituted or unsubstituted (e.g., with R groups as described above))). ores of drugs are masked by reductively cleavable groups has The present invention is not limited by the type of den been widely explored by many research groups and pharma drimer conjugate (e.g., comprising a trigger) for use in treat ceutical companies (see, e.g., Beall, H. D., et al., Journal of ing a subject. In some embodiments, the dendrimer conju Medicinal Chemistry, 1998. 41(24): p. 4755-4766; Ferrer, S., gates of the present invention (see, e.g., FIGS. 32 and 33) are 10 D. P. Naughton, and M. D. Threadgill, Tetrahedron, 2003. used as delivery agents for pain relief agents and pain relief 59(19): p.3445-3454; Naylor, M.A., et al., Journal of Medici agent antagonists. Such dendrimer conjugates are not limited nal Chemistry, 1997.40(15): p. 2335-2346; Phillips, R. M., et to uses within particular settings. Indeed, the dendrimer con al., Journal of Medicinal Chemistry, 1999. 42(20): p. 4071 jugates of the present invention (see, e.g., FIGS. 32 and 33) 4080; Zhang, Z. et al., Organic & Biomolecular Chemistry, may be used in any setting requiring treatment and/or man 15 2005. 3(10): p. 1905-1910; each of which are herein incor agement of pain (e.g., battlefield, ambulance, hospital, clinic, porated by reference in their entireties). Several such hypoxia rescue, etc.). In addition, the present invention contemplates activated prodrugs have been advanced to clinical investiga dendrimer conjugates comprising one or more pain relief tions, and work in relevant oxygen concentrations to prevent agent prodrugs and/or pain relief agent antagonist prodrugs cerebral damage. The present invention is not limited to par developed for site specific conversion to drug based on tumor ticular hypoxia activated trigger agents. In some embodi associated factors (e.g., hypoxia and pH, tumor-associated ments, the hypoxia activated trigger agents include, but are enzymes, and/or receptors). In some embodiments, den not limited to, indoquinones, nitroimidazoles, and nitrohet drimer conjugates of the present invention are configured erocycles (see, e.g., Damen, E. W. P. et al., Bioorganic & Such that a prodrug (e.g., pain relief agent prodrug, pain relief Medicinal Chemistry, 2002. 10(1): p.71-77; Hay, M.P., et al., agent antagonist prodrug) is conjugated to a linker that is 25 further conjugated to a targeting moiety (e.g., that targets the Journal of Medicinal Chemistry, 2003.46(25): p. 5533-5545: conjugate to a particular body region (e.g., CNS)). Although Hay, M. P. et al., Journal of the Chemical Society-Perkin an understanding of the mechanism is not necessary for the Transactions 1, 1999(19): p. 2759-2770; each herein incor present invention, and the present invention is not limited to porated by reference in their entireties). The mechanism of any particular mechanism of action, in Some embodiments, a 30 re-dox triggered release of drugs from these linkers is shown trigger component serves as a precursor for site-specific acti in Scheme 2. vation. For example, in some embodiments, once the trigger recognizes a particular condition (e.g., hypoxia), cleavage and/or processing of the trigger is induced, thereby releasing Scheme 2. Mechanism of re-dox triggered the pain relief agent and/or pain relief antagonist. 35 release of drug from dendritic polymers. The present invention is not limited to a particular trigger agent or to any particular cleavage and/or processing of the pus trigger agent. In some embodiments, the present invention O O O provides pain relief agents and/or pain relief agent antago 40 MeO Bioreduction nists coupled to dendrimers with a linkage agent connected to O Her a trigger agent that slowly degrades in a biological system N Me (e.g., amide linkage, ester linkage, ether linkage) (as shown in FIG. 32A, the trigger agent is an ester bond that is released by N serum esterases to mediate Sustained-release analgesia). \ In some embodiments, the present invention provides a 45 O dendrimer conjugate comprising a trigger agent that is sensi tive to (e.g., is cleaved by) hypoxia (e.g., as described in OH (1NO Ous Example 8). Hypoxia is a feature of several disease states, including cancer, inflammation and rheumatoid arthritis, as MeO V O U Drug -- well as an indicator of respiratory depression (e.g., resulting 50 from analgesic drugs). Advances in the chemistry of biore Me HO ductive drug activation have led to the design of various Q hypoxia-selective drug delivery systems in which the phar \ macophores of drugs are masked by reductively cleaved OH groups. In some embodiments, a dendrimer conjugate of the 55 OH OH present invention utilizes a quinone, N-oxide and/or (hetero) aromatic nitro groups. For example, a quinone present in a MeO dendrimer conjugate of the present invention is reduced to N Me phenol under hypoxia conditions, with spontaneous forma N tion of lactone that serves as a driving force for drug release 60 \ (e.g., as shown in FIG. 18). In some embodiments, a het OH eroaromatic nitro compound present in a dendrimer conju gate of the present invention is reduced to either an amine or Bioreduction a hydroxylamine, thereby triggering the spontaneous release of a therapeutic agent/drug (e.g., as shown in FIG. 19). In 65 Some embodiments, the present invention provides pain relief agents and/or pain relief agent antagonists coupled to den US 8,980,907 B2 23 24 -continued tive to (e.g., is cleaved by) and/or that associates with plas N t min. The serine protease plasmin is over expressed in many (UW). human tumor tissues. Tripeptide specifiers (e.g., including, r yO O O -e- -- but not limited to, Val-Leu-Lys) have been identified and HOHN N NM HO linked to anticancer drugs through elimination or cyclization O linkers. N In some embodiments, the present invention provides a (cy-OH dendrimer conjugate comprising a trigger agent that is sensi HN N tive to (e.g., is cleaved by) and/or that associates with a matrix 10 metalloproteases (MMPs). In some embodiments, the present invention provides a dendrimer conjugate comprising a trig 14 ger that is sensitive to (e.g., is cleaved by) and/or that associ H ates with B-Lactamase (e.g., a B-Lactamase activated cepha Drug losporin-based prodrug). 15 In some embodiments, the present invention provides a HO dendrimer conjugate comprising a trigger agent that is sensi tive to (e.g., is cleaved by) and/or activated by a receptor (e.g., expressed on a target cell (e.g., a tumor cell)). Thus, in some HO O O embodiments, a dendrimer conjugate comprises a receptor binding motif conjugated to a therapeutic agent (e.g., cyto Naloxone toxic drug) thereby providing target specificity. Examples include, but are not limited to, a dendrimer conjugate com In some embodiments, the present invention provides a prising a prodrug (e.g., of doxorubicin and/or paclitaxel) tar dendrimer conjugate comprising a trigger agent that is sensi geting integrin receptor, a hyaluronic acid receptor, and/or a 25 hormone receptor tive to (e.g., is cleaved by) and/or that associates with a tumor In some embodiments, the present invention provides a associated enzyme. In some embodiments, the present inven dendrimer conjugate comprising a trigger agent that is sensi tion provides a dendrimer conjugate comprising a trigger that tive to (e.g., is cleaved by) and/or activated by a nucleic acid. is sensitive to (e.g., is cleaved by) and/or that associates with Nucleic acid triggered catalytic drug release can be utilized in a glucuronidase. Glucuronic acid can be attached to several 30 the design of chemotherapeutic agents. Thus, in some anticancer drugs via various linkers. These anticancer drugs embodiments, disease specific nucleic acid sequence is uti include, but are not limited to, doxorubicin, paclitaxel, doc lized as a drug releasing enzyme-like catalyst (e.g., via com etaxel, 5-fluorouracil, 9-aminocamtothecin, as well as other plex formation with a complimentary catalyst-bearing drugs under development. These prodrugs are generally nucleic acid and/or analog). In some embodiments, the stable at physiological pH and are significantly less toxic than 35 release of a therapeutic agent is facilitated by the therapeutic the parent drugs. In some embodiments, dendrimer conju component being attached to a labile protecting group. Such gates comprising anticancer prodrugs find use for treating as, for example, cisplatin or methotrexate being attached to a necrotic tumors (e.g., that liberate B-glucuronidase) or for photolabile protecting group that becomes released by laser ADEPT with antibodies that can deliver B-glucuronidase to light directed at cells emitting a color of fluorescence (e.g., in target tumor cells. 40 addition to and/or in place of target activated activation of a In some embodiments, the present invention provides a trigger component of a dendrimer conjugate). In some dendrimer conjugate comprising a trigger agent that is sensi embodiments, the therapeutic device also may have a com tive to (e.g., is cleaved by) and/or that associates with brain ponent to monitor the response of the tumor to therapy. For enzymes. For example, trigger agents such as indolequinone example, where atherapeutic agent of the dendrimer induces are reduced by brain enzymes such as, for example, diapho 45 apoptosis of a target cell (e.g., a cancer cell (e.g., a prostate rase (see, e.g., Damen, E. W. P. et al., Bioorganic & Medici cancer cell)), the caspase activity of the cells may be used to nal Chemistry, 2002. 10(1): p.71-77; herein incorporated by activate a green fluorescence. This allows apoptotic cells to reference in